Zhongping Liao Address

The Role of Vacuolar Protein Sorting 4 Homolog B in Regulating Epidermal Growth Factor Receptor Signaling in Breast Cancer

Item Type dissertation

Authors Liao, Zhongping

Publication Date 2011

Abstract Breast cancer is the most common cancer among American women. Aberrant signaling by epidermal growth factor receptor (EGFR) is causally involved in the abnormal cell growth, proliferation, invasion and metastasis that occur in breast cancer. Endoso...

Keywords liquid chromatography mass spectrometry; vacuolar protein sorting 4 homolog B; Breast--Cancer; Proteomics; Receptor, Epidermal Growth Factor

Download date 02/10/2021 22:10:47

Link to Item http://hdl.handle.net/10713/790 Curriculum vitae

Name: Zhongping Liao

Address: BRB-7-040, 655 W. Baltimore Street, Baltimore, MD, 21201

Phone: 410-328-7824

Email: [email protected]

Degree and Date to be Conferred: Ph.D., 2011

Collegiate Institutions Attended: University of Maryland, Baltimore August 2006 – December 2011 Doctor of Philosophy, December 2011 Major: Molecular Medicine Minor: Cancer Biology

China Pharmaceutical University September 2000 – June 2003 Master of Science, June 2003 Major: Microbial Pharmaceuticals

China Pharmaceutical University September 1996 – June 2000 Bachelor of Science, June 2000 Major: Microbial Pharmaceuticals

Professional Publications: 1. Liao Z, Thomas SN, Wan Y, Lin HH, Shapiro P, Ann DK, Yang AJ. Phosphoproteomic analysis reveals activation of EGFR/EPS8/SH3BP1 complex and stabilization of cell adhesion in VPS4B-mediated endosomal EGFR signaling. PLoS ONE. In revision.

2. Liao Z, Wan Y, Thomas SN, Yang AJ. IsoQuant: a software tool for SILAC- based mass spectrometry quantitation. Anal Chem. In revision.

3. Liao Z, Thomas SN, Wan Y, Lin HH, Ann DK, Yang AJ. An internal standard-assisted synthesis and degradation proteomic approach reveals up- regulated fatty acid β-oxidation and down-regulated glycolysis in SKBR3 breast cancer cells with decreased VPS4B expression. In preparation.

4. Thomas SN, Wan Y, Liao Z, Hanson PI, Yang AJ. Stable isotope labeling with amino acids in cell culture based mass spectrometry approach to detect

transient protein interactions using substrate trapping. Anal Chem. 2011. Jul 15;83(14):5511-8.

5. Thomas SN, Funk KE, Wan Y, Liao Z, Davies P, Kuret J, Yang AJ. Dual modification of Alzheimer's disease PHF-tau protein by lysine methylation and ubiquitylation: a mass spectrometry approach. Acta Neuropathologica. 2011. In press. 6. Liao Z, Thomas SN, Wan Y, Thompson SM, Yang AJ. Using ex vivo SILAC- labeled brain slice cultures to address the dynamic assembly of synaptic protein complexes. USHUPO 7th Annual Conference, 2011, Raleigh, NC.

7. Liao Z, Wan Y, Thomas, SN, Ann DK, Yang AJ. Quantitative synthesis/degradation proteomic analysis of VPS4B-mediated perturbations in EGFR signaling in breast cancer cells under hypoxic conditions. 58th ASMS Conference, 2010, Salt lake city, UT.

8. Liao Z, Wan Y, Rynarzewski S, Thomas, SN, Yang AJ. Use of iTRAQ and MudPIT to quantify synaptic protein expression changes in post-synaptic density isolated from individual mouse brain. 57th ASMS Conference, 2009, Philadelphia, PA.

Professional Positions Held: Graduate Research Assistant University of Maryland, Baltimore Baltimore, MD September 2006 to present

Research Scientist in Monoclonal Antibody Shanghai Kehua Bio-Engineering Co., Ltd Shanghai, China July 2004 – June 2006

Genetic Engineering Research Scientist Shanghai Asia United Antibody Medical Co., Ltd Shanghai, China July 2003 – June 2004

Current Committee Memberships: Austin J. Yang, Ph.D. Associate Professor, Department of Anatomy and Neurobiology School of Medicine, University of Maryland, Baltimore

Angela Brodie, Ph.D. Professor, Department of Pharmacology and Experimental Therapeutics School of Medicine, University of Maryland, Baltimore

David K. Ann, Ph.D. Professor, Department of Molecular Pharmacology Beckman Research Institute, City of Hope Medical Center

Dudley K. Strickland, Ph.D. Professor, Center for Vascular and Inflammatory Diseases School of Medicine, University of Maryland, Baltimore

Iris Lindberg, Ph.D. Professor, Department of Anatomy and Neurobiology School of Medicine, University of Maryland, Baltimore

Yun Qiu, Ph.D. Professor, Department of Pharmacology and Experimental Therapeutics School of Medicine, University of Maryland, Baltimore

Abstract

Title of Dissertation: The Role of Vacuolar Protein Sorting 4 Homolog B in Regulating

Epidermal Growth Factor Receptor Signaling in Breast Cancer

Zhongping Liao, Doctor of Philosophy, 2011

Dissertation Directed by: Austin J. Yang, Associate Professor, Department of Anatomy and Neurobiology, School of Medicine, University of Maryland, Baltimore

Breast cancer is the most common cancer among American women. Aberrant signaling by epidermal growth factor receptor (EGFR) is causally involved in the abnormal cell growth, proliferation, invasion and metastasis that occur in breast cancer.

Endosomal sorting complexes required for transport (ESCRTs) have been suggested to play an important role in regulating EGFR signaling. Dysfunction of vacuolar protein sorting 4 homolog B (VPS4B), an ESCRT-III associated protein, inhibits EGFR degradation and prolongs intracellular EGFR signaling. Although EGFR signaling is one of the most well characterized signaling pathways, it is not known how changes of the endosomal/lysosomal system can affect EGFR signaling in breast cancer.

Here, we analyzed the role of VPS4B-mediated ESCRT dysfunction in altering

EGFR signaling in breast cancer cells by a proteomics-based systems biology approach.

By using a stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative phosphoproteomics approach coupled with an attenuated VPS4B expression

breast cancer cell culture model, we found that EGFR activates epidermal growth factor

receptor kinase substrate 8 (EPS8)/SH3 domain-binding protein 1 (SH3BP1) signaling

and subsequently stabilizes various cell adhesion and junction molecules. In addition,

EGFR activates extracellular signal-regulated kinase (ERK) signaling, and attenuates

signal transducer and activator of transcription (STAT) signaling. These results suggest

that dysfunction of VPS4B differentially activates and attenuates EGFR signaling in

breast cancer cells.

An internal standard-assisted synthesis and degradation mass spectrometry

(iSDMS) method was developed to determine the consequences of altered EGFR signaling as related to protein synthesis, degradation and relative protein abundance. We found that VPS4B down-regulation results in up-regulation of proteins with roles in fatty acid β-oxidation and down-regulation of proteins involved in glycolysis and fatty acid synthesis. The adoption of fatty acid β-oxidation as an alternative energy source could be

an unrevealed survival mechanism for breast cancer cells with VPS4B dysfunction.

In summary, our study provides the first systems biology evidence demonstrating

the potential pathological relevance of altered EGFR signaling caused by endosomal

dysfunction. The study casts a new light on the functional mechanisms underlying breast

cancer survival, which will aid the development of new breast cancer treatment strategies.

The Role of Vacuolar Protein Sorting 4 Homolog B in Regulating Epidermal Growth Factor Receptor Signaling in Breast Cancer

by Zhongping Liao

Dissertation submitted to the faculty of the Graduate School of the University of Maryland, Baltimore in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2011

Acknowledgements

I would like to thank my mentor Dr. Austin J. Yang for guiding me throughout my dissertation research and helping me mature as a researcher. I would also like to thank Dr. Stefani N. Thomas, Dr. Yunhu Wan, and Noble Nemieboka of the Proteomics

Core Facility, for their continued support and assistance. Without Dr. Yang, his lab and the Proteomics Core Facility, I would not have developed my critical thinking skills nor my passion for proteomics and breast cancer research. I would also like to acknowledge my thesis committee members, Dr. Angela Brodie, Dr. Alan E. Tomkinson, Dr. David K.

Ann, Dr. Dudley K. Strickland, Dr. Iris Lindberg, and Dr. Yun Qiu for providing valuable insight for my project. I would like to thank the following collaborators who have contributed to my work: Dr. David K. Ann, Dr. Scott M. Thompson, Dr. Frank

Yang and Helen H. Lin. Finally I would like to thank my wife Xinli Xu for keeping me grounded and being so patient with me throughout my graduate career.

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Table of Contents

I. Introduction ...... 1 Breast cancer...... 2 Epidermal growth factor receptor (EGFR) ...... 2 EGFR and breast cancer ...... 3 EGFR structure, activation and signaling ...... 4 EGFR endocytosis, sorting and degradation ...... 8 EGFR signaling during endocytosis and endosomal trafficking ...... 11 Vacuolar protein sorting 4 (VPS4) ...... 12 Tumor hypoxia ...... 16 Tumor hypoxia and EGFR ...... 17 Hypoxia and VPS4B ...... 18 Goals of the present study ...... 19

II. Phosphoproteomic Analysis Reveals Activation of EGFR/EPS8/SH3BP1 Complex and Stabilization of Cell Adhesion in VPS4B-Mediated Endosomal EGFR Signaling ... 21 ABSTRACT ...... 22 MATERIALS AND METHODS ...... 26 Cell culture and SILAC labeling ...... 26 Cell stimulation, lysis and in solution protein digestion ...... 27 Peptide fractionation by strong cation exchange ion chromatography ...... 28 Phosphopeptide enrichment using titanium dioxide micro-columns ...... 29 Analysis of phosphopeptides by LC-MS/MS ...... 29 Quantitation of SILAC-labeled phosphopeptides...... 30 RESULTS...... 32 Decreased expression of VPS4B in EGF-stimulated SKBR3 cells prolongs EGFR activation ...... 32 Quantitative phosphoproteomic analysis of aberrant EGF signaling mediated by down-regulation of VPS4B ...... 34

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Signal transduction pathway and network analysis reveals activation of EGFR/EPS8/SH3BP1 complex in EGF-stimulated SKBR3_shVPS4B cells ...... 44 DISCUSSION ...... 50 Increased activation of MAPKs in the EGFR signal transduction pathway...... 51 Decreased STAT activation in the EGFR signal transduction pathway ...... 52 Chromosomal structure, gene transcription and translation ...... 53 Regulation of actin and cytoskeleton remodeling ...... 55 CONCLUSION ...... 58

III. An Internal Standard-Assisted Synthesis and Degradation Proteomic Approach Reveals Up-Regulated Fatty Acid β-Oxidation and Down-Regulated Glycolysis in SKBR3 Breast Cancer Cells with Decreased VPS4B Expression ...... 61 ABSTRACT ...... 62 INTRODUCTION ...... 63 MATERIALS AND METHODS ...... 66 Cell culture ...... 66 Cell lysis and protein digestion ...... 67 Peptide fractionation ...... 69 LC-MS/MS analysis ...... 69 Peptide identification ...... 71 Peptide relative abundance ...... 71 Peptide degradation rate constant ...... 72 Peptide synthesis rate...... 72 Protein synthesis rate, degradation rate constant and relative abundance ...... 74 RESULTS...... 74 iSDMS analysis of dynamic protein profiles ...... 74 Overall protein synthesis rate, degradation rate constant and relative abundance .... 81 Correlation between altered protein expression in SKBR3_shVPS4B cells and protein synthesis and degradation...... 87 VPS4B down-regulation alters energy metabolism in SKBR3 cells ...... 94 DISCUSSION ...... 97

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IV. IsoQuant: A Software Tool for SILAC-Based Mass Spectrometry Quantitation ... 104 ABSTRACT ...... 105 INTRODUCTION ...... 106 MATERIALS AND METHODS ...... 109 SILAC labeling and hippocampal slice culture ...... 109 Preparation of cell lysates and protein digestion ...... 109 LC-MS/MS analysis ...... 111 RESULTS AND DISCUSSION ...... 112 Overview of IsoQuant SILAC-based quantitation ...... 112 IsoQuant minimizes under-sampling problem when a data-dependent method is used to acquire MS2 spectra ...... 116 Peptide ratios calculated by weighted average method significantly improve the reproducibility of peptide quantitation ...... 119 Quantitative accuracy of IsoQuant using sPRG2009 SILAC peptide standards and complex biological sample ...... 121 CONCLUSIONS ...... 126

V. Conclusion and Future Directions...... 127

Appendix 1. Quantified phosphopeptides...... 136 Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells...... 148 Appendix 3. Protein synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells...... 180 Appendix 4. Source codes for peptide and protein quantitation in IsoQuant...... 200

References ...... 233

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List of Figures

Figure 1. The structure of EGFR...... 5 Figure 2. EGFR tyrosine phosphorylation sites and binding proteins...... 7 Figure 3. EGFR down-regulation and VPS4B...... 10 Figure 4. The structure of VPS4B...... 15 Figure 5. The mechanism of VPS4-mediated dissociation of ESCRT-III complex from the endosomal membrane...... 15 Figure 6. VPS4B is down-regulated in hypoxic breast cancer cells...... 19 Figure 7. Central hypothesis: Down-regulation of VPS4B promotes the survival of breast cancer cells by modulating EGFR signaling...... 20 Figure 8. VPS4B down-regulation prolongs EGFR activation...... 33 Figure 9. Quantitative phosphoproteomics workflow to analyze VPS4B-mediated EGFR signal transduction events...... 35 Figure 10. Solution charge separation of phosphopeptides identified in each SCX fraction...... 37 Figure 11. Phosphopeptide enrichment in SCX fractions...... 37 Figure 12. Reproducibility of phosphopeptide quantitation ratios in two technical replicates...... 38 Figure 13. Representative MS and MS/MS spectra of up-regulated and down-regulated phosphopeptides...... 43 Figure 14. Network and pathway analysis of EGFR signaling in SKBR3_shVPS4B cells...... 46 Figure 15. Protein signaling pathways with significant changes in constituent protein phosphorylation levels in response to VPS4B down-regulation in EGF-stimulated SKBR3 cells...... 49 Figure 16. Internal standard assisted synthesis and degradation mass spectrometry (iSDMS) workflow to analyze EGF-mediated protein synthesis and degradation rates. . 76 Figure 17. VPS4B down-regulation decreases the relative abundance of fatty acid synthase peptide SLLVNPEGPTLMR in SKBR3 cells...... 79 Figure 18. Average relative protein abundance...... 82 Figure 19. Dynamic protein profiles of SKBR3_shVPS4B cells and parental SKBR3 cells...... 84 Figure 20. Protein expression is positively correlated with protein synthesis and is negatively correlated with protein degradation...... 88 Figure 21. Altered protein expression in SKBR3_shVPS4B cells correlates with altered protein synthesis and degradation rates...... 89 Figure 22. VPS4B down-regulation up-regulates the expression of fatty acid β-oxidation related proteins and down regulates the expression of glycolysis related proteins in SKBR3 cells...... 96

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Figure 23. Framework of IsoQuant ...... 114 Figure 24. User interface of IsoQuant...... 115 Figure 25. IsoQuant overcomes the limitation of under-sampling in data-dependent instrument methods, thereby increasing the accuracy of SILAC-based quantitation. .... 118 Figure 26. Peptide ratios calculated by weighted average method significantly improves the reproducibility of peptide quantitation ratios...... 120 Figure 27. Interface of IsoQuant visualization module permits users to validate quantitation ratios...... 125 Figure 28. A model of the mechanisms underlying altered EGFR signaling caused by VPS4B dysfunction...... 130

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List of Tables

Table 1. Phosphopeptides with increased abundance in SKBR3_shVPS4B cells...... 40 Table 2. Phosphopeptides with decreased abundance in SKBR3_shVPS4B cells...... 41 Table 3. KEGG pathway enrichment analysis...... 45 Table 4. Changes of protein synthesis, degradation and relative abundance induced by VPS4B down-regulation in SKBR3 cells...... 86 Table 5. Proteins with increased relative abundance in SKBR3_shVPS4B cells at 24 h...... 91 Table 6. Proteins with decreased relative abundance in SKBR3_shVPS4B cells at 24 h...... 92 Table 7. Increased protein expression in SKBR3_shVPS4B cells is mostly the result of increased protein synthesis...... 93 Table 8. Decreased protein synthesis and increased protein degradation contribute to decreased protein expression in SKBR3_shVPS4B cells...... 93 Table 9. IsoQuant peptide quantitation results from LC-MS/MS analysis of ABRF sPRG09 quantitative proteomics standard...... 122

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List of Abbreviations

ACN Acetonitrile BCA Bicinchoninic acid cDNA Complementary deoxyribonucleic acid CV Coefficient of variation DMEM Dulbecco's modified eagle medium DPBS Dulbecco’s phosphate-buffered Saline DTT Dithiothreitol FBS Fetal bovine serum HPLC High performance liquid chromatography LC-MS/MS Liquid chromatography-tandem mass spectrometry MS/MS Tandem mass spectrometry PMSF Phenylmethylsulfonyl fluoride SILAC Stable isotopic labeling with amino acid in cell culture SDS Sodium dodecyl sulfate SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis TCEP Tris (2-carboxyethyl) phosphine TFA Trifluoroacetic acid

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I. Introduction

Breast cancer

Breast cancer is the most common cancer among American women and it is the second leading cause of cancer death in women. One of eight women will develop invasive breast cancer at some time during her life [1]. According to statistics from the

American Cancer Society, in the year 2011, 230,480 new cases of breast cancer will be diagnosed among American women and approximately 39,520 women are expected to die from breast cancer [2]. Although great progress in breast cancer diagnostic methods and therapy have been made in past decades, half of late stage breast cancer patients die from metastasis and relapse.

Epidermal growth factor receptor (EGFR)

The epidermal growth factor receptor (EGFR) is a 170-kDa glycosylated trans-

membrane protein that belongs to the ErbB family of receptor tyrosine kinases (RTK). It

was first discovered by Cohen and co-workers when they were studying 32P incorporation

in response to epidermal growth factor (EGF) stimulation in A431 tumor cells [3]. Since

the early 1980s when the cDNA sequence of human EGFR was isolated from normal

placental cells and A431 tumor cells and was characterized [4], intensive research efforts

have led to important insights into the molecular structure and function of EGFR.

It is well known that EGFR plays a key role in signaling pathways that regulate fundamental cellular processes such as proliferation, migration, metabolism, differentiation and survival. EGFR is also essential for epithelial development in vivo.

Knockout of egfr gene in mice results in embryonic lethality or severe failure of epithelial 2

development in several organs, including skin, lung, intestine and placenta [5-8]. In normal, resting cells, EGFR activity is tightly regulated. However, when mutated, structurally altered, or over-expressed, EGFR becomes a potent oncoprotein. Abnormal

EGFR activity is causally involved in the development and progression of many human cancers.

EGFR and breast cancer

EGFR was first implicated in cancer in the early 1980s when the avian erythroblastosis tumor virus was found to encode an aberrant form of human EGFR [4].

Mutations, gene amplification and protein over-expression of EGFR have been reported in many types of cancers including brain, head and neck, thyroid, lung, colon, kidney, prostate, ovarian, bladder, and breast cancer [9][10].

In breast cancer, somatic mutations of EGFR are rarely identified [11-14], while over-expression of EGFR is more common [15-18]. The largest and most comprehensive study to date analyzing EGFR expression in 2,567 breast cancer patients reveals that

EGFR is over-expressed in 18% of breast carcinomas [19]. EGFR over-expression is associated with an aggressive phenotype which is characterized by higher proliferation rate, greater genomic instability, and poor prognosis [19][20]. More importantly, breast cancer patients with EGFR over-expressing tumors have worse disease-free survival and overall survival after receiving either chemotherapy, tamoxifen, or both, as compared to breast cancer patients with normal levels of EGFR expression [19][21]. These data

indicate that EGFR over-expression is involved in the development of resistance to systemic therapy.

EGFR over-expression is the result of multiple genomic and proteomic processes, of which EGFR gene amplification is only one. EGFR over-expression can occur in the absence of gene amplification [13]. It has been shown that only 34% of EGFR over- expressing breast carcinomas harbor increased egfr gene copy number [22]. Recently, defective EGFR degradation and increased EGFR protein translation have emerged as alternative mechanisms of EGFR over-expression in breast cancer cells [23][24].

EGFR structure, activation and signaling

EGFR contains a large extracellular ligand-binding domain, a short transmembrane domain and a cytoplasmic tyrosine kinase domain that is flanked by a C- terminal non-catalytic regulatory region (Figure 1) [25]. The extracellular domain is divided into four subdomains (I-IV). Subdomains I and III are leucine-rich repeats that function in ligand binding, while subdomains II and IV are laminin-like, cysteine-rich domains [26][27]. In the resting state, subdomain II interacts with subdomain IV, which separates the ligand binding subdomain I from subdomain III and sequesters the dimer loop within subdmain II. Ligand binding brings subdomains I and III together, which disrupts the inhibitory interaction between subdomain II and IV and exposes the dimer loop within subdomain II to permit dimerization with another receptor [25][28].

Receptor activation promotes the formation of EGFR dimers, trimers and tetramers [29].

The dimerization of the extracellular domains brings two isolated kinase domains

together to form an active head-to-tail asymmetric dimer through the cooperation of the juxtamembrane segment that connects the transmembrane domain to the kinase domain

[30][31].

Extracelluar domain II III

IV Trans-membrane domain Membrane

Kinase domain

C-terminal tail

Figure 1. The structure of EGFR. The large extracellular domain of EGFR contains subdomains I, II, III, and IV. The interaction of subdomain II and subdomain IV sequesters the dimer loop within subdomain II. Upon ligand binding, the interaction among subdomain I, III and ligand exposes the dimer loop for dimerization with another receptor. The dimerization of extracellular domains brings two kinase domains in close proximity in order to form an active dimer, which leads to C-terminal tyrosine phosphorylation and receptor activation.

EGFR activation results in the transphosphorylation of tyrosine residues in its C- terminal tail. The phosphotyrosines recruit a number of SH2 or phosphotyrosine binding

(PTB) domain-containing adaptors or enzymes to EGFR, such as growth factor receptor bound-2 (Grb2), phosphatidylinositol 3-kinase p85 (PI3K-p85), signal transducer and activator of transcription-5 (STAT5), adaptor molecule crk (Crk), Src/Src tyrosine kinase

(Src/CSk), Src homology 2 domain-containing-transforming protein (Shc), E3 ubiquitin- protein ligase Cbl (Cbl), protein tyrosine phosphatase 2C (PTP-2C), and SH3BGRL

(Figure 2) [32]. The recruitment of these enzymes and adaptor molecules further activates various EGFR downstream signaling pathways, for example, Ras/mitogen- activated protein kinase (MAPK)/extracellular signal-regulated kinases (ERK), phospholipase Cγ (PLCγ), phosphatidylinositol-3 kinase (PI3K)/AKT, and signal transduction and activator of transcription (STAT) pathways [33][34].

Figure 2. EGFR tyrosine phosphorylation sites and binding proteins. EGFR binding proteins were screened by incubation of proteins with synthetic phosphopeptides corresponding to EGFR tyrosine phosphorylation sites. Figure adapted from [32].

A variety of growth factors serve as ligands for EGFR including EGF, transforming growth factor-α (TGF-α), heparin-binding EGF, betacellulin, amphiregulin, epiregulin, and epigen [35]. The diversity of EGFR ligands, together with the strength and duration of stimulation, the intracellular microenvironment, and more importantly, other signaling molecules that EGFR cross-talks with, results in a broad range of biological outcomes including cell growth, proliferation, migration, metabolism, differentiation and cell survival. As opposed to the traditional view of the EGFR signaling network being composed of simple non-interconnected pathways, the more commonly accepted view is that of a complex web of highly interconnected pathways

[25][35][36]. Recent advances in high throughput proteomics and phosphoproteomics have permitted the study of EGFR signaling on global and systematic levels, thereby allowing the intricacies of the EGFR signaling network to be revealed [37-44].

EGFR endocytosis, sorting and degradation

Ligand binding not only activates EGFR signaling, but also promotes receptor endocytosis and degradation (Figure 3). Phosphorylated EGFR recruits Cbl directly through phosphotyrosine 1045 (pY1045) or indirectly through Grb2 [45]. Subsequently,

Cbl modifies multiple lysines in the EGFR kinase domain with monomeric [46] and polymeric ubiquitin molecules [47]. Ubiquitinated EGFR is recognized by ubiquitin binding domain (UBD)-containing proteins, such as epidermal growth factor receptor pathway substrate 15 (EPS15) and Epsins, which are associated with adaptor

protein complex 2 (AP-2) and clathrin heavy chain. EGFR is then sequestered in clathrin-coated pits and endocytosed. As clathrin-coated pits fuse with endosomes,

EGFR is released while remaining ligand-bound, dimerized, phosphorylated, associated with Shc, Grb2, SOS and Cbl, and ubiquitinated [48-52]. After entering early or intermediate endosomes, receptors can be sorted back to the cell surface via recycling endosomes [53]. Alternatively, receptors are directed into the intralumenal vesicles

(ILVs) of multivesicular bodies (MVBs) through the endosomal sorting complex required for transport (ESCRT) machineries, ESCRT-0, -I, -II and –III [53]. Initially, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) and signal transducing adapter molecule (STAM), two main components of ESCRT-0, recognize ubiquitinated EGFR through their UBDs [54][55]. Then, ESCRTs-I, -II and –III sequentially form concentric rings around ESCRT-0 complexes, which ultimately results in inward membrane invagination [56]. Finally, oligomeric vacuolar protein sorting 4 (VPS4) dissociates

ESCRT-III from the endosome membrane and promotes ILV formation in MVBs [56].

Mature MVBs fuse with lysosomes, which leads to receptor degradation.

MAPK Cascades Clathrin coated pits Recycling

ATP VPS4B ADP

Endosomes Multivesicular bodies (MVBs)

Lysosomes Nucleus

EGF VPS4B Signaling molecules Clathrin EGFR ESCRT-0 ESCRT-I ESCRT-II ESCRT-III

Figure 3. EGFR down-regulation and VPS4B. Ligand binding promotes EGFR endocytosis through clathrin-coated pits. On endosomes, EGFR can be recycled back to the plasma membrane, or committed to MVB-lysosome degradation by ESCRT-0, -I, -II, -III and VPS4B.

Ligand-induced receptor lysosomal degradation is the major cellular degradation pathway responsible for the attenuation of the strength and duration of EGFR signals.

Upon EGF stimulation, the half-life of EGFR decreases from 20-24 hours to 8-10 hours

[57][58]. Active receptors, functional endocytosis and endosomal sorting machinery components are required for the proper down-regulation of EGFR. Loss of kinase activity by mutation or kinase inhibitors decreases the EGFR endocytosis rate and inefficiently recruits the receptors into clathrin-coated pits [59-61]. Disruption of Grb2 binding sites, Tyr1068 and Tyr1086, depletion of clathrin heavy chain, or depletion of dynamin, strongly inhibits EGFR internalization [62-64]. The STAM, TSG101, VPS36 and VPS24 components of ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III, respectively, are necessary for EGFR lysosomal degradation [65]. Expression of functionally inactive

VPS4, the last protein required for MVB maturation, results in the accumulation of

EGFR within abnormally enlarged endosomes and prolonged EGFR half-life [66-68].

EGFR signaling during endocytosis and endosomal trafficking

The termination of extracellular signals that are initiated by cell surface receptors has traditionally been viewed as the main function of endocytosis. However, growing evidence suggests that receptor endocytosis and endosomal trafficking also play central roles in signal propagation and specificity by regulating both the dynamics and localization of signaling [69][70]. The large and dynamic tubulovesicular network of the endosomal system, which extends throughout the cytoplasm, provides an optimal platform for the multitude of steps involved in signal initiation and transduction [71].

Endosomal signaling composes a major part of EGFR signaling, which determines the temporal, spatial, and specificity aspects of signaling. Although EGFR is activated from the cell membrane, it remains ligand-bound, phosphorylated and active in endosomes until it is fully sequestered in the intralumenal vesicles of MVBs [72]. Many signaling proteins are found or recruited to endosomes upon EGFR activation, such as

Grb2, Shc, Sos and Ras [50][51], which can prolong the duration of EGFR endosomal signaling. For example, as the late stage of EGFR signaling, full activation of ERK1/2 requires the recruitment of ERK kinase (MEK) scaffold protein, MP1, to the late endosomes by late endosome adaptor proteins p14 and p18 [73-75]. MP1 forms a complex with ERK and MEK, which can be activated by EGFR-associated or Raf1- associated Ras [76][77]. Activated EGFR can recruit signaling factors (Shc, Grb2, p85 subunit of PI3K) to endosomes, and lead to the activation of AKT [78]. The endosomal/late endosomal localization of AKT and mammalian target of rapamycin

(mTOR) determines their substrate specificity and leads to cell survival signaling

[79][80]. The mechanisms that control EGFR coupling to signaling machineries and subcellular localization of signaling molecules tightly regulate EGFR endosomal signaling. Selective disruption of EGFR signaling in endosomes may provide novel therapeutic strategies for breast cancer.

Vacuolar protein sorting 4 (VPS4)

Vacuolar protein sorting 4 (VPS4) is a highly conserved, ubiquitously expressed, type 1 AAA+ ATPase (ATPase associated with diverse cellular activities) protein.

Humans and other mammals have two closely related orthologous VPS4 proteins, 12

VPS4A and VPS4B/suppressor of K+ transport defect 1 (SKD1), which share 80%

sequence identity [81]. Yeasts have a single VPS4 protein, the sequence of which is ~60%

identical to both human proteins [81]. In mammals, most tissues show a strong bias for

the expression of only one of the VPS4 orthologs. For example, the highest expression of

VPS4A is found in the testis, whereas the highest expression of VPS4B is in the liver

[82].

The crystal structure of VPS4 reveals that it has an N-terminal microtubule

interacting and trafficking (MIT) domain, a canonical AAA+ ATPase domain, followed

by a β domain and a C-terminal helix (Figure 4) [83]. VPS4 plays an important role in

sorting endocytosed cargo to MVBs. It functions to dissociate ESCRT-III from the

endosomal membrane and promote ILV formation (Figure 5). During this process, VPS4

shuttles between an inactive monomeric or dimeric state and an active dodecameric state,

which is composed of two stacked hexameric rings [84]. In the absence of adenosine

triphosphate (ATP), VPS4 can form a dimer in a concentration-dependent manner [84],

which can be further facilitated by vacuolar protein sorting-associated protein VTA1

(VTA1) through interaction with the VPS4 β domain [85-87]. The interaction with

VTA1 also exposes the flexible MIT domains of VPS4 [87][88]. The helix-helix

interaction within its C-terminal domain stabilizes the VPS4 dimer [89]. ATP binding

promotes the assembly of dodecameric VPS4, which is recruited to the endosomal membrane by ESCRT-III proteins via the interaction between the MIT domains of VPS4 and the C-terminal MIT domain interaction motifs, MIM1 or MIM2, of ESCRT-III proteins [90][91]. This interaction abolishes the auto-inhibition of VPS4 and activates

the AAA+ ATPase activity of VPS4 [92][93]. Upon ATP hydrolysis, VPS4 dissociates

ESCRT-III from the endosomal membrane thereby making these proteins available for further rounds of cargo sorting [84][94]. It has been hypothesized that the dissociation of

ESCRT-III involves the partial denaturing of ESCRT-III proteins as they pass through the central pore of the two stacked hexameric rings of the VPS4 dodecamer, a process which is directed by the VPS4 MIT domain and VTA1 [88][95].

Large Small β Helix MIT AAA ATPase AAA ATPase domain domain

1 6 79 123 130 301 360 407 423 444 K180 E235

Figure 4. The structure of VPS4B. The MIT domain is flexible and responsible for the interaction of VPS4B with ESCRT- III complex. The ATPase domain contains a large subdomain for ATP binding and hydrolysis and a small structural subdomain. Mutation of K180 or E235 in ATPase domain results in the loss of ATP binding ability or ATP hydrolysis activity, respectively. The β domain interacts with vacuolar protein sorting-associated protein VTA1 and facilitates dimerization and oligomerization. The interaction of helix domains within two VPS4B molecules stabilizes the dimer. Figure modified from [83].

VPS4 MIT domain

ATP

Endosomal ESCRT-III ESCRT-III membrane

VTA1

Figure 5. The mechanism of VPS4-mediated dissociation of ESCRT-III complex from the endosomal membrane. In the absence of ATP, VPS4 can form an inactive dimer, which is facilitated by VTA1 and the C-terminal helix domain of VPS4. ATP binding promotes the assembly of active dodecameric VPS4, which is recruited to the endosomal membrane by ESCRT-III. Upon ATP hydrolysis, VPS4 dissociates ESCRT-III from the endosomal membrane, which may involve the concurrent transport of ESCRT-III proteins through the central pore of the VPS4 dodecamer. Figure modified from [83].

The VPS4-mediated dissociation of ESCRT-III from the endosomal membrane is important for normal endocytosed cargo MVB sorting and trafficking. Expression of functionally inactive VPS4, either loss of ATP binding ability (K173Q mutation for

VPS4A and K180Q mutation for VPS4B) or loss of ATP hydrolysis activity (E228Q mutation for VPS4A and E235Q mutation for VPS4B), precludes MVB formation, resulting in the formation of abnormally enlarged endosomes (class E compartments) and the accumulation of endocytosed cargo on endosomal membranes or within class E compartments [96]. Loss of function of VPS4B blocks the dynamic recycling of transferrin receptors and low density lipoprotein receptors [66][68]. The dominant negative E235Q mutation of VPS4B can lead to the endosomal accumulation of Src [97], which traffics between the late endosome/lysosome and the plasma membrane [98-100].

More importantly, MVB-lysosome degradation is the major pathway for ligand-induced receptor down-regulation. Loss of function of VPS4B results in the delayed degradation of EGFR and extended activation following EGF stimulation [66-68].

Tumor hypoxia

Within growing tumors, the cells that are located at a distance greater than 75 µm away from a blood vessel suffer from hypoxia due to oxygen tension levels of less than

10 mm Hg (as compared with >60 mm Hg in normal tissue) [101][102]. The hypoxic microenvironment represents nearly 50% of locally advanced breast cancers that are heterogeneously distributed within the tumor mass [103]. Hypoxia is a frequent cause of treatment failures by radiation and chemotherapy in most aggressive tumors [104].

Hypoxia can cause normal tissue necrosis or apoptosis; however, in solid tumors, hypoxia 16

can lead tumor cells to develop a more aggressive phenotype by tremendous genomic and proteomic changes that facilitate proliferation, local evasion, adaptation to nutritive deprivation, and metastatic spread [105][106]. One of the most important promoters of tumor cell response to hypoxic stress is the transcription factor hypoxia induced factor

(HIF). HIF is elevated in hypoxic tumors and activates genes with roles in oxygen delivery, angiogenesis, energy preservation, and other processes essential to tumor cell survival, propagation, and invasion [107]. In addition to diminishing therapeutic efficacy, hypoxia plays a pivotal role in malignant progression.

Tumor hypoxia and EGFR

The hypoxic microenvironment can induce the up-regulation and activation of

EGFR in many tumors [108][109]. Elevated EGFR levels in hypoxic tumors increase cell motility and survival, consequently aiding the development of apoptotic resistance

[110][111], and contributing to poor disease prognosis [112]. It has been shown that hypoxia-induced EGFR up-regulation can be mediated by HIF through two approaches: decreasing EGFR degradation [23] and increasing EGFR mRNA translation [24]. Tumor hypoxia–mediated activation of HIF represses the translation of rabaptin-5 protein, a key effector of Rab5-mediated early endosome fusion [23]. As a result, endocytosis slows and degradation of internalized EGFR is inhibited [23]. In the latter case, hypoxia/HIF activation may result in the expression of a hypoxia-inducible activator of EGFR translation or alternatively, may inhibit a negative regulator of receptor synthesis, which leads to increased EGFR protein translation [24]. These results indicate that tumors can

over-express EGFR by controlling EGFR protein synthesis and degradation without altering egfr gene levels.

Hypoxia and VPS4B

In order to determine the relationship between VPS4B expression and hypoxia in breast cancer, we performed a preliminary study whereby SKBR3 breast cancer cells were grown as 2D monolayer cultures for desired time periods and exposed to hypoxia

(0.5% O2). To mimic the in vivo tumor microenvironment, SKBR3 cells were also

cultured as 3D spheroids [113]. In 2D monolayers, hypoxia caused a time-dependent

decrease of VPS4B in SKBR3 cells (Figure 6). A marked down-regulation of VPS4B

was observed upon hypoxic exposure in 3D spheroids (Figure 6). Notably, VPS4B

expression level was lower in cells cultured as 3D spheroids than in cells cultured in 2D

monolayers, even in the absence of hypoxic exposure (Figure 6, lane 1 vs. lane 4). Due

to the limitation of oxygen diffusion, cells in the inner core of 3D spheroids suffer from

low oxygen concentration. When exposed to hypoxic conditions, the pathological effects

are more severe. These results clearly demonstrate a negative correlation between

hypoxic conditions in breast cancer and VPS4B expression.

2D monolayers 3D spheroids

0.5% O2 0 24 48 02448(h)

VPS4B 100 97 83 64 34 2 (%)

Actin

Figure 6. VPS4B is down-regulated in hypoxic breast cancer cells. SKBR3 cells were cultured as 2D monolayers or 3D spheroids and exposed to a hypoxic environment, 0.5% O2, for the indicated times. Quantification of VPS4B expression is represented as the percentage of actin expression. Figure kindly provided by co- investigator, Dr. David K. Ann.

Goals of the present study

As summarized in Figure 7, hypoxia induces down-regulation of VPS4B.

Functionally inactive VPS4B can inhibit EGFR degradation. Considering the important role of EGFR in signaling pathways that are involved in cancer cell survival, growth, proliferation and metastasis, I hypothesized that down-regulation of VPS4B promotes the survival of breast cancer cells by modulating EGFR signaling. This hypothesis was tested by the following two aims.

Aim 1. To determine the role of VPS4B in aberrant EGFR signal transduction pathway phosphorylation in breast cancer cells. We will use a global, quantitative phosphoproteomic approach to address how VPS4B dysfunction modulates

EGFR-mediated signaling in breast cancer cells.

Aim 2. To determine the protein dynamics of VPS4B down-regulation mediated aberrant EGFR signaling in breast cancer. We will use a novel synthesis/degradation mass spectrometry approach to address how VPS4B dysfunction affects global protein synthesis and degradation in breast cancer cells.

Hypoxia

Contribute to tumor survival? Down-regulation

VPS4B EGFR Delayed degradation

Figure 7. Central hypothesis: Down-regulation of VPS4B promotes the survival of breast cancer cells by modulating EGFR signaling.

II. Phosphoproteomic Analysis Reveals Activation of EGFR/EPS8/SH3BP1 Complex and Stabilization of Cell Adhesion in VPS4B-Mediated Endosomal EGFR Signaling

ABSTRACT

The aberrant expression, activation, and down-regulation of the epidermal growth factor receptor (EGFR) are causal factors in the abnormal cell growth, proliferation, invasion and metastasis that occur in breast cancer. Increasing evidence has suggested that the endosomal/lysosomal system, in particular the endosomal sorting complexes required for transport (ESCRTs), could play an essential role in regulating this abnormal

EGFR signaling. Dysfunction of vacuolar protein sorting 4 homolog B (VPS4B), an

ESCRT-III associated protein, leads to the inhibition of EGFR degradation and prolonged intracellular EGFR signaling in abnormally enlarged endosomal compartments.

Although EGFR-mediated signaling is one of the best characterized cell signaling pathways and many elegant large scale EGFR-mediated functional proteomic analyses have been reported recently, there is a lack of information addressing how changes of the endosomal/lysosomal system can affect EGFR signaling in breast cancer. Therefore, the goal of this study was to use a global phosphoproteomic analysis to specifically determine the role of VPS4B-mediated ESCRT dysfunction in altering intracellular and endosomal EGFR signaling. By using a stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomic approach coupled with an attenuated

VPS4B expression breast cancer cell culture model, we have determined that intracellular

EGFR activates an EGFR/epidermal growth factor receptor kinase substrate 8

(EPS8)/SH3 domain-binding protein 1 (SH3BP1) signaling pathway and subsequently stabilizes various cell adhesion and junction molecules. EPS8 and SH3BP1 are known to function in the reorganization of the actin cytoskeleton network, assembly of cell adhesion modules and regulation of cell motility.

This report provides the first systems biology evidence demonstrating the potential pathological relevance of altered EGFR signaling by endosomal dysfunction.

Our data suggests that differential down-regulation of VPS4B could be one of the possible mechanisms involved in modulating cell adhesion modules in metastatic breast cancer cells.

INTRODUCTION

The epidermal growth factor receptor (EGFR) is of considerable interest in breast cancer research due to its ability to induce tumorigenesis when its signaling functions are deregulated. Results from the largest and most comprehensive clinical study to date analyzing EGFR expression in 2,567 breast cancer patients indicate that EGFR was over- expressed in 18% of breast carcinomas [19]. EGFR over-expression in breast carcinomas is associated with reduced survival [20] and studies have indicated that EGFR is involved in the development of hormone-resistant breast cancer [21]. Thus, the determination of

EGFR’s role in the development of breast cancer malignancy is of clinical importance.

The EGFR signal transduction pathway is one of the best characterized cell signaling pathways. It plays a causal role in cell growth, proliferation, differentiation, cell survival, cell cycle progression and angiogenesis [35]. EGFR does not act as an autonomous entity, but rather as part of a complex interconnected regulatory system.

EGF-stimulated EGFR homo- or hetero-dimerization with other receptor tyrosine kinases results in the trans-phosphorylation of C-terminal EGFR tyrosine residues which then serve as docking sites for the recruitment and activation of multiple downstream

signaling pathways including the Ras/mitogen-activated protein kinase (MAPK), phospholipase Cγ (PLCγ), phosphatidylinositol-3 kinase (PI3K)/AKT, and signal transduction and activator of transcription (STAT) pathways [33][34]. Consequently, the abnormal activation, expression and down-regulation of EGFR can have multiple pathological effects. Several elegant, large-scale proteomic and phosphoproteomic analyses have been conducted in order to elucidate the intricacies of the EGFR signal transduction pathway. A comprehensive discussion of these analyses is beyond the scope of the current manuscript; therefore, the readers are encouraged to refer to several recently published studies and review articles for further information [37-44].

Although EGFR signaling is initiated from the cell membrane, activated EGFR continues to signal from within endosomes after it has been endocytosed following EGF binding until it is sequestered in the intralumenal vesicles of multivesicular bodies en route to being degraded in the lysosome [45]. Thus, lysosomal degradation-mediated

EGFR down-regulation has a critical role in attenuating the propagation of extracellular signals, thereby terminating cell proliferation signals and preventing uncontrollable cell growth. In the absence of EGF binding-triggered endocytosis, the half-life of EGFR is

20-24 hours. EGFR half-life decreases to 8-10 hours following EGF stimulation [57][58].

As a consequence, impaired endocytosis and degradation result in the elevation of intracellular EGFR protein levels and cause the abnormal activation of endosomal EGFR signaling pathways [55][114] .

Recently it has been shown that the dysfunction of protein complexes involved in regulating endosomal trafficking, such as the endosomal sorting complexes required for transport (ESCRT), causes mis-trafficking and abnormal activation of membrane receptors. For example, functionally deficient vacuolar protein sorting 4 homolog B

(VPS4B) – a protein that plays a critical role in the endosomal sorting pathway where it mediates the down-regulation of many cell surface membrane receptors – has been shown to cause the abnormal accumulation of EGFR in endosomal/lysosomal compartments

[66-68]. However, to the best of our knowledge, currently there is no information in the literature at the systems biology level addressing the role of ESCRT dysfunction in

EGFR signaling. Therefore, in order to determine the role of ESCRT dysfunction in

EGFR signaling and its pathological consequences underlying breast cancer malignancy, we analyzed differential changes in the EGFR signal transduction pathway phosphoproteome in response to EGF treatment using SKBR3 breast cancer cells with decreased VPS4B expression.

Our data suggest that EGFR-mediated epidermal growth factor receptor kinase substrate 8 (EPS8)/Son of sevenless homolog 1 (Sos-1)/Abl interactor 1 (Abi1) signaling pathways are differentially activated in VPS4B down-regulated breast cancer cells. EPS8 is part of the Rho GTPase EPS8/Sos-1/Abi1 complex, which has been shown to play a critical role in propagating signals initiated by receptor tyrosine kinases such as EGFR.

Activation of the Rho GTPase EPS8/Sos-1/Abi1 complex can subsequently lead to the reorganization of the actin cytoskeleton network [115]. Numerous reports have demonstrated that increased EPS8 expression and phosphorylation are directly related to

poor disease prognosis and increased metastatic states of breast cancer [115-118].

Although it is not clear how the multi-molecular EPS8/Sos-1/Abi1 complex regulates cell motility in breast cancer cells, our results suggest that EPS8/Sos-1/Abi1 could be regulated by SH3 domain-binding protein 1 (SH3BP1), a Rho GAP-associated protein, since the phosphorylation states of both EPS8 and SH3BP1 are elevated in VPS4B expression-attenuated cells. Recently, it has been shown that one of the functions of

SH3BP1 is to inactivate Rac1 at the leading edge of motile cells, consequently blocking the assembly of cell adhesion molecules and increasing cell motility [119]. This model is further supported by our observation that the phosphorylation levels of several key cell connection and adhesion molecules, such as tight junction protein 1 (TJP1) and tight junction protein 2 (TJP2), are drastically reduced in VPS4B expression-attenuated cells.

Taken together, the work reported here provides the initial systems biology information regarding how endosomal system dysfunction can potentially affect EGFR signaling. Our data also provide a conceptual framework for the design of future hypothesis-driven experiments to address the role of endosomal/lysosomal dysfunction in

EGFR signaling under various pathological conditions.

MATERIALS AND METHODS

Cell culture and SILAC labeling

SKBR3 cells were obtained from the American Type Culture Collection

(Rockville, MD). VPS4B down regulated SKBR3 (SKBR3_shVPS4B) cells were generated by transducing SKBR3 cells with a lentivirus harboring shRNA against VPS4B

(SA Biosciences/Qiagen, Frederick, MD). Lentiviral stock was made as previously described [120].

SKBR3_shVPS4B cells were cultured in SILAC DMEM (Thermo

Scientific/Pierce). The medium was deficient in arginine and lysine and was supplemented with 10% dialyzed FBS (Invitrogen) and 1% penicillin/streptomycin.

SKBR3_shVPS4B cells were cultured in “light” SILAC labeling medium supplemented

12 12 with C6 L-Lysine-HCl (100 mg/L; Thermo Scientific/Pierce) and C6 L-Arginine-HCl

(100 mg/L; Thermo Scientific/Pierce). Parental SKBR3 cells were cultured in “heavy”

13 SILAC medium: SILAC DMEM supplemented with C6 L-Lysine-HCl (100 mg/L;

13 purity 97-99 %; Cambridge Isotope Laboratories) and C6 L-Arginine-HCl (100 mg/L;

purity 97-99 %; Cambridge Isotope Laboratories). After five passages, the incorporation

13 12 of C6 Lysine (Lys6)/ C6 Arginine (Arg6) was ~98% as determined by mass

spectrometric analysis of tryptic peptides isolated from the Lys6/Arg6-labeled parental

SKBR3 cells.

Cell stimulation, lysis and in solution protein digestion

Five 15-cm dishes of 80% confluent SILAC-labeled SKBR3 and

SKBR3_shVPS4B cells were prepared. Following 16 h of serum starvation, cells were

stimulated with EGF (100 ng/ml) for 30 min and washed with DPBS. Cells were

harvested and lysed by sonication in 8 M urea, 75 mM NaCl, 50 mM Tris-HCl, pH 8.2,

protease inhibitor cocktail (Roche), 1 mM NaF, 1 mM β-glycerophosphate, 1 mM sodium

orthovanadate, 10 mM sodium pyrophosphate, and 1 mM PMSF. Insoluble material was

pelleted by centrifugation at 10,000 x g for 10 min at 4 °C. The concentration of the soluble proteins was determined by the BCA assay kit (Thermo Scientific/Pierce) and 3 mg of proteins from each SILAC condition were combined prior to reduction, alkylation and protein digestion. Disulfide bonds were reduced by incubation with 5 mM DTT for

25 min at 56 °C followed by alkylation with 15 mM iodoacetamide for 30 min at room temperature in the dark. Un-reacted iodoacetamide was quenched by addition of DTT to

5 mM final concentration for 30 min at room temperature in the dark. The protein mixture was diluted by 25 mM Tris-HCl, pH 8.2 at a 1:5 ratio to reduce the final concentration of urea to 1.6 M. Reduced and alkylated proteins were digested with trypsin (2% wt/wt) overnight at 37 °C. The digestion was terminated by acidification with TFA to 1% (vol/vol) followed by desalting with a C18 SepPak cartridge (Waters).

The desalted peptide samples were then lyophilized and stored in -80 °C prior to analysis.

Peptide fractionation by strong cation exchange ion chromatography

Peptides were fractionated by strong cation exchange (SCX) ion chromatography using a Waters 2695 dual pump HPLC Separation module (Waters) operated at a flow rate of 1 ml/min. Two mg of peptides were loaded onto a PolySulfoethyl-A column (4.6 mm × 200 mm, 5 µm particles, 200Å pores, PolyLC) in 100% Buffer A (35% ACN, 5 mM KH2PO4, pH 2.65) and were eluted using the following gradient profile: 0 – 10 min:

100% Buffer A; 10 – 45 min: 0 – 25% Buffer B (35% ACN, 350 mM KH2PO4, pH 2.65);

45 – 50 min: 25 – 100% B; 50 – 55 min: 100% B; 55 – 60 min: 0 – 100% A. Twenty

fractions were collected and were de-salted using C18 SepPak cartridges (Waters) before

the enrichment of phosphopeptides using titanium dioxide (TiO2) micro-columns.

Phosphopeptide enrichment using titanium dioxide micro-columns

Phosphopeptide enrichment was performed according to the procedure detailed in

Rappsilber et al. [121]. Briefly, 3 mg of 10 µm Titansphere beads (GL Sciences, Cat #

1352B500) were added to in-house prepared C8-stop and go extraction (STAGE) tips in

200 µl pipet tips (Empore, 3M). The TiO2 micro-columns were washed and equilibrated with Buffer A (80% ACN, 0.1 % TFA and 300 mg/ml lactic acid). Dried SCX- fractionated peptides were re-constituted in Buffer A and loaded onto the TiO2 columns.

The columns were washed with Buffer A followed by Buffer B (80% ACN, 0.1 % TFA) and the phosphopeptides were eluted with freshly prepared 0.5% NH4OH into microcentrifuge tubes containing 2% TFA. To elute any peptides bound to the C8 membrane, the TiO2 micro-columns were washed with Buffer B; this eluate was combined with the 0.5% NH4OH eluate. The volume of the samples was reduced in a

SpeedVac concentrator (Thermo Scientific) followed by de-salting with C18 STAGE

tips. De-salted phosphopeptides were re-suspended in 0.1% formic acid immediately

prior to LC-MS/MS analysis.

Analysis of phosphopeptides by LC-MS/MS

TiO2-enriched phosphopeptides were separated by nano-scale reversed-phase

liquid chromatography using a nano-flow Xtreme Simple liquid chromatography system

(CVC/Micro-Tech) equipped with a C18 column (100 µm x 15 mm, 3 µm particles, 300

Å pores, Michrom Bioresources) and coupled online to a linear ion trap-obitrap mass

spectrometer (LTQ-Orbitrap, Thermo Electron). Peptides were loaded onto the C18

column with 0.1% formic acid in water using a Surveyor autosampler (Thermo Electron).

A 60 min LC gradient method from 5 – 40% B (95% ACN, 4.9% water, 0.1% formic acid) at a flow rate of 0.5 µl/min was used to elute the peptides into the mass spectrometer.

The mass spectrometer was equipped with a nanospray ionization source containing an uncoated 10 µm i.d. SilicaTipTM PicoTipTM nanospray emitter (New

Objective) and was operated in positive ion mode. The spray voltage was 1.8 kV and the

heated capillary temperature was 200 °C. MS1 data were acquired in the profile mode in

the Orbitrap with a resolution of 60,000 at 400 m/z and the top five most intense ions in each MS1 scan were selected for collision-induced dissociation in the linear ion trap.

Dynamic exclusion was enabled with repeat count 2 and exclusion duration 15 sec.

Other mass spectrometry data generation parameters were as follows: collision energy

35%, ion selection threshold for MS/MS 1000 counts, isolation width 3 and default charge state 3. Each sample was analyzed twice by LC-MS/MS for two technical replicates.

Quantitation of SILAC-labeled phosphopeptides

DTA files were extracted from RAW data by DeconMsn [122]. The systematic parent ion mass measurement errors were corrected by DtaRefinery [123]. Resulting

DTA files were searched against a UniProt human database (downloaded on Oct. 18,

2010) using SEQUEST (Bioworks 3.3.1 SP1, Thermo Scientific) with the following search criteria – mass tolerance: 5 ppm; fragment tolerance: 0.5 Da; digestion method: fully tryptic. Carbamidomethyl cysteine was set as a fixed modification and oxidized

methionine was set as a variable modification. Additional variable modifications included arginine +6.02013 and lysine +6.02013. The peptides were filtered by the following criteria: Xcorr ≥ 2.5, 3.0 and 3.5 for 2+, 3+, and 4+ peptides, respectively (1+ peptides were ignored); and ∆Cn > 0.1. After database search, the phosphopeptide

SILAC-pairs corresponding to SEQUEST results were re-calculated using an in-house software program, named RebuildXML. The recalibrated MS1 SILAC pairs were then quantified using an in-house developed quantitation software tool, named IsoQuant.

IsoQuant is written in C# language under the Microsoft .Net framework 4.0 Windows environment. The software is freely available for non-commercial users and can be downloaded at http://www.proteomeumb.org/MZw.html.

Phosphopeptide data visualization and network analysis

The search for significantly enriched signaling pathways in SKBR3_shVPS4B cells was carried out using the Database for Annotation, Visualization and Integrated

Discovery (DAVID) database of the National Institute of Allergy and Infectious Diseases

[124]. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway was considered significantly enriched if its p value was less than 0.05.

The protein-protein interaction network of all identified proteins was extracted from the Search Tool for the Retrieval of Interacting Genes (STRING) database version 8

[125]. The interaction confidence is defined as a confidence score in STRING with a highest value of 1 and a lowest value of 0. All interactions chosen had medium or high confidence (score > 0.4). The interaction network was spatially represented using the

Spring-embedded layout in Cytoscape version 2.7 [126]. In the spring embedded layout, the connection between two nodes is treated like a metal spring [127]. The layout algorithm sets the positions of the nodes in a way that minimizes the sum of forces in the network. The relative abundance ratios of phosphopeptides were used as the values of the nodes, or proteins, in the network analysis.

RESULTS

Decreased expression of VPS4B in EGF-stimulated SKBR3 cells prolongs EGFR activation

In order to determine the role of VPS4B-mediated ESCRT dysfunction in altering intracellular and endosomal EGFR signaling, stable cell lines, SKBR3_shVPS4B, with reduced expression of VPS4B were established by transducing SKBR3 cells with a lentivirus harboring shRNA against VPS4B (Figure 8). To stimulate ligand-induced

EGFR down-regulation and the potentiation of EGFR signal transduction,

SKBR3_shVPS4B cells and parental SKBR3 cells were treated with EGF. Figure 8 demonstrates that the active and phosphorylated form of EGFR was significantly accumulated in SKBR3_shVPS4B cells. EGF-stimulated Tyr1092 EGFR phosphorylation (pTyr1092), which is associated with growth factor receptor-bound protein 2 (Grb2) binding and activation of MAPK signaling [128], was evaluated by

Western blotting (Figure 8). In parental SKBR3 cells, phosphorylation of Tyr1092 reached a maximum after 10 min of EGF stimulation, followed by marked attenuation –

70% decrease in relative expression – after 30 min of EGF stimulation, which is similar to the kinetics of EGFR degradation reported previously [57][58]. By comparison, the

phosphorylation of Tyr1092 in SKBR3_shVPS4B cells was more sustained. After 30 min of stimulation with EGF, the relative expression of pTyr1092-EGFR decreased by 18% as compared to its maximum expression level at 10 min. Thus, this experiment clearly demonstrates that decreased VPS4B expression in SKBR3 cells prolongs EGF-mediated

EGFR signal transduction events.

SKBR3 SKBR3_shVPS4B EGF (100 ng/ml) 0 10 30 120 0 10 30 120 (min)

pY1092-EGFR 1 5.3 2.3 0.50.4 6.5 4.3 1.0 EGFR 1 0.6 0.7 0.40.6 0.5 0.7 0.6

VPS4B 1 1.0 0.9 0.80.1 0.1 0.1 0.1

Actin

Figure 8. VPS4B down-regulation prolongs EGFR activation. VPS4B expression was reduced by shRNA against VPS4B in SKBR-3 cells (SKBR3_shVPS4B). Cells were stimulated with 100 ng/ml EGF for the indicated times after overnight serum starvation. Quantification of EGFR, pTyr1092-EGFR and VPS4B expression are represented as fold-changes relative to their expression at 0 min in SKBR3 cells.

Quantitative phosphoproteomic analysis of aberrant EGF signaling mediated by down-regulation of VPS4B

In order to determine the effects of reduced VPS4B expression on the phosphorylation status of EGF signal transduction cascade proteins, a quantitative proteomics approach was taken. The workflow of our quantitative phosphoproteomics approach to delineate VPS4B-mediated EGF signal transduction is outlined in Figure 9.

13 13 Parental SKBR3 cells were labeled with C6-lysine (Lys6) and C6-arginine (Arg6).

12 12 SKBR3_shVPS4B cells were labeled with C6-lysine (Lys0) and C6-arginine (Arg0).

Both cell lines were serum-starved overnight and stimulated with EGF for 30 min. Cell

lysates were prepared and combined 1:1 (wt/wt). After digestion with trypsin, peptides

were fractionated by strong cation exchange (SCX) HPLC [129]. Phosphopeptides in each fraction were enriched using TiO2 stop and go extraction (STAGE) Tips [130]. The

enriched phosphopeptides were analyzed by LC-MS/MS using an LTQ-Orbitrap mass

spectrometer followed by identification using the SEQUEST algorithm.

Lys0, Arg0

P SKBR3_shVPS4B P EGF P P Lys6, Arg6 P P P 30min P P SKBR3 P 1:1 mix Trypsin digestion SCX fractionation

MS/MS Phosphopeptide identification P

relative intensity P P P P m/z P P P P L P 6Da H P IsoQuant MS LC-MS/MS TiO2 phosphopeptide quantitation enrichment

relative intensity m/z

Relative Ratio = SKBR3_shVPS4B(L) vs. SKBR3(H)

Figure 9. Quantitative phosphoproteomics workflow to analyze VPS4B-mediated EGFR signal transduction events. 13 13 SKBR3 cells were labeled with C6-lysine (Lys6) and C6-arginine (Arg6). 12 12 SKBR3_shVPS4B cells were labeled with C6-lysine (Lys0) and C6-arginine (Arg0). Both cell lines were serum-starved overnight and stimulated with EGF for 30 min. Lysates from each cell line were pooled followed by protein digestion and SCX peptide fractionation. Phosphopeptides in each SCX fraction were enriched using TiO2 stop and go extraction (STAGE) Tips and analyzed by online LC-MS/MS using an LTQ-Orbitrap mass spectrometer. IsoQuant, an in-house developed software tool, was used for peptide and protein quantitation

Fractionation by SCX chromatography is an efficient means of differentially enriching tryptic phosphopeptides based on net solution charge state [125]. When pH is blow 3, phosphopeptides retain an additional negative charge and have reduced retention on polysulfoethyl aspartamide stationary phase, which accounts for their earlier elution as compared to non-phosphorylated peptides. As indicated in Figure 10, singly charged phosphopeptides were well separated from 2+ and 3+ phosphopeptides by SCX with the longer and higher charge-state phosphopeptides eluting in the later portion of the liquid chromatography gradient. Adjacent SCX fractions were combined yielding 20 fractions that were subsequently enriched for phosphopeptides by TiO2 STAGE tips. The overall

phosphopeptide enrichment for each of the SCX fractions is presented in Figure 11.

To quantitatively determine changes in the EGF-induced phosphoproteome in

relationship to the down-regulation of VPS4B, an in-house software tool termed IsoQuant

was utilized. IsoQuant uses the MS1 peak intensities of heavy and light SILAC-labeled

peptides for ratio calculation. To determine the reproducibility of phosphorylation site identification and quantitation, each phosphopeptide-enriched SCX fraction was analyzed twice by LC-MS/MS. Using IsoQuant, 80% of the identified phosphopeptides were quantified. The overall peptide quantitation ratio coefficient of variation (CV) was 4%.

All quantitation ratios are expressed as SKBR3_shVPS4B (Light-Lys0/Arg0) vs. parental

SKBR3 (Heavy-Lys6/Arg6). Figure 12 illustrates the reproducibility of the phosphopeptide quantitation from two technical replicates. A correlation coefficient (r) of 0.67 was achieved.

100%

90%

80%

70% > +3

60% +3

50% +2

40% +1 Percentage 30% < +1 20%

10%

0% 4_1 4_2 5_1 5_2 6_1 6_2 7_1 7_2 8_1 8_2 9_1 9_2 11_1 11_2 10_1 10_2 12_1 12_2 13_1 13_2 14_1 14_2 15_1 15_2 16_1 16_2 17_1 17_2 18_1 18_2 19_1 19_2 20_1 20_2 SCX fraction

Figure 10. Solution charge separation of phosphopeptides identified in each SCX fraction. Separation of peptides at pH 2.7 by SCX chromatography permits increased separation between phosphopeptides with charges +1 and +2. The results from both technical replicate LC-MS/MS analyses for SCX fractions 4 – 20 are shown.

1000 Total peptide 900 Phosphopeptide 800 700 600 500 400 300 Number of peptides 200 100 0 4_1 4_2 5_1 5_2 6_1 6_2 7_1 7_2 8_1 8_2 9_1 9_2 11_1 11_2 10_1 10_2 12_1 12_2 13_1 13_2 14_1 14_2 15_1 15_2 16_1 16_2 17_1 17_2 18_1 18_2 19_1 19_2 20_1 20_2 SCX fraction

Figure 11. Phosphopeptide enrichment in SCX fractions. Phosphopeptides in the SCX fractions were enriched using TiO2 STAGE tips.

r = 0.67 2

2 Replicate No.2 Replicate Log (Ratio) 1

0 -3 -2 -1 0 123

-1 Replicate No.1 Log (Ratio) 2 -2 Ratio = SKBR3_shVPS4B vs. SKBR3 -3

Figure 12. Reproducibility of phosphopeptide quantitation ratios in two technical replicates. Quantitative analysis was performed using our in-house software tool, IsoQuant. Ratios are SKBR3_shVPS4B vs. SKBR3. Linear regression analysis of the correlation between the phosphopeptide ratios from both technical replicates yielded a correlation coefficient (r) of 0.67.

In total, 677 unique phosphorylated peptides (Appendix 1) were quantified and the mean was 1.00 ± 0.30. We defined increased and decreased phosphopeptides as those with a fold change of more than 1.60 (determined by twice the standard deviation).

Seventy five phosphopeptides showed specific changes in response to the down- regulation of VPS4B expression: 22 phosphopeptides had increased abundances (Table 1) and 53 phosphopeptides had decreased abundances (Table 2). Figures 13A and C are representative MS1 spectra of down-regulated and up-regulated phosphopeptides from tight junction protein 1 (TJP1/ZO1) and epidermal growth factor receptor kinase substrate 8-like protein 3 (EPS8L3) (ratios = 0.44 ± 0.09 and 2.29 ± 0.71, respectively).

MS/MS spectra of the light-labeled phosphopeptides from these proteins are shown in

Figures 13B and D.

Table 1. Phosphopeptides with increased abundance in SKBR3_shVPS4B cells. Ratio = Gene UniProt Delta Protein Name Phosphopeptide Sequence* Position Known Xcorr SKBR3_shVPS4B Name Accession Cn vs. SKBR3 SH3BP1 Q9Y3L3 SH3 domain‐binding protein 1 TESEVPPRPAS@PK S544 Yes 2.98 0.45 2.06 ± 0.08 C9orf142 Q9BUH6 Uncharacterized protein C9orf142 LAAAEETAVS@PR S148 Yes 3.36 0.31 2.80 ± 0.38 TBC1D4 O60343 TBC1 domain family member 4 HAS@APSHVQPSDSEK S341 Yes 3.66 0.15 2.58 ± 0.76 TRIM3 O75382 Tripartite motif‐containing protein 3 VKS@PGGPGSHVR S437 No 2.75 0.34 2.31 ± 0.00 Epidermal growth factor receptor kinase substrate 8‐like EPS8L3 Q8TE67 MQVLY@EFEARNPR Y459 No 2.55 0.39 2.29 ± 0.71 protein 3 HIST1H1E P16403 Histone H1.2 KAS@GPPVSELITK S36 Yes 4.87 0.36 2.04 ± 0.29 TACC2 O95359 Transforming acidic coiled‐coil‐containing protein 2 HPGASEAADGCS@PLWGLSK S1025 Yes 3.7 0.18 1.98 ± 0.38 MAPK3 P27361 Mitogen‐activated protein kinase 3 IADPEHDHTGFLTEY@VATR Y204 Yes 4.34 0.13 1.95 ± 0.34 MYO19 Q96H55 Myosin‐XIX FKAS@LEQLLQVLHSTTPHYIR S3 No 3.46 0.16 1.86 ± 0.29 LIG1 P18858 DNA ligase 1 VLGS@EGEEEDEALSPAK S66 Yes 4.85 0.53 1.79 ± 0.40 ZFYVE19 Q96K21 Zinc finger FYVE domain‐containing protein 19 EHQTSAYS@PPR S463 Yes 2.99 0.11 1.79 ± 0.38 SRRM2 Q9UQ35 Serine/arginine repetitive matrix protein 2 AQT@PPGPSLSGSK T1003 Yes 3.06 0.37 1.76 ± 0.03

40 WDR77 Q9BQA1 Methylosome protein 50 KET@PPPLVPPAAR T5 Yes 2.68 0.41 1.74 ± 0.30 TRIM3 O75382 Tripartite motif‐containing protein 3 REDS@PGPEVQPMDK S7 Yes 4.33 0.68 1.73 ± 0.32 T670, Yes, NCAPG Q9BPX3 Condensin complex subunit 3 IKTLHCEGT@EINSDDEQES@KEVEETATAK 3.05 0.12 1.67 ± 0.37 S680 No SEC16A O15027 Protein transport protein Sec16A LLPSAPQTLPDGPLAS@PAR S1786 No 5.75 0.49 1.67 ± 0.20 PSMD2 Q13200 26S proteasome non‐ATPase regulatory subunit 2 APVQPQQS@PAAAPGGTDEKPSGK S16 Yes 3.63 0.36 1.64 ± 0.45 PTDSS2 Q9BVG9 Phosphatidylserine synthase 2 DAGGPRPES@PVPAGR S16 Yes 2.66 0.5 1.64 ± 0.15 Epidermal growth factor receptor kinase substrate 8‐like EPS8L1 Q8TE68 DRS@PAAETPPLQR S182 Yes 3.77 0.5 1.62 ± 0.22 protein 1 THRAP3 Q9Y2W1 Thyroid hormone receptor‐associated protein 3 SPALKS@PLQSVVVR S253 Yes 3.44 0.31 1.62 ± 0.00 BAT2 P48634 Large proline‐rich protein BAT2 EGTLTQVPLAPPPPGAPPS@PAPAR S1147 Yes 3.38 0.55 1.61 ± 0.88 H1FX Q92522 Histone H1x AGGSAALS@PSK S31 Yes 3.61 0.11 1.61 ± 0.15 *: Phosphorylation sites are indicated by "@" after the amino acid in the peptide sequence.

Table 2. Phosphopeptides with decreased abundance in SKBR3_shVPS4B cells. Ratio = Gene UniProt Delta Protein Name Phosphopeptide Sequence* Position Known Xcorr SKBR3_shVPS4B Name Accession Cn vs. SKBR3 PD52 P55327 Tumor protein D52 ASAAFSSVGS@VITK S119 No 4.7 0.18 0.30 ± 0.06 CGN Q9P2M7 Cingulin KVS@LVLEK S332 Yes 3.52 0.46 0.32 ± 0.03 STAT5A P42229 Signal transducer and activator of transcription 5A AVDGY@VKPQIK Y694 Yes 2.84 0.39 0.32 ± 0.03 C1orf226 A1L170 Uncharacterized protein C1orf226 ASS@PSLIER S223 Yes 2.76 0.19 0.33 ± 0.00 TNS3 Q68CZ2 Tensin‐3 KLS@LGQYDNDAGGQLPFSK S776 Yes 4.41 0.26 0.35 ± 0.09 CLTA P09496 Clathrin light chain A LQS@EPESIR S105 Yes 2.57 0.11 0.38 ± 0.03 HSPB1 P04792 Heat shock protein beta‐1 QLSS@GVSEIR S83 Yes 2.79 0.18 0.38 ± 0.06 MTOR P42345 FKBP12‐rapamycin complex‐associated protein T@LDQSPELR T1162 Yes 2.62 0.2 0.38 ± 0.03 TMSB10 P63313 Thymosin beta‐10 TET@QEKNTLPTK T23 Yes 3.96 0.1 0.38 ± 0.03 CGN Q9P2M7 Cingulin SHS@QASLAGPGPVDPSNR S131 Yes 4.07 0.11 0.39 ± 0.13 REPS2 Q8NFH8 RalBP1‐associated Eps domain‐containing protein 2 HQAS@LIR S254 Yes 2.55 0.51 0.39 ± 0.09 HSPB1 P04792 Heat shock protein beta‐1 QLS@SGVSEIR S82 Yes 2.78 0.19 0.42 ± 0.04 SQSTM1 Q13501 Sequestosome‐1 SRLT@PVSPESSSTEEK T269 Yes 2.79 0.14 0.42 ± 0.07 41 HSPB1 P04792 Heat shock protein beta‐1 GPS@WDPFR S15 Yes 3.09 0.36 0.43 ± 0.06 EPB41L1 Q9H4G0 Band 4.1‐like protein 1 HQAS@INELK S510 Yes 3.08 0.22 0.44 ± 0.08 MARCKSL1 P49006 MARCKS‐related protein AAAT@PESQEPQAK T148 Yes 3.96 0.23 0.44 ± 0.14 TJP1 Q07157 Tight junction protein ZO‐1 IDS@PGFKPASQQK S912 Yes 3.28 0.26 0.44 ± 0.09 CGN Q9P2M7 Cingulin QPAQSQNLS@PLSGFSR S252 Yes 2.93 0.24 0.45 ± 0.07 ENAH Q8N8S7 Protein enabled homolog QNS@QLPAQVQNGPSQEELEIQR S125 Yes 4.79 0.56 0.45 ± 0.09 MARCKSL1 P49006 MARCKS‐related protein GEVPPKET@PK T85 Yes 2.68 0.49 0.45 ± 0.03 CDK7 P50613 Cell division protein kinase 7 AYT@HQVVTR T170 Yes 2.99 0.12 0.46 ± 0.19 BNIP2 Q12982 BCL2/adenovirus E1B 19 kDa protein‐interacting protein 2 KGS@ITEYTAAEEK S114 Yes 4.31 0.1 0.48 ± 0.10 TPD52L1 Q16890 Tumor protein D53 HSIS@MPAMR S137 Yes 2.65 0.13 0.48 ± 0.10 ETV3 P41162 ETS translocation variant 3 SSGVVPQS@APPVPTASSR S139 Yes 4.18 0.27 0.49 ± 0.00 SSH3 Q8TE77 Protein phosphatase Slingshot homolog 3 RQS@FAVLR S37 Yes 3.01 0.32 0.49 ± 0.07 CTNND1 O60716 Catenin delta‐1 VGGS@SVDLHR S268 Yes 2.97 0.16 0.50 ± 0.18 Dual adapter for phosphotyrosine and 3‐phosphotyrosine DAPP1 Q9UN19 SRS@FIFK S276 No 2.64 0.2 0.51 ± 0.12 and 3‐phosphoinositide RPS28 P62857 40S ribosomal protein S28 TGS@QGQCTQVR S23 Yes 3.3 0.44 0.52 ± 0.07 ITPR3 Q14573 Inositol 1,4,5‐trisphosphate receptor type 3 VAS@FSIPGSSSR S1832 Yes 3.5 0.26 0.54 ± 0.14

Table 2. Phosphopeptides with decreased abundance in SKBR3_shVPS4B cells (continued). LIMA1 Q9UHB6 LIM domain and actin‐binding protein 1 ASSLSES@SPPK S373 Yes 2.64 0.19 0.54 ± 0.05 ANK3 Q12955 Ankyrin‐3 RQS@FASLALR S1459 Yes 3.08 0.29 0.55 ± 0.19 C11orf52 Q96A22 Uncharacterized protein C11orf52 VLQQQGS@QER S62 No 3.04 0.5 0.55 ± 0.06 DENND4C Q5VZ89 DENN domain‐containing protein 4C RSS@LYGIAK S948 Yes 2.78 0.2 0.56 ± 0.03 ETV6 P41212 Transcription factor ETV6 RLS@PAER S213 Yes 2.8 0.36 0.56 ± 0.17 Calcium/calmodulin‐dependent protein kinase type II CAMK2B Q13555 QET@VECLR T287 Yes 2.69 0.41 0.56 ± 0.04 subunit gamma LIMA1 Q9UHB6 LIM domain and actin‐binding protein 1 ASS@LSESSPPK S369 Yes 3.08 0.19 0.56 ± 0.06 TJP3 O95049 Tight junction protein ZO‐3 S@PGGGSEANGLALVSGFK S164 Yes 5.66 0.23 0.56 ± 0.09 EIF4G1 Q04637 Eukaryotic translation initiation factor 4 gamma 1 ITKPGS@IDSNNQLFAPGGR S1077 Yes 4.56 0.17 0.57 ± 0.05 TPD52L1 Q16890 Tumor protein D53 VGGTNPNGGS@FEEVLSSTAHASAQSLAGGSR S174 Yes 4 0.2 0.57 ± 0.10 TNKS1BP1 Q9C0C2 182 kDa tankyrase‐1‐binding protein S@QEADVQDWEFR S836 Yes 4.31 0.49 0.58 ± 0.09

42 LIM and calponin homology domains‐containing protein LIMCH1 Q9UPQ0 SINHQIES@PSER S973 No 3.49 0.2 0.59 ± 0.12 1 NAV1 Q8NEY1 Neuron navigator 1 TPPVAVT@SPITHTAQSALK T540 No 3.21 0.12 0.59 ± 0.05 PPL O60437 Periplakin GKYS@PTVQTR S14 No 2.81 0.12 0.59 ± 0.12 TNS3 Q68CZ2 Tensin‐3 GVGSGPHPPDTQQPS@PSK S660 Yes 3.18 0.11 0.59 ± 0.15 TJAP1 Q5JTD0 Tight junction‐associated protein 1 KDS@LTQAQEQGNLLN S545 Yes 3.95 0.14 0.59 ± 0.05 BET1 O15155 BET1 homolog SLS@IEIGHEVK S50 Yes 3.47 0.16 0.60 ± 0.14 MYH9 P35579 Myosin‐9 GAGDGS@DEEVDGK S1943 Yes 3.82 0.73 0.60 ± 0.08 PEA15 Q15121 Astrocytic phosphoprotein PEA‐15 DIIRQPS@EEEIIK S116 Yes 3.74 0.41 0.60 ± 0.13 MARCKS P29966 Myristoylated alanine‐rich C‐kinase substrate AEDGATPSPSNET@PKK T150 Yes 3.72 0.22 0.61 ± 0.03 BTG4 Q9NY30 Protein BTG4 VS@NPKSIYQVENLK S163 No 2.5 0.22 0.62 ± 0.10 SGPP1 Q9BX95 Sphingosine‐1‐phosphate phosphatase 1 RNS@LTGEEGQLAR S112 Yes 3.74 0.24 0.62 ± 0.06 STAT3 P40763 Signal transducer and activator of transcription 3 FICVTPTTCSNTIDLPMS@PR S727 Yes 5.03 0.41 0.62 ± 0.05 USP14 P54578 Ubiquitin carboxyl‐terminal hydrolase 14 AS@GEMASAQYITAALR S143 Yes 3.88 0.26 0.62 ± 0.40 *: Phosphorylation sites are indicated by "@" after the amino acid in the peptide sequence.

A B b TJP1(ZO1)(IDSpPGFKPASQQK) TJP1(ZO1) I DSpPGFKPASQQK Ratio = 0.44 ± 0.09 y x3 x3 +2 SKBR3 y Neutral loss 747.8651 10 100 100 544.5 692.8

y+1 Xcorr=3.28 +1 80 y y+2 6 ΔCn=0.26 80 3 11 658.3 403.3 627.8 y+1 b+2 +2 y+1 SKBR3_shVps4b y b+1 9 60 741.8450 60 5 8 990.4 10 275.9 7 1087.6 467.5 825.2 +1 b+1 +1 748.3660 b+1 y b 742.3463 7 10 11 +1 40 40 3 1080.0 y

Relative Abundance 786.4 1209.0 396.0 y+1 +1 11 Relative Abundance y 4 1254.6 b+1 8 747.3446 490.0 933.4 20 20 2 b+1 742.8483 748.8696 229.3 12 741.3702 1336.4 0 0 741 743 745 747 749 200 400 600 800 1000 1200 1400 m/z m/z

C EPS8L3(M^QVLYpEFEARNPR) D b Ratio = 2.29 ± 0.71 EPS8L3 M^QVLYpEFEARNPR y SKBR3_shVps4b x3 x3 100 874.8878 Neutral loss 100 826.2

80 b+2 Xcorr=2.55 80 11 ΔCn=0.39 y+2 b+2 739.8 875.3868 5 10 60 y+2 307.4 682.5 SKBR3 60 b+1 4 b+2 881.4100 271.7 6 875.8906 5 860.7 +1 366.1 40 y +1 Relative Abundance 880.9075 40 2 +1 b 272.2 y +1 10 Relative Abundance b 3 +1 1363.6 386.3 b 8 20 7 1207.5 b+1 20 1007.1 11 1477.4

0 0 874 876 878 880 882 400 600 800 1000120014001600 m/z m/z

Figure 13. Representative MS and MS/MS spectra of up-regulated and down- regulated phosphopeptides. A) The light/heavy ratio of the TJP1 (ZO1) phosphopeptide was 0.44 ± 0.09 indicating the down-regulation of this phosphopeptide in SKBR3_shVPS4B cells. B) MS/MS spectrum of TJP1 (ZO1) phosphopeptide. C) The light/heavy ratio of the EPS8L3 phosphopeptide was 2.29 ± 0.71 indicating the up-regulation of this phosphopeptide in SKBR3_shVPS4B cells. D) MS/MS spectrum of EPS8L3 phosphopeptide. ^ = oxidation.

Signal transduction pathway and network analysis reveals activation of

EGFR/EPS8/SH3BP1 complex in EGF-stimulated SKBR3_shVPS4B cells

To identify the potential protein-protein interaction networks affected by altered

EGFR signaling in SKBR3 cells with decreased VPS4B expression, the protein-protein interaction networks of the entire differential VPS4B phosphoproteome dataset were first mapped by the online database source Search Tool for the Retrieval of Interacting

Genes (STRING) [131] and major protein-protein interacting pathways (Table 3) were subsequently visualized by Cytoscape version 2.7 [126] (Figure 14). There are a total of

347 nodes and 1,158 potential protein-protein interactions. The following four major protein-protein interacting pathways have the highest STRING and MCODE scores: 1)

ErbB/mTOR/gonadotropin-releasing hormone (GnRH) signaling (EGFR signaling), 2) regulation of actin cytoskeleton/adhesion junction/tight junction/gap junction

(cytoskeleton/cell junctions), 3) spliceosome, and 4) glycolysis/gluconeogenesis.

Table 3. KEGG pathway enrichment analysis. KEGG pathway KEGG pathway name Count p‐Value Proteins id CAMK2B ERBB2 MAP2K2 MAPK1 MAPK3 MTOR PAK2 hsa04012 ErbB signaling pathway 8 0.00598 STAT5A hsa04150 mTOR signaling pathway 6 0.00943 EIF4B MAPK1 MAPK3 MTOR PDPK1 RPS6KA1 hsa04912 GnRH signaling pathway 7 0.03622 CAMK2B GNAS ITPR3 MAP2K2 MAPK1 MAPK3 PRKCD hsa05221 Acute myeloid leukemia 6 0.01475 MAP2K2 MAPK1 MAPK3 MTOR STAT3 STAT5A hsa05215 Prostate cancer 7 0.02397 ERBB2 HSP90AA1 MAP2K2 MAPK1 MAPK3 MTOR PDPK1 hsa05213 Endometrial cancer 5 0.04095 ERBB2 MAP2K2 MAPK1 MAPK3 PDPK1 hsa05216 Thyroid cancer 5 0.00559 CCDC6 MAP2K2 MAPK1 MAPK3 TPR hsa05223 Non‐small cell lung cancer 5 0.04606 ERBB2 MAP2K2 MAPK1 MAPK3 PDPK1

45 ARHGEF6 ARHGEF7 BAIAP2 ENAH GRLF1 IQGAP1 IQGAP3 Regulation of actin hsa04810 16 0.00029 MAP2K2 MAPK1 MAPK3 MYH7B MYH9 PAK2 PPP1R12A cytoskeleton SLC9A1 SSH3 hsa04520 Adherens junction 7 0.01247 BAIAP2 CTNND1 ERBB2 IQGAP1 MAPK1 MAPK3 TJP1 CGN CLDN7 CTTN EPB41L1 F11R MYH7B MYH9 PARD6B hsa04530 Tight junction 13 0.00012 PRKCD TJAP1 TJP1 TJP2 TJP3 hsa04540 Gap junction 7 0.02397 CDK1 GNAS ITPR3 MAP2K2 MAPK1 MAPK3 TJP1 hsa00010 Glycolysis / Gluconeogenesis 7 0.00372 ALDOA GAPDH PDHA1 PDHA2 PGK1 PGM1 TPI1 ACIN1 HNRNPC HNRNPK PCBP1 PRPF38A RBM25 RBMXL1 hsa03040 Spliceosome 13 0.00007 SLU7 SNRNP70 SRSF6 SRSF9 TRA2A TRA2B CAMK2B ITPR3 MAP2K2 MAPK1 MAPK3 PPP1R12A hsa04720 Long‐term potentiation 7 0.00691 RPS6KA1

TJP1 S912_0.44 TJP3 S164_0.56 ENAH S125_0.45 CLTA S105_0.38 CTNND1 S268_0.5 CGN S131_0.39 TJAP1 S545_0.59CCDC6 S244_1.48 MAP2K2 T394_1.08 PRKCD S304_0.90 TNS3 S660_0.59EPS8L3 Y459_2.29 EPS8L1 S182_1.62 EPB41L1 S510_0.44 BAIAP2 T360_1.53 ITPR3 S1832_0.54 2.58 TRIM3 S437_2.31 TBC1D4 S341_ ERBB2 S1054_1.31 CAMK2B T287_0.56 PAK2 S197_0.7 MARCKS T150_0.61 LIG1 S66_1.79 MYH9 S1943_0.6 TACC2 S1025_1.98 MARCKSL1 T148_0.44 MAPK3 Y204_1.95 PSMD2 S16_1.64 MAPK1 Y187_1.29 STAT5A Y694_0.32 HSPB1 S15_0.43 WDR77 T5_1.74 EIF4G1 S1077_0.57 STAT3 S727_0.62 SEC16A S1786_1.67 ZFYVE19 S463_1.79 CDK7 T170_0.46 RPS28 S23_0.52 H1FX S31_1.61 EIF4B S207_1.54 HIST1H1E S36_2.04 SQSTM1 T269_0.42 TRAP T201_1.45 SRSF6 S303_1.41 MTOR T1162_0.38 TMSB10 T23_0.38 0>20.62 0.77 1.30 1.60

ErbB signaling pathway/mTOR signaling pathway/GnRH signaling pathway Regulation of actin cytoskeleton/Tight junction/Gap junction/Adhesion junction Glycolysis/Gluconeogenesis Spliceosome

Figure 14. Network and pathway analysis of EGFR signaling in SKBR3_shVPS4B cells. The protein interaction network was extracted from STRING and is represented using the spring embedded layout in Cytoscape. Nodes of proteins in enriched pathways are represented using different shapes with enlarged node size: circles – ErbB/ mTOR/GnRH signaling; diamonds – regulation of actin cytoskeleton/adhesion junction/tight junction/gap junction; triangles – spliceosome; and round rectangles – glycolysis/gluconeogenesis. Nodes in non-enriched pathways are represented by small dots. All nodes are color-coded according to their phosphopeptide ratios (see color bar). Representative phosphosites that showed significant increases (light/heavy ratio > 1.60) or decreases (light/heavy ratio < 0.625) in SKBR3_shVPS4B cells are labeled with their protein name followed by phosphosite position and ratio.

Based on the pathways with the highest STRING and MCODE scores, we developed a potential model for the classes of proteins in the EGFR signal transduction pathway whose phosphorylation levels are significantly changed under conditions of EGF stimulation in SKBR3_shVPS4B cells (Figure 15). Our model indicates that the phosphorylation levels of proteins that interact with EGFR are significantly increased, whereas the phosphorylation levels of proteins further downstream in the signal transduction pathway are significantly decreased. Phosphorylation levels of proteins with functions related to actin and cytoskeleton remodeling, gene transcription and translation, cell junction integrity and protein degradation were largely decreased in conditions of down-regulated VPS4B expression. The phosphorylation levels of proteins with functions related to the maintenance of chromosome structure such as histone H1X, DNA

Ligase 1, histone H1.2, methylosome protein 50 (MEP50) and transforming acidic coiled coil-containing protein 2 (TACC2) were up-regulated (Table 1). In contrast, the phosphorylation levels of proteins involved in DNA transcription and protein translation were significantly decreased in SKBR3_shVPS4B cells: ETS translocation variants 3 and 6 (ETV3, ETV6), signal transducer and activator of transcription 3 and 5 (STAT3,

STAT5), cyclin-dependent kinase 7 (CDK7) and eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) (Figure 15B, Table 2).

Of particular interest in our pathway and network analysis was the up-regulation of the phosphorylation of the EGFR/EPS8/SH3BP1 complex in SKBR3_shVPS4B cells

(Figure 15A). It is well documented that the EPS8 GTPase complex plays an essential role in propagating extracellular stimuli from receptor tyrosine kinases such as EGFR to

the reorganization of the actin cytoskeleton network [132-134]. Interestingly, elevated

EPS8 expression is directly related to poor disease prognosis and increased metastatic states of breast cancer [115-118]. Our results suggest that EPS8/Sos -1/Abi1 could be regulated by SH3BP1 - a RhoGAP associated protein - since the phosphorylation states of both EPS8 and SH3BP1 are elevated in SKBR3_shVPS4B cells. The observation that the functions of SH3BP1 include serving as a RhoGAP protein and inactivating Rac1 at the leading edge of migratory cells to block the assembly of cell adhesion modules and increase cell motility [119] is consistent with our data indicating that the phosphorylation levels of many cell adhesion and junction molecules such as myristoylated alanine-rich C kinase substrate (MARCKS), tight junction proteins ZO-1 and ZO-3, and catenin delta-1

(CTNND1), are decreased in response to EGF treatment of SKBR3_shVPS4B cells

(Figure 15A and Table 2) [135][136].

A B Altered EGFR accumulation Altered EGFR accumulation caused by VPS4B down-regulation caused by VPS4B down-regulation

EGF EGF EGFR S259 EGFR p14 EGFR PI3K RAS p18 MEKK EGFR SOS-1 SOS-1 MEK SOS-1 RAS S183 Grb2 Grb2 ABI1 EPS8Ls S225 S649 ERK2 ERK1

Plasma membrane S488 S182 Y459 RAC-GEFs S694 Y187 Y204 S174 S107 Protein translation

TFIIH complex RAC CDK7 T170 S131 S322 SH3BP1 S544 S912 S164 Y694 CING STAT5 STAT3 S727 EIF4G1 ZO1 T1187 Cadherin PAK2 ETV6 S213 ETV3 S139 Cadherin p120 IRSp53 S197 ZO3 Catenin MEP50 T 5 S268 T360 S252 WAVE2 PRMT5 DNA transcription S15 S82 Histone arginine Cell junctions T140 T85 S83 HSBP1 Arp2/3 MRP methyltransferase complex Actin LIMA1 MARCKS S373 MYH9 S369 T1943 T150 Actin dynamics

Identified protein Up-regulated phosphorylation Activation Inhibition

Phosphorylation not changed Down-regulated phosphorylation Attenuated activation

Figure 15. Protein signaling pathways with significant changes in constituent protein phosphorylation levels in response to VPS4B down-regulation in EGF- stimulated SKBR3 cells. VPS4B down-regulation leads to the accumulation of EGFR within abnormally enlarged endosomes upon EGF binding. The differential activation or attenuation of pathways downstream of EGFR further affects the phosphorylation levels of proteins with functions related to cell junction and actin integrity (A) in addition to protein translation and DNA transcription (B). Gray, identified protein; Red, up-regulated phosphorylation; Green, down-regulated phosphorylation; Yellow, unchanged phosphorylation.

DISCUSSION

EGF-mediated signaling is one of the best characterized signal transduction pathways affecting cell growth, proliferation, invasion and metastasis in breast cancer

[35]. Several recent studies have indicated that aberrant endocytic trafficking and delayed turnover of EGFR could have drastic effects on its intracellular signaling with consequences related to oncogenesis [137-142]. Toward gaining a more thorough understanding of the consequences of delayed EGFR signal attenuation in the context of abnormal endosomal trafficking mediated by decreased VPS4B expression, we applied quantitative phosphoproteomics to identify changes in phosphorylation events in

SKBR3_shVPS4B breast cancer cells. Our results suggest an abnormal endocytic pathway could lead to the differential activation or attenuation of EGFR downstream signaling. In particular, down-regulation of VPS4B could regulate the activation or phosphorylation of several Rho- and Rac-associated proteins, which subsequently stabilizes the association of cell adhesion and junction modules.

Recently, several comprehensive phosphoproteomic analyses of EGF-induced cell signaling have been reported [37-44]. These studies provide numerous new insights and important information on the consequences of protein phosphorylation in regulating the dynamics of EGFR-mediated cell signaling. However, our network analysis suggests that one of the causes underlying altered EGFR signaling, including changes in the phosphorylation levels of EGFR signal transduction pathway proteins, is the perturbation of EGFR degradation or turnover resulting from abnormal endosome formation associated with VPS4B down-regulation.

Increased activation of MAPKs in the EGFR signal transduction pathway

It has been shown that EGFR remains ligand-bound following endocytosis and that EGFR can be phosphorylated and remains active within endosomes until late stages of endosomal trafficking [143]. This is consistent with results from our studies indicating that down-regulation of VPS4B by shRNA leads to increased EGFR half-life and prolonged phosphorylation and activation of EGFR within the endosomal compartment in response to EGF stimulation (Figure 8). Among the EGFR downstream signaling proteins that are found within endosomes are Grb2, SH2 domain-containing transforming protein (Shc), son of sevenless (Sos) and Ras proteins [50][51]. In addition to the accumulation of EGFR, VPS4B down-regulation may also lead to the accumulation of signaling molecules required for MAPK activation within abnormal endosomes, consequently increasing MAPK activation. As expected, we found that phosphorylation of Tyr204 on extracellular signal-regulated kinase 1 (ERK1, also known as mitogen- activated protein kinase 3, MAPK3) was significantly increased in SKBR3_shVPS4B cells (ratio = 1.95 ± 0.34, Table 1). The phosphorylation of Tyr204 on ERK1, which is mediated by MAPK kinase (MEK), stimulates the kinase activity of ERK1 [144][145].

Interestingly, it has been demonstrated recently that late endosome proteins, p14 and p18, act as endosome adaptors and recruit the MEK scaffold protein, MP1, to form the p14/p18/MP1/MEK signaling complex on endosomes, which is required to activate

MAPKs during the late stage of EGFR signaling [73-75]. It would be critical in the near future to determine whether the down-regulation of VPS4B could lead to the accumulation of p14/p18/MP1/MEK within abnormal endosomal compartments and subsequently decrease the activity of MAPK-mediated signaling.

Decreased STAT activation in the EGFR signal transduction pathway

Signal transducers and activators of transcription (STATs) are commonly activated in response to EGF stimulation in many malignancies [146]. Recently, Tu et al. reported that STAT3 activation requires localization of activated Src at focal adhesions, which requires the function of ESCRT proteins [97]. Expression of functionally inactive

VPS4B leads to the accumulation of Src within abnormal endosomes and results in a reduction in the levels of activated STAT3 [97]. This result strongly suggests that

VPS4B and ESCRTs are essential in maintaining the fidelity of protein trafficking among membrane compartments.

We found that the phosphorylation levels of STAT5A Tyr694 (or Tyr699 in

STAT5B) and STAT3 Thr727 were decreased in SKBR3_shVPS4B cells (ratio = 0.32 ±

0.03 and 0.62 ± 0.05, respectively, Table 2). STAT5A Tyr694 phosphorylation, which is mediated by Src [147] or EGFR [148], is required for dimerization and confers DNA binding activity and transcriptional activation [149]. In our studies, the abnormal accumulation of EGFR did not increase the phosphorylation of STAT5A Tyr694, suggesting that the trafficking and dynamic recycling of EGFR is clearly disrupted under conditions of decreased VPS4B expression. Our observation is also supported by several previous observations that functionally inactive VPS4B impairs the dynamic recycling of transferrin receptors and low density lipoprotein receptors from late endosomes to the cell surface [66-68].

ERK1 is one of kinases known to phosphorylate STAT3 Ser727 in response to

EGF stimulation [147]. However, we found that phosphorylation of STAT3 Ser727 was decreased in SKBR3_shVPS4B cells even in the presence of active ERK1, as indicated by increased Tyr204 phosphorylation (Table 1). Similar to its effect on Src, abnormal endosomal trafficking mediated by down-regulated VPS4B may cause activated MAPKs, such as ERK1, to be retained within abnormal endosomes [97], and thus decrease the ability of MAPKs to activate downstream proteins that are localized to subcellular compartments other than endosomes. Taken together, it is plausible to hypothesize that down-regulation of VPS4B can attenuate EGF/EGFR-mediated signal transduction by confining signaling molecules to certain subcellular compartments.

Chromosomal structure, gene transcription and translation

The altered EGFR signaling in SKBR3_shVPS4B cells could ultimately affect downstream cellular processes such as gene expression (Figure 15B). Chromosomal structure, especially, post-translational histone modification-related structure, plays an important role in gene transcription and repression [150]. We found increased phosphorylation levels of histone 1 family protein H1x (H1FX) Ser31 and histone H1.2

(HIST1H1E) Ser36 in SKBR3_shVPS4B cells (ratio = 1.61 ± 0.15 and 2.04 ± 0.29, respectively, Table 1). Phosphorylation of histone 1 by cyclin-dependent kinase 2

(CDK2)/cyclin A has been shown to result in a local alteration of chromatin structure to a more relaxed for modification [151][152] and facilitates transcription by RNA polymerase I and II [153].

We observed increased phosphorylation of Thr5 in methylosome protein 50

(MEP50), a non-catalytic component of the 20S protein arginine methyltransferase 5

(PRMT5)-containing methyltransferase complex [154], in SKBR3_shVPS4B cells (ratio

= 1.74 ± 0.30, Table 1). Methylosome protein 50 Thr5 phosphorylation is mediated by cyclin D1/cyclin-dependent kinase 4 (CDK4) and increases PRMT5 activity, which can lead to histone arginine di-methylation (histones H3R8 and H4R3) and subsequently lead to cullin 4 (CUL4) transcriptional repression, chromatin silencing, DNA replication factor 1 (CDT1) over-expression, and DNA replication [155]. Increased phosphorylation was also seen in transforming acidic coiled-coil-containing protein 2 (TACC2) Thr1025

(ratio = 1.98 ± 0.38, Table 1), which interacts with histone acetyltransferase and contributes to the modification of chromatin structure [156]. Together, the phosphorylation changes in chromatin and its modifying proteins suggest a global altered gene expression pattern in SKBR3_shVPS4B cells in response to treatment with EGF.

Two ETS transcription factors that are down-stream nuclear targets of the growth signal transduction cascades were identified in our phosphoproteome dataset: ETS translocation variant 6 (ETV6) and ETS translocation variant 3 (ETV3). Increased phosphorylation of ETSs is important for their DNA binding, transcriptional activities, association with cellular partners, subcellular localization and protein stabilities [157].

We found that phosphorylation of Ser213 in ETV6 was decreased in SKBR3_shVPS4B cells (ratio = 0.56 ± 0.17, Table 2). ETV6 Ser213 phosphorylation is mediated by activated MAPKs and leads to the abolishment of the repression of MAPK-related proliferation of gene transcription [158]. However, phosphorylation of ERK1 Tyr204

was increased in SKBR3_shVPS4B cells (Table 1), which indicates an increased activity of ERK1. As discussed earlier, this decreased ETV6 phosphorylation in VPS4B down regulated cells could be due to the impaired endosomal trafficking. The abnormal sequestration of MAPKs within abnormal endosomes decreases their substrate phosphorylation levels. Decreased phosphorylation was also seen in ETV3 on Ser139.

ETV3 blocks Ras-MAPK dependent proliferation by replacing other ETS transcriptional activators from the cell cycle control genes, such as c-Myc and cell division control protein 2 (Cdc2) [159]. Thus, the decreased phosphorylation of these transcription repressors suggests that proliferation genes may be repressed in SKBR3_shVPS4B cells as compared to parental SKBR3 cells.

Regulation of actin and cytoskeleton remodeling

In a number of cell types, EGF evokes dramatic morphological changes, cortical actin polymerization, and stress fiber breakdown [160]. Many proteins involved in actin remodeling showed phosphorylation changes in SKBR3_shVPS4B cells (Figure 15A).

The myristoylated alanine-rich C kinase substrate (MARCKS, MARCS) is involved in cytoskeleton rearrangement through interacting with actin filaments [161-163]. We found that phosphorylation on Thr150 was decreased in VPS4B down regulated cells

(ratio = 0.61 ± 0.03, Table 2). Thr150 is adjacent to the effector domain of MARCKS and is phosphorylated by CDK2 [164]. It has been demonstrated that phosphorylation of

MARCKS effector domain by protein kinase C (PKC) leads to its release from the membrane actin filaments to the cytoplasm [165-167], dissociation of cell-cell adhesions

[168], and down-regulation of MARCKS through proteasomal degradation [169]. As a

result, the decreased phosphorylation of Thr150 suggests PKC-mediated phosphorylation of the effector domain is attenuated in SKBR3_shVPS4B cells and this decreased

MARCKS phosphorylation could cause the dissociation of MARCKS from actin.

Non-muscle myosin II (NMII) is an actin cross-linking and contractile protein involved in cell adhesion and cell migration [170]. Phosphorylation of NMII Ser1943 was decreased in SKBR3_shVPS4B cells (ratio = 0.60 ± 0.08, Table 2). Phosphorylation of amino acids within the C-terminal “nonhelical tailpiece”, such as Ser1943 in myosin 9

(MYH9), favors filament disassembly [171], while a non-phosphorylatable S1943A mutation leads to cortical over-assembly [172]. In cancer cell lines, EGF stimulation triggers phosphorylation of MYH9 Ser1943 [173], however, in SKBR3_shVPS4B cells, the phosphorylation of this site was decreased, which suggests the signaling from EGF to

MYH9 may be inhibited by VPS4B down-regulation.

LIM domain and actin-binding protein 1 (LIMA1) is an actin-binding protein which links the cadherin/catenin complex to F-actin to form and stabilize the circumferential actin belt structure [174]. Phosphorylation of the C-terminal region of

LIMA1 by MAPKs reduces its affinity for actin filaments and promotes growth factor- induced stress filament disassembly, membrane ruffling and cell migration [175]. We identified several C-terminal phosphorylation sites (Appendix 1), among which phosphorylation of Ser369 and Ser373 were decreased in VPS4B down regulated cells

(ratio = 0.56 ± 0.06 and 0.54 ± 0.05, respectively, Table 2). The decreased

phosphorylation may increase LIMA1’s affinity for actin and reduce actin filament disassembly upon EGF stimulation.

Heat shock protein beta-1 (HSPB1), a stress response protein, acts as an ATP- independent chaperone in protein folding, but it is also implicated in cell motility and migration through its interaction with actin [176]. As compared to parental SKBR3 cells, phosphorylation levels of HSPB1 Ser15, Ser82, Ser83 were decreased in VPS4B down regulated cells upon EGF stimulation (ratio = 0.43 ± 0.06, 0.42 ± 0.04 and 0.38 ± 0.06, respectively, Table 2). In response to stress, growth factor and cytokine stimulation,

HSPB1 Ser15, Ser78, and Ser82 are phosphorylated by various kinases, including PKC

[177], which increases cell motility by regulating actin remodeling [176][178]. The inhibition of HSBP1 phosphorylation by its upstream kinase inhibitors decreases the invasiveness of cancer cells [179].

A large number of cell adhesion and junction proteins showed changes in their phosphorylation levels in VPS4B down regulated SKBR3 cells (Figure 15A). We found that phosphorylation levels of tight junction protein ZO-1 (TJP1, ZO1) Ser912 and tight junction protein ZO-3 (TJP3, ZO3) Ser164 were decreased in SKBR3_shVPS4B cells upon EGF stimulation (ratio = 0.44 ± 0.09 and 0.56 ± 0.08, respectively, Table 2). TJP1 and TJP3 form the cytoplasmic plaques beneath the plasma membrane of tight junctions

[180]. Phosphorylation is involved in the regulation of tight junction permeability through junction assembly and disassembly [181-183], and the interaction with other junction proteins or cytoskeleton proteins [184][185]. Cingulin (CGN) is another tight

junction protein that interacts with TJPs and actin [186][187]. In response to various environmental changes, such as calcium depletion, the phosphorylation levels of serine residues in CGN are highly increased [188]; however, in our study, the phosphorylation levels of Ser252, Ser131 and Ser322 in CGN were decreased in VPS4B down-regulated cells (ratio = 0.45 ± 0.07, 0.39 ± 0.13 and 0.32 ± 0.03, respectively, Table 2).

Catenin delta-1 (CTNND1, p120-catenin) is a critical component of cell-cell adhesion. It regulates cadherin presentation and stability by binding to the cadherin tail and selectively preventing cadherin endocytosis and degradation [189]. We observed decreased CTNND1 Ser268 phosphorylation upon EGF stimulation in SKBR3_shVPS4B cells (ratio = 0.50 ± 0.18, Table 2). Phosphorylation of CTNND1 has distinct roles at different times during the dynamic process of adhesion junction assembly and disassembly [190]. CTNND1 Ser268 phosphorylation is mediated by protein kinase C- alpha (PKC) and casein kinase 1 epsilon (CK1ε), which leads to the disruption of its interaction with E-cadherin and its release into the cytoplasm [191][192]. This phosphorylation is not only implicated in cell-cell adhesion [193], but also in the regulation of the activity of Kaiso transcriptional factor [194]. The decreased phosphorylation of Ser268 in CTNND1 indicates that cell-cell adhesion might not be disrupted in SKBR3_shVPS4B cells in response to EGF stimulation.

CONCLUSION

In summary, by using a SILAC-based quantitative phosphoproteomic analysis, we revealed an important role for VPS4B in EGFR signal transduction in SKBR3 cells. The multiple functions of VPS4B render it an important regulator for the fine-tuning of cell

signaling mediated by endocytosed receptors such as EGFR. VPS4B down-regulation results in the deficiency of the endosomal trafficking of receptors targeted for degradation, consequently prolonging the time that EGFR remains on the limiting endosomal membrane and abnormally potentiating its signaling. VPS4B-mediated impaired endosomal trafficking may play an important role in altering downstream EGFR signal transduction, in which specific signaling is activated, attenuated or maintained in the context of loss of VPS4B function, for example, increasing ERK1 phosphorylation while decreasing STAT5/STAT3 phosphorylation. The altered signal transduction leads to perturbations of a variety of cellular processes, such as gene expression, cytoskeleton remodeling, and cell junction integrity (Figure 15).

Our unpublished observations indicate that hypoxia induces VPS4B down- regulation in SKBR3 cells. The altered signaling in VPS4B down-regulated cells may contribute to cell survival under harsh conditions. Instead of activating growth and proliferation-related signaling, such as STAT signaling, VPS4B down-regulation attenuates their activities to conserve cellular resources, while preserving the function of essential signaling pathways, such as MAPK signaling, for cell survival under harsh conditions. Also, increased cell motility and invasion, which are related to cytoskeleton remodeling and cell junction integrity, may not occur when cells experience harsh conditions. When cells are exposed to adverse conditions, their activities may be decreased by VPS4B down-regulation, which supports the supposition of this study that the differential down-regulation of VPS4B could be one of the potential “protective”

mechanisms whereby the assembly of cell adhesion modules is perturbed in metastatic breast cancer cells.

In conclusion, our studies provide a potential new mechanism for SKBR3 cells to control EGF-related signaling in response to environmental changes. The delineation of the functional relationship between VPS4B down-regulation, altered EGFR signaling and breast cancer cell survival can shed light upon potential mechanisms of anti-cancer therapy.

ABSTRACT

The endosomal/lysosomal system, in particular the endosomal sorting complexes required for transport (ESCRTs), plays an essential role in regulating the trafficking and destination of endocytosed receptors and their associated signaling molecules. It has been shown that dysfunction of vacuolar protein sorting 4 homolog B (VPS4B), an

ESCRT-III associated protein, leads to delayed degradation of epidermal growth factor receptor (EGFR) and prolonged intracellular EGFR signaling in abnormally enlarged endosomal compartments [66-68]. Our previous phosphoproteomic study also indicates that EGFR downstream signaling is differentially activated or attenuated in breast cancer cells. However, the consequences of EGFR accumulation and altered EGFR signaling caused by ESCRT dysfunction have not been well addressed. Thus, the goal of this study was to determine the role of VPS4B in dynamic protein expression in the context of altered EGFR signaling in breast cancer cells. We developed an internal standard- assisted synthesis and degradation mass spectrometry (iSDMS) method, which permits the direct measurement of protein synthesis and degradation and consequently, dynamic protein expression. The dynamic expression profiles of more than 700 proteins were compared between a VPS4B down regulated SKBR3 breast cancer cell line and parental cells. We found that increased protein expression in VPS4B down regulated cells relied more on increased protein synthesis, while decreased protein expression was more related to both decreased synthesis and increased degradation. More importantly, VPS4B down- regulation resulted in differential expression of proteins involved in energy metabolism; proteins involved in glycolysis were down-regulated while proteins with roles in fatty acid β-oxidation were up-regulated. The adoption of fatty acid β-oxidation as an

alternative energy source could be an unrevealed survival mechanism for breast cancer cells with VPS4B dysfunction, and could be a new drug target for cancer treatment.

INTRODUCTION

VPS4B, a member of the AAA (ATPases associated with diverse cellular activities) protein family, plays an important role in the lysosomal degradation pathway, which functions in ligand-induced membrane receptor down-regulation. In lysosomal degradation, endocytosed receptors are sorted into multivesicular bodies (MVBs), which requires the sequential assembly of endosomal sorting complex required for transport I, II, and III (ESCRT-I, -II and –III) on the endosomal membrane [195]. VPS4B functions to dissociate ESCRT-III from endosomes for further rounds of endosomal sorting [92].

Expression of functionally inactivate VPS4B results in the accumulation of receptors on abnormally enlarged endosomes (class E compartments) [66-68]. As an essential

ESCRT-III interacting protein, VPS4B is also involved in protein trafficking among membrane compartments and in signal transduction [66][68][80][97].

EGF-mediated signaling is one of the most important signaling pathways for cell growth, proliferation, invasion and metastasis in breast cancer [35]. Upon ligand binding, the EGF receptor (EGFR) is phosphorylated, promoting downstream signal transduction, while it is internalized and degraded within the MVB-lysosome [45]. EGFR signaling is initiated at the cell membrane; however, late signaling propagation occurs on the endosomes [51]. Dysfunction of VPS4B not only leads to delayed degradation of EGFR but also to prolonged intracellular EGFR signaling in abnormally enlarged endosomal

compartments [66-68]. Our recent systems biology phosphoproteomic study indicates that EGFR downstream signaling is differentially activated or attenuated in SKBR3 cells with down regulated VPS4B. To understand the role of VPS4B dysfunction in breast cancer, we set out to determine the consequences of altered EGFR signaling: changes in protein synthesis and degradation, as well as protein dynamics in VPS4B down regulated breast cancer cells. Altered protein synthesis and degradation of cancer-related proteins are involved in cellular transformation and cancer progression [196-200]. Understanding protein synthesis and degradation is essential to fully appreciate cellular dynamics and develop more effective strategies to treat cancer.

Traditional protein synthesis and degradation studies rely on radiolabel tracer labeling (pulse) followed by exposure to non-radioactive medium (chase). However, these approaches are limited to total protein or only a few individual proteins. Recently, high throughput synthesis and degradation mass spectrometry (SDMS) has become a novel approach to study protein synthesis and degradation, in which protein synthesis and degradation can be measured simultaneously, unbiasedly, and unambiguously with minimal cell perturbation [201]. In this approach, proteins are metabolically labeled by stable isotope labeling with amino acids in cell culture (SILAC) followed by chasing in normal medium. The pre-existing proteins are labeled with stable isotope amino acids, while the newly synthesized proteins only incorporate regular amino acids. These proteins can be discriminated from each other and quantified by mass spectrometry based on the differences in masses of their peptides following enzymatic digestion. Since

Beynon and co-workers introduced the use of mass spectrometry in protein synthesis and

degradation studies [202], this approach, with various modifications, has been applied to numerous systems, such as Escherichia coli [203], Mycobacterium tuberculosis [204],

Mycoplasma pneumoniae [205], Streptomyces coelicolor [206], Saccharomyces cerevesiae [202], HeLa cell nucleoli [207], human adenocarcinoma cells [208], chicken skeletal muscle [209][210] and, recently, in mouse [211].

In SILAC-based protein synthesis and degradation experiments, the degradation rate constant is derived from the relative isotope abundance (RIA), or the percentage of pre-existing labeled protein [202], which is sufficient to estimate protein degradation rates under steady state conditions, or no net synthesis and degradation, for every protein.

In order to bypass the steady-state assumption in dynamic systems, such as cell proliferation, Jayapal et al. introduced isobaric tags for relative and absolute quantitation

(iTRAQ) into SILAC-based protein turnover experiments, which permitted the inclusion of the gain or loss of total protein into RIA calculations [206]. In this study, the authors showed that in a non steady-state system, the degradation rates for fast turnover proteins calculated by incorporating the total protein abundance changes into RIA differed from the degradation rates calculated by RIA alone [206].

Dysfunction of VPS4B leads to EGFR accumulation and prolonged activation

[66-68], which could contribute to cell proliferation and growth. To estimate protein synthesis and degradation rates in the context of altered EGFR signaling caused by down- regulation of VPS4B, we developed a new approach, internal standard-assisted synthesis and degradation mass spectrometry (iSDMS), to measure protein synthesis and

degradation rates, as well as protein expression, simultaneously in a dynamic system. We compared the synthesis and degradation rates of more than 700 proteins between VPS4B down regulated SKBR3 cells and the parental SKBR3 cells. We found that VPS4B down-regulation resulted in differential protein expression in energy metabolism pathways, in which glycolysis proteins were down-regulated, while fatty acid β-oxidation proteins were up-regulated. The adoption of fatty acid β-oxidation as an alternative energy source could be an unrevealed survival mechanism for breast cancer cells with

VPS4B dysfunction. Our findings could provide new strategies for the development of innovative cancer treatments. This is the first time, to the best of our knowledge, that a global protein synthesis and degradation approach has been used to study the role of

VPS4B/ESCRT dysfunction in EGFR signaling.

MATERIALS AND METHODS

Cell culture

SKBR3 cells were obtained from the American Type Culture Collection

(Rockville, MD). VPS4B knock-down SKBR3 (SKBR3_shVPS4B) cells were generated by transducing SKBR3 cells with a lentivirus harboring shRNA against VPS4B (SA

Biosciences/Qiagen, Frederick, MD). Lentiviral stock was made as previously described

[120].

SKBR3_shVPS4B cells and parental SKBR3 cells were cultured in DMEM

13 “medium” SILAC medium (Thermo Scientific/Pierce) supplemented with 100 mg/L C6

L-Arginine-HCl (purity 97-99 %, Cambridge Isotope Laboratories) and 100 mg/L D4 L-

Lysine-HCl (purity 96-98 %, Cambridge Isotope Laboratories), 10 % dialyzed FBS

(Invitrogen) and 1 % antibiotic antimycotic solution (Invitrogen). For SKBR3_shVPS4B cells, 2 µg/ml puromycin was added to maintain their phenotype. After five passages, cells were starved in serum free “medium” SILAC medium for 18 h. Following three washes with DPBS (Invitrogen), cells were stimulated with 100 ng/ml EGF in DMEM

“light” SILAC medium (Thermo Scientific/Pierce) supplemented with 100 mg/L arginine and 100 mg/L lysine (Sigma) and 1 % antibiotic antimycotic solution (Invitrogen) for 0,

2, 6, 12 and 24 h. Cells were harvested at each time point. The cell pellets were frozen in liquid nitrogen and stored in -80 ºC. For the internal standard, SKBR3_shVPS4B cells

13 15 13 15 were metabolically labeled with C6 N4-arginine and C6 N2-lysine by culturing in

13 15 DMEM “heavy” SILAC medium supplemented with 100 mg/L C6 N4 L-Arginine-HCl

13 15 (purity 97-99 %, Cambridge Isotope Laboratories) and 100 mg/L C6 N2 L-Lysine-HCl

(purity 97-99 %, Cambridge Isotope Laboratories), 10 % dialyzed FBS and 1 % antibiotic

13 15 13 15 antimycotic solution (Invitrogen). The incorporation of C6 N4-arginine and C6 N2-

lysine in the internal standard SKBR3_shVPS4B cells was ~98% as determined by mass

13 15 spectrometric analysis of tryptic peptides isolated from the C6 N4-arginine and

13 15 C6 N2-lysine labeled SKBR3_shVPS4B cells. Cells were harvested and stored as

indicated above.

Cell lysis and protein digestion

Cell pellets were lysed in 4% SDS in 100 mM Tris-HCl, pH 7.6 with 5 U/ml

benzonase nuclease (Novagen) and complete EDTA-free protease inhibitor cocktail

(Roche) and were well sonicated. After removal of cell debris by centrifugation at

10,000×g at room temperature for 10 min, protein concentration was measured in

triplicate using the BCA protein assay kit (Thermo Scientific/Pierce). 180 µg of protein

13 15 from each time point were mixed with 60 µg of protein labeled with C6 N4-arginine

13 15 and C6 N2-lysine. DTT (Sigma) and TCEP (Thermo Scientific/Pierce) were added to

the mixture to final concentrations of 100 mM and 10 mM, respectively. Mixtures were

kept at 90 °C for 10 min.

After cooling to room temperature, the cell lysates were processed by the Filter

Aided Sample Preparation (FASP) procedure [212] using 30k VIVACON 500 filtration

units (Sartorius Biolab) with some modifications. Briefly, cell lysates were mixed with

200 µl 8 M urea in 100 mM Tris-HCl, pH 8.5 (UA buffer) with 5 mM TCEP, loaded into

the filtration units, and centrifuged at 14,000×g for 15 min. All following centrifugation steps were the same. 200 µl of UA buffer was added to the concentrates in the filtration units and centrifuged again. Alkylation was performed by adding 100 µl of 50 mM iodoacetamide in UA buffer and incubating at room temperature for 30 min. Excess iodoacetamide was eliminated by centrifugation, followed by three washes with 200 µl of

UB buffer (8 M urea in 100 mM Tris-HCl, pH 8.0).

Samples were digested by sequential addition of enzymes in the filtration units.

Briefly, 2 µg of endoproteinase LysC (Roche) in 40 µl water were added to the filtration units and kept at room temperature in a humidified chamber overnight. Samples were then diluted with 200 µl of 50 mM NH4HCO3. 4 µg of trypsin (Promega) were added

and kept at 37 ºC for another 6 h. After digestion, the peptides were collected by

centrifugation of the filtration units, followed by two washes with 50 µl of 50 mM

NH4HCO3. All filtrates were combined and acidified by adding TFA (Sigma) to a final concentration of 1 %. Peptides were desalted by Sep-Pak C18 cartridges (Waters) and dried by SpeedVac (Thermo Scientific). Dried peptides were stored at -80 °C.

Peptide fractionation

Peptides were fractionated using stop and go extraction (STAGE) tips made in- house with anion exchange disks as described elsewhere [213-215]. The STAGE tips were assembled by stacking 6 layers of Empore anion exchange membrane disks (3M) into a 200 µl micropipet tip. Two stacked tips were used to separate 100 µg peptides.

Britton & Robinson buffer (BR buffer), composed of 20 mM acetic acid, 20 mM phosphoric acid, and 20 mM boric acid and titrated with NaOH to the desired pH, was used in peptide separation. The tip was wet with methanol and washed with 1 M NaOH, followed by equilibration with 100 µl of BR buffer pH 11. Peptides were loaded at pH

11 and fractions were subsequently eluted with BR buffer of pH 11, 8, 6, 5, 4 and 3,

respectively. The flow through and all the fractions were acidified by adding TFA to a

final concentration of 1 % and desalted by a STAGE tip containing three layers of C18

membrane disks (3M). Peptides were dried and stored as mentioned above.

LC-MS/MS analysis

Peptides were dissolved in 0.5 % acetic acid (solvent A) and were separated on a

10-cm reverse-phase PicoFrit spray tip (New Objective, Woburn, MA) packed in-house

with sub-2 µm C18 resin (Prospereon Life Science, IL), using a nano-flow Xtreme

Simple liquid chromatography system (CVC/Microtech) coupled to a linear ion trap-

orbitrap mass spectrometer (LTQ Orbitrap, Thermo Electron). Peptides were loaded onto the column with solvent A at a flow rate of 0.6 µl/min, and eluted with a 180 min linear gradient at a flow rate of 0.2 µl/min. A gradient of 2-60 % solvent B (40 % ACN, 0.5 % acetic acid) was applied to the SAX flow through and fractions eluted with pH 11 and pH

8 buffer, a 2-65 % solvent B gradient was used for the pH 6 and pH 5 fractions, and a 5-

70 % solvent B gradient was used for the pH 4 and pH 3 fractions. After the linear gradient, the column was washed with 95 % solvent B and re-equilibrated with 95 % solvent A. Seven fractions from one sample were run sequentially followed by a 40 min wash with 80 % ACN with 0.5 % acetic acid.

Mass spectra were acquired in the positive ion mode applying a data-dependent automatic switch between survey scan and MS/MS acquisition. The survey scans were acquired in the orbitrap using a mass range of m/z 400-1,600 at a resolution of 60,000 at

400 m/z. The target value was 100,000. MS/MS scans were acquired in the linear ion trap on the 5 most intense ions in each survey scan with dynamic exclusion of previously selected ions; repeat count 1 and exclusion duration 15 s. The fragmentation was performed by collision-induced dissociation (normalized collision energy 35%) with a target value of 30,000. Ion selection threshold was 5000 counts. Charge state screening was enabled and +1 ions were excluded from fragmentation.

Other mass spectrometric parameters were: spray voltage 1.35 kV; no sheath and auxiliary gas flow; ion transfer tube temperature 180 ºC; activation q = 0.25 and activation time of 30 ms were applied in MS2 acquisitions.

Peptide identification

Data were searched against a UniProt human database (downloaded on Oct. 18,

2010) using SEQUEST (Bioworks 3.3.1 SP1, Thermo Scientific). Enzyme specificity was set to trypsin and allowed two missed cleavages. Database search parameters were: precursor peptide mass tolerance 50 ppm and fragment ion tolerance 1 amu.

Carbamidomethyl cysteine was set as a fixed modification and oxidized methionine was set as a variable modification. Additional variable modifications included arginine

+6.02013, arginine +10.00827, lysine +4.02510 and lysine +8.01420. The peptides were filtered by the following criteria: Xcorr ≥ 2.5, 3.0, and 3.5 for 2+, 3+, and 4+ peptides, respectively, and ∆Cn > 0.1.

Peptide relative abundance

By adding the internal standard, three populations of a given peptide were present in the mixture: the peptide containing regular arginine or lysine (unlabeled peptide), the

13 peptide containing C6-arginine or D4-lysine (labeled peptide), and the peptide

13 15 13 15 containing C6 N4-arginine or C6 N2-lysine (internal standard peptide). The relative abundance of unlabeled peptide (Al) was defined as the ratio of the peak intensities of

unlabeled peptide (Il) to the intensities of internal standard peptide (Ih), Eq.1. Similarly,

the relative abundance of labeled peptide (Am) was defined as the ratio of the peak

intensities of labeled peptide (Im) to Ih, Eq.2. An in-house software program, IsoQuant,

was used to automatically integrate the isotopic peak intensities of each peptide and

calculate Al and Am. The software is freely available for non-commercial use and can be

downloaded from http://www.proteomeumb.org/MZw.html. The relative abundance of

total peptide (Atot) was calculated by summing the relative abundance of unlabeled

peptide (Al) and labeled peptide (Am), Eq.3.

Al = Il / Ih Eq.1

Am = Im / Ih Eq.2

Atot = Al + Am Eq.3

Peptide degradation rate constant

Since the labeled peptides were only subjected to degradation, the degradation

rate constant could be derived from the changes in relative abundance of labeled peptides.

Protein degradation rate follows first-order kinetics [216]. The relative abundances of the

pre-existing labeled peptides over time (Am_t) were fit to exponential decay curves to

derive the degradation rate constant (λ). At least four time points were required to find

the best fitting curve and only curves with a coefficient (R2) > 0.81 were selected to

derive the degradation rate constant from the exponential decay equation, Eq.4,

–λt Am_t = a × e Eq.4

where a was the corrected initial relative peptide abundance.

Peptide synthesis rate

Peptide synthesis rate was defined as the rate of change of the relative abundance of the newly synthesized peptide over time. Due to cell type and status, and the purity of the stable isotope-labeled amino acids, the basal label efficiency was not 100 %, which

consequently resulted in the detection of unlabeled peptides at the beginning of the chase period. In this study, at the beginning of the chase period, ~10 % and 30 % of the peptides were unlabeled in SKBR3_shVPS4B cells and parental SKBR3 cells, respectively. Since both the pre-existing unlabeled and labeled peptides were subjected to degradation, the remaining relative abundance of pre-existing unlabeled peptide at each time point (Apre_l_t ) was calculated by

–λ t Apre_l_t = Al_0 × e Eq.5

where Al_0 was the relative abundance of unlabeled peptide at 0 h.

’ The relative abundance of the newly synthesized peptide (A l_t) was then

calculated by subtracting the relative abundance of the remaining pre-existing unlabeled

peptide from the relative abundance of the total unlabeled peptide (Al_t).

’ A l_t = Al_t - Apre_l_t Eq.6

Protein synthesis follows zero-order kinetics [216]. The relative abundances of

’ newly synthesized peptides over time (A l_t ) were fit to linear curves to derive the

synthesis rates (γ). At least four time points were required to find the best fitting curve

and only curves with a coefficient (R2) > 0.81 were selected to derive the synthesis rate from the linear equation, Eq.7,

’ A l_t = γ × t + b Eq.7

where b was the corrected initial relative peptide abundance.

Protein synthesis rate, degradation rate constant and relative abundance

The protein synthesis rate, degradation rate constant and relative protein abundance were calculated by the mean of their identified peptides’ synthesis rates, degradation rate constants and total relative peptide abundance.

RESULTS iSDMS analysis of dynamic protein profiles

To reveal the role of VPS4B dysfunction in global protein dynamics, we measured the protein synthesis rates and degradation rate constants of proteins in VPS4B down regulated SKBR3 (SKBR3_shVPS4B) cells and parental SKBR3 cells by an internal standard assisted synthesis and degradation mass spectrometry (iSDMS) approach. As shown in Figure 16, in our iSDMS approach, cells were metabolically

13 labeled with stable isotopic arginine and lysine, C6-arginine / D4-lysine (Arg6/Lys4),

and then chased in medium containing regular arginine and lysine (Arg0/Lys0), in the

presence of EGF. During the chase period, the Arg6/Lys4-labeled proteins were

undergoing degradation, while newly synthesized proteins only incorporated Arg0/Lys0.

13 15 The inclusion of an internal standard – cell lysates metabolically labeled with C6 N4-

13 15 arginine / C6 N2-lysine (Arg10/Lys8) – permitted the relative abundance of Arg6/Lys4-

labeled peptides (Am) to be defined as the ratio of the intensities of Arg6/Lys4-labeled

peptides to the intensities of the internal standard peptides detected by mass spectrometry

(Eq.1, Materials and methods). Similarly, the relative abundance of unlabeled peptides

(Al) was defined as the ratio of the intensities of unlabeled peptides to the intensities of

the internal standard peptides (Eq.2, Materials and methods).

Arg6, Lys4

EGF stimulation Arg10, Lys8 Chase in Arg0, Lys0 medium

Arg0, Lys0 0h 6h 2h 12h 24h Cell lysate

Internal standard

Mix with internal standard

Trypsin digestion Newly synthesized peptide Filter Aided Sample Preparation (FASP) A l = I l/ I h Pre-existing peptide SAX STAGE Tip fractionation A m = I m / I h

A l LC-MS/MS A m

M M M M L H H H L H H M L L Relative intensity m/z m/z m/z m/z m/z 0h 2h 6h 12h 24h

Figure 16. Internal standard assisted synthesis and degradation mass spectrometry (iSDMS) workflow to analyze EGF-mediated protein synthesis and degradation rates. 13 Cells were labeled with C6-arginine (Arg6) and D4-lysine (Lys4). After overnight serum-starvation, cells were stimulated with 100 ng/ml EGF in medium supplemented with regular arginine (Arg0) and lysine (Lys0) for 0, 2, 6, 12 and 24 h. Protein isolated 13 15 13 15 from SKBR3 cells labeled with C6 N4-arginine (Arg10) and C6 N2-lysine (Lys8) – internal standard – was spiked into each sample at a ratio of 1:3 (wt/wt). The mixture was digested by the Filter Aided Sample Preparation (FASP) procedure followed by strong anion exchange (SAX) peptide fractionation. Peptides were analyzed by online LC-MS/MS using an LTQ-Orbitrap mass spectrometer. The relative abundance of the newly synthesized (Al) or pre-existing peptides (Am) was defined as the ratio of mass spectrometric peak intensities of the unlabeled peptides (Il) or Arg6/Lys4-labeled peptides (Im) to the intensities of the Arg10/Lys8-labeled peptides (Ih), respectively. The same workflow was followed for the analysis of EGF-mediated protein synthesis and degradation rates in SKBR3_shVPS4B cells and parental SKBR3 cells.

Figures 17A and B are representative MS spectra of fatty acid synthase peptide,

SLLVNPEGPTLMR, in parental SKBR3 cells (Figure 17A) and SKBR3_shVPS4B cells

(Figure 17B) identified in chase time periods of 0, 2, 6, 12 and 24 h. The decreasing relative abundance of the pre-existing Arg6/Lys4-labeled peptides in both cells indicates the degradation of this peptide. Since protein degradation follows first-order kinetics

[216], we fit the time-course of relative abundance of pre-existing Arg6/Lys4-labled peptides to exponential curves (Figure 17C). The best fitting curves had R2 values of

0.90 and 0.91 in parental SKBR3 cells and SKBR3_shVPS4B cells, respectively. As

shown in Figure 17C, peptide SLLVNPEGPTLMR had a faster degradation rate in

SKBR3_shVPS4B cells (squares) than in parental SKBR3 cells (diamonds) with

degradation rate constants of 0.045 h-1 and 0.015 h-1 in SKBR3_shVPS4B cells and

parental SKBR3 cells, respectively.

The newly synthesized peptides were unlabeled. However, the pool of unlabeled

peptides consisted not only of newly synthesized peptides, but also pre-existing unlabeled

peptides, which were present at 0 h. Since pre-existing unlabeled and labeled peptides

were being degraded with time, we calculated the relative abundance of the remaining

(non-degraded) pre-existing unlabeled peptides at each time point by using the

degradation rate constant obtained as described previously (Eq.5, Materials and methods).

The relative abundance of the newly synthesized peptide was calculated by Eq.6

(Materials and methods).

Protein synthesis follows zero-order kinetics [216]. We fit the time-course profile of the relative abundance of newly synthesized peptides to linear curves (Figure 17D).

The best fitting curves had R2 values of 0.98 and 0.99 for parental SKBR3 cells (Figure

17D), diamonds and SKBR3_shVPS4B cells (Figure 17D, squares), respectively. As

shown in Figure 17D, peptide SLLVNPEGPTLMR had a slower synthesis rate in

SKBR3_shVPS4B cells (0.051 h-1) as compared to parental SKBR3 cells (0.133 h-1).

The relative abundance of a given peptide (Atot) was calculated by summing the

relative abundance of unlabeled peptide (Al) and labeled peptide (Am) (Eq.3, Materials

and methods). Figure 17E shows the changes of relative abundance of the

SLLVNPEGPTLMR peptide in parental SKBR3 cells and SKBR3_shVPS4B cells. The relative amount of the SLLVNPEGPTLMR peptide decreased with time in

SKBR3_shVPS4B cells (Figure 17E, squares) and increased in parental SKBR3 cells

(diamonds), which was consistent with its higher degradation rate constant (Figure 17C) and lower synthesis rate (Figure 17D) in SKBR3_shVPS4B cells.

A Fatty acid synthase (FASN) peptide SLLVNPEGPTLMR in parental SKBR3 cells (SKBR3) 0 h 2 h 6 h 12 h 24 h Al=1.85±0.24 Al=1.67±0.21 Al=2.79±0.15 Al=2.79±0.15 Al=4.30±0.45

Am=3.43±0.65 Am=3.07±0.44 Am=2.81±0.18 Am=2.85±0.08 pre-existing Am=2.27±0.10 pre-existing newly-synthesized pre-existing pre-existing pre-existing newly-synthesized pre-existing 716.893 100 100 716.888 100 716.875 100 713.889 716.899 100 713.914 714.367 80 80 717.390 80 80 80 714.416 717.377 pre-existing pre-existing pre-existing 717.401 pre-existing newly-synthesized 714.391 716.924 60 60 60 newly-synthesized 60 60 713.884 713.878 717.395 713.867 717.878 714.385 internal standard internal standard 717.426 40 40 714.380 internal standard 40 internal standard 40 40 Relative Abundance Relative Abundance Relative Abundance Relative Abundance 718.888 718.869 718.893 Relative Abundance internal standard 718.882 714.892 717.902 719.395 717.897 719.371 714.917 718.918 20 719.383 20 20 717.891 20 714.868 20 719.419 714.887 719.390 714.881 719.871 717.927 719.890 719.881 719.897 715.420 719.921 0 0 0 0 0 714 716 718 720 714 716 718 720 714 716 718 720 714 716 718 720 714 716 718 720 m/z m/z m/z m/z m/z

B Fatty acid synthase (FASN) peptide SLLVNPEGPTLMR in VPS4B down regulated SKBR3 cells (SKBR3_shVPS4B) 0 h 2 h 6 h 12 h 24 h Al=0.17±0.03 Al=0.21±0.03 Al=0.47±0.06 Al=0.66±0.07 Al=1.28±0.11 Am=4.91±0.89 Am=2.86±0.06 Am=2.84±0.52 Am=2.15±0.03 Am=1.56±0.03 79 pre-existing pre-existing pre-existing pre-existing pre-existiing 100 716.916 100 716.911 100 716.866 100 716.903 100 716.927 pre-existing newly-synthesized 717.428 80 80 80 80 80 713.917 internal standard 718.921 717.412 717.404 717.4179 717.368 714.418 60 60 60 60 60 internal standard internal standard 717.919 714.359 718.860 pre-existing 718.897 714.402 internal standard 719.423 40 internal standard 40 40 40 newly-synthesized 40 Relative Abundance Relative Abundance Relative Abundance Relative Abundance Relative Abundance 714.905 718.905 719.362 718.910 pre-existing 717.869 713.893 719.399 pre-existing 714.407 719.407 newly-synthesized 719.865 714.39433 717.906 714.920 717.930 20 719.412 20 newly-synthesized 717.914 20 713.857 20 20 719.924 pre-existing 719.900 713.900 719.909 713.905 719.913 714.890 714.89631 0 0 0 0 0 714 716 718 720 714 716 718 720 714 716 718 720 714 716 718 720 714 716 718 720 m/z m/z m/z m/z m/z

Figure 17. VPS4B down-regulation decreases the relative abundance of fatty acid synthase peptide SLLVNPEGPTLMR in SKBR3 cells. Representative MS spectra of fatty acid synthase peptide SLLVNPEGPTLMR in SKBR3 (A) and SKBR3_shVPS4B cells (B). The relative abundance of unlabeled peptides (Al) or labeled peptides (Am), expressed as mean ± standard deviation, was calculated by IsoQuant.

C D E Newly synthesized peptide Total peptide Pre‐existing peptide 7 7 7 6 SKBR3 6 SKBR3 SKBR3_shVPS4B 6 SKBR3_shVPS4B 5 5 5 y = 0.1331x ‐ 0.2807 y = 3.261e‐0.015x 4 4 4 R² = 0.9791

abundance R² = 0.9021

abundance 3 3

3 abundance

y = 0.0511x ‐ 0.014 2 2 R² = 0.9947 2 SKBR3 1 y = 4.2057e‐0.045x 1 1 80 Relative

Relative SKBR3_shVPS4B R² = 0.9099 Relative 0 0 0 0 5 10 15 20 25 ‐1 0 5 10 15 20 25 0 5 10 15 20 25 Time Time Time

Figure 17. VPS4B down-regulation decreases the relative abundance of fatty acid synthase peptide SLLVNPEGPTLMR in SKBR3 cells (continued). VPS4B down-regulation increased the degradation rate of the SLLVNPEGPTLMR fatty acid synthase peptide (C), and decreased its synthesis rate (D) in SKBR3 cells, which was related to the decrease of its relative abundance (E). The relative abundance of pre-existing peptides was derived from the relative abundance of labeled peptides (Am). The relative abundance of the newly synthesized peptides was calculated using Eq.6 (Materials and methods). The relative abundance of total peptides was the sum of relative abundance of unlabeled peptides (Al) and labeled peptides (Am) (Eq.3, Materials and methods).

Overall protein synthesis rate, degradation rate constant and relative abundance

We identified more than 15,000 total peptides assigned to more than 4,500 proteins in both SKBR3_shVPS4B cells and parental SKBR3 cells. To compare the degradation rate constants and synthesis rates of each peptide in SKBR3_shVPS4B cells and parental SKBR3 cells, we required that peptides must be quantified at each time point in each cell line, and that the best fitting curves for the time-dependent relative abundance profiles of each peptide must have at least four points with an R2 > 0.81. We

obtained the degradation rate constants and synthesis rates, as well as total peptide

profiles of 1623 peptides (Appendix 2), which were assigned to 723 proteins (Appendix

3). The protein degradation rate constants, synthesis rates, as well as expression profiles, were calculated based on the means of the degradation rate constants, synthesis rates, and total relative abundance for each protein’s constituent peptides.

An internal standard was spiked into each sample at a ratio of 1 to 3 (wt/wt). As expected, the average relative protein abundance at each time point (SKBR3_shVPS4B

cells or parental SKBR3 cells vs. the internal standard) was ~ 3.0 (Figure 18). The distributions of protein synthesis rates in parental SKBR3 cells and SKBR3_shVPS4B cells are shown in Figures 19A and 19B, respectively. The mean protein synthesis rate in

SKBR3_shVPS4B cells was 0.058 ± 0.010 h-1, which was similar to that of the parental

SKBR3 cells, 0.057 ± 0.012 h-1. The distributions of protein degradation rate constants

in parental SKBR3 cells and SKBR3_shVPS4B cells are shown in Figures 19D and 19E,

respectively. The mean protein degradation rate constant was 0.033 ± 0.012 h-1 in

SKBR3_shVPS4B cells, which was slightly higher than that of the parental SKBR3 cells,

0.028 ± 0.010 h-1.

7 SKBR3 6 SKBR3_shVPS4B 5 abundance 4 3 2 1 Relative protein 0 0261224 Time (h)

Figure 18. Average relative protein abundance. Lysate from SKBR3_shVPS4B cells (grey bars) or parental SKBR3 cells (white bars) following the 0, 2, 6, 12 and 24 h chase periods were mixed with the internal standard cell lysate at a ratio of 3:1. The average relative protein abundance was close to the expected value of 3. Error bars represent one standard deviation.

The half-life of most proteins ranges from minutes to tens of hours [208]. To analyze the affect of VPS4B down-regulation on EGF-induced protein expression, we chose to only use the longest time point, 24 h, to compare the relative protein abundance between the parental SKBR3 cells and SKBR_shVPS4B cells. The distributions of relative protein abundance at 24 h in parental SKBR3 cells and SKBR3_shVPS4B cells are shown in Figures 19H and 19I. The mean relative protein abundance at 24 h in

SKBR3_shVPS4B cells was 3.0 ± 0.5, which was similar to that of the parental SKBR3 cells, 3.0 ± 0.7. The ratios of protein synthesis rates, degradation rate constants, and relative protein abundance (SKBR3_shVPS4B cells vs. SKBR3 cells) were calculated and their distributions are shown in Figures 19C, 19F and 19I, respectively.

To address the effect of VPS4B down-regulation on dynamic protein profiles, we defined increased and decreased protein synthesis, degradation and relative protein abundance in SKBR3_shVPS4B cells as those values greater than 1.5-fold that of the parental SKBR3 cells (SKBR3_shVPS4B vs. SKBR3 ratio < 0.67 or > 1.5). Among 723 proteins, the majority did not show changes in protein synthesis (80.9 % of proteins), degradation (77.2 % of proteins) or relative protein abundance (90.2 % of proteins)

(Table 4). Less than 10 % of proteins had increased or decreased protein synthesis, degradation or relative abundance. However, 20 % of proteins had increased degradation in SKBR3_shVPS4B cells (Table 4). These data suggest that VPS4B down-regulation does not change the dynamic protein profiles of most proteins in SKBR3 cells. The altered dynamic protein profiles induced by down-regulation of VPS4B are likely protein-specific.

A B C

200 4 3 3 200 ‐ ‐ SKBR3) rate

×10 ×10

150 150 3 vs.

100 100 2 (SKBR3) synthesis

50 (SKBR3_shVPS4B) 50 1 Synthesis rate Synthesis rate Ratio of

0 0 (SKBR3_shVPS4B 0 0 100 200 300 400 500 600 700 0 100 200 300 400 500 600 700 0 100 200 300 400 500 600 700 Protein Protein Protein

84 F D E ‐ 3 ‐ 200 200 4 ×10

×10

constant

150 150 SKBR3) 3

rate

vs.

constant

constant 100 100 2 rate

rate

(SKBR3) 50 50 1 (SKBR3_VPS4B) degradation

0 0 0

0 100 200 300 400 500 600 700 0 100 200 300 400 500 600 700 (SKBR3_shVPS4B 0 100 200 300 400 500 600 700 Degradation Degradation

Ratio of Protein Protein Protein

Figure 19. Dynamic protein profiles of SKBR3_shVPS4B cells and parental SKBR3 cells. The distributions of the synthesis rates in parental SKBR3 cells and SKBR3_shVPS4B cells and the ratios of their synthesis rates are shown in panels A, B, and C respectively. The distribution of the degradation rate constants in parental SKBR3 cells and SKBR3_shVPS4B cells and the ratios of their degradation rate constants are shown in panels D, E and F, respectively.

G H I

7 7 4 6 6 SKBR3) 5 5 3 vs. 4 4 2 3 protein abundance 3 (SKBR3)

protein abundance 2

2 protein abundance 1 1 1

(SKBR3_shVPS4B) Relative

0 0 0 Relative 85 (SKBR3_shVPS4B 0 100 200 300 400 500 600 700 Relative 0 100 200 300 400 500 600 700 0 100 200 300 400 500 600 700

Protein Protein Ratio of Protein

Figure 19. Dynamic protein profiles of SKBR3_shVPS4B cells and parental SKBR3 cells (continued). The distributions of the relative protein abundance at 24 h in parental SKBR3 cells and SKBR3_shVPS4B cells and the ratios of their relative protein abundance are shown in panels G, H and I, respectively. Proteins are listed in ascending order according to their relative protein abundance ratios at 24 h.

Table 4. Changes of protein synthesis, degradation and relative abundance induced by VPS4B down-regulation in SKBR3 cells.

# of proteins # of proteins # of proteins with with unchanged with increased decreased ratio* (0.66 ≤ ratio* (> 1.5) ratio* (< 0.67) Ratio ≤ 1.5)

Synthesis rate 69 (9.5%) 69 (9.5%) 585 (80.9%) Degradation rate constant 141 (19.5%) 24 (3.3%) 558 (77.2%) Relative protein abundance at 24 h 33 (4.65) 38 (5.3%) 652 (90.2%) Numbers were calculated from a total of 723 proteins. Ratio = SKBR3_shVPS4B vs. SKBR3

Correlation between altered protein expression in SKBR3_shVPS4B cells and protein synthesis and degradation

We then determined the relationship between protein expression and protein synthesis and degradation. The relative protein abundance ratios between the two cell lines (SKBR3_shVPS4B vs. SKBR3) were plotted against the ratios of protein synthesis rates (Figure 20A) and the ratios of protein degradation rate constants (Figure 20B). The data in Figure 20 clearly show that overall protein expression is positively correlated with protein synthesis, while being negatively correlated with protein degradation. Proteins with higher relative abundance in SKBR3_shVPS4B cells (Figure 21, dark and light red diamonds) were found to have higher synthesis rates and lower degradation rate constants

(Figure 21, 2nd quadrant). Conversely, lower relative protein abundance (Figure 21, dark and light green diamonds) was correlated with lower synthesis rates and higher degradation rate constants (Figure 21, 4th quadrant). These data indicate that protein expression changes in SKBR3_shVPS4B cells are correlated with protein synthesis and degradation as measured by our iSDMS approach.

A 2

t R=0.79 of

relavant

1 abundance protein 24h) Log2(Ratio

0 ‐2 ‐10 1 2

‐1 Log2(Ratio of protein synthesis rate) Ratio = SKBR3_shVPS4B vs. SKBR3 ‐2

B 2

R=0.71

24h)

relevant 1 protein abundane at Log2(Ratio

0 ‐2 ‐1012

‐1

Ratio = SKBR3_shVPS4B Log2(Ratio of protein vs. SKBR3 degradation rate ‐2 constant)

Figure 20. Protein expression is positively correlated with protein synthesis and is negatively correlated with protein degradation. Log2 ratios of protein expression at 24 h between SKBR3_shVPS4B cells and SKBR3 cells are plotted against log2 ratios of protein synthesis rates (A) and protein degradation rate constants (B).

1 2 Log (Ratio of protein synthesis rate) 0 -2 -1 0 1 2

-1

Log (Ratio of protein -2 2 degradation rate constant )

Ratio = SKBR3_shVPS4B <-0.66 -0.66 -0.33 0.33 0.66 >0.66 vs. SKBR3 Log (Ratio of relative protein abundance at 24 h) 2

Figure 21. Altered protein expression in SKBR3_shVPS4B cells correlates with altered protein synthesis and degradation rates. Log2 ratios of synthesis rates of SKBR3_shVPS4B cells vs. SKBR3 cells (y axis) are plotted against log2 ratios of degradation rate constants of SKBR3_shVPS4B cells vs. SKBR3 cells (x axis). Diamonds represent log2 ratios of protein expression at 24 h between the two cell lines, the relative values of which are indicated on the color scale.

Next, we wanted to identify the synthesis and degradation patterns of proteins with altered relative abundance. Proteins with increased (Table 5) or decreased (Table 6) relative abundance at 24 h were selected. We found that among thirty-three proteins with increased relative abundance, 63.6 % had increased synthesis only, 9.1 % had decreased degradation only, and 9.1 % had both (Table 7). Among thirty-eight proteins with decreased relative abundance, 18.4 % had decreased synthesis only, 15.8 % had increased degradation only, and 65.8 % had both (Table 8). These results indicate that different regulatory mechanisms are used by SKBR3_shVPS4B cells to up-regulate or down- regulate specific proteins. Although both synthesis and degradation can regulate protein expression, in SKBR3_shVPS4B cells, increased protein expression was frequently associated with increased synthesis, while decreased protein expression was related to decreased synthesis and increased degradation.

Table 5. Proteins with increased relative abundance in SKBR3_shVPS4B cells at 24 h. Ratio* of Ratio* of Ratio* of Relative Protein Gene UniProt Protein Protein Name Protein Degradation Name Accession Synthesis Abundance Rate Rate at 24 h Constant GPI P06744 Glucose‐6‐phosphate isomerase 1.93 2.07 2 CNP P09543 2',3'‐cyclic‐nucleotide 3'‐phosphodiesterase 1.92 2.36 1.67 DSG2 Q14126 Desmoglein‐2 1.91 2.84 1.42 PSAP P07602 Proactivator polypeptide 1.85 2.13 1.31 ANXA2 P07355 Annexin A2 1.82 1.96 0.89 ACBD3 Q9H3P7 Golgi resident protein GCP60 1.77 2 0.47 Hydroxyacyl‐coenzyme A dehydrogenase, HADH Q16836 1.74 1.43 0.51 mitochondrial CD59 P13987 CD59 glycoprotein 1.67 1.59 0.89 Isocitrate dehydrogenase [NAD] subunit beta, IDH3B O43837 1.67 1.03 0.58 mitochondrial KRT79 Q5XKE5 Keratin, type II cytoskeletal 79 1.67 2.15 1 TPM1 B7Z722 Tropomyosin 1 (Alpha), isoform CRA_i 1.65 1.86 1.34 FLNA P21333 Filamin‐A 1.61 1.83 1.04 CLTB P09497 Clathrin light chain B 1.59 1.89 1.09 Dolichyl‐diphosphooligosaccharide‐‐protein DAD1 P61803 1.59 1.68 0.7 glycosyltransferase subunit DAD1 KRT7 P08729 Keratin, type II cytoskeletal 7 1.59 1.91 1.07 P4HA1 P13674 Prolyl 4‐hydroxylase subunit alpha‐1 1.59 1.6 0.89 Isocitrate dehydrogenase [NAD] subunit IDH3A P50213 1.58 1.89 0.62 alpha, mitochondrial LGALS3BP Q08380 Galectin‐3‐binding protein 1.58 1.24 1.32 RTN4 Q9NQC3 Reticulon‐4 1.58 1.9 0.91 EPB41L1 Q9H4G0 Band 4.1‐like protein 1 1.57 1.54 1.12 CALD1 Q05682 Caldesmon 1.56 1.76 1.09 LMNA P02545 Prelamin‐A/C 1.56 1.46 0.93 Trifunctional enzyme subunit beta, HADHB P55084 1.55 1.4 0.95 mitochondrial GLB1 P16278 Beta‐galactosidase 1.53 1.55 0.76 GSTK1 Q9Y2Q3 Glutathione S‐transferase kappa 1 1.53 1.67 0.59 KRT14 P02533 Keratin, type I cytoskeletal 14 1.53 1.74 0.79 KH domain‐containing, RNA‐binding, signal KHDRBS1 Q07666 1.52 1.43 0.63 transduction‐associated protein 1 RCC1 P18754 Regulator of chromosome condensation 1.52 1.3 0.89 Calcium‐binding mitochondrial carrier protein SLC25A24 Q6NUK1 1.52 2.13 1.16 SCaMC‐1 Trifunctional enzyme subunit alpha, HADHA P40939 1.51 1.17 0.92 mitochondrial CKMT1A P12532 Creatine kinase U‐type, mitochondrial 1.51 1.25 0.91 Major facilitator superfamily domain‐ MFSD10 Q14728 1.51 1.52 0.72 containing protein 10 RALA P11233 Ras‐related protein Ral‐A 1.51 1.7 0.81

*: Ratio = SKBR3_shVPS4B vs. SKBR3

Table 6. Proteins with decreased relative abundance in SKBR3_shVPS4B cells at 24 h. Ratio* of Ratio* of Ratio* of Relative Protein Gene UniProt Protein Protein Name Protein Degradation Name Accession Synthesis Abundance Rate Rate at 24 h Constant S100A9 P06702 Protein S100‐A9 0.41 0.45 2.11 S100A8 P05109 Protein S100‐A8 0.42 0.43 1.55 HSPB1 P04792 Heat shock protein beta‐1 0.51 0.45 1.94 Na(+)/H(+) exchange regulatory cofactor NHE‐ SLC9A3R1 O14745 0.52 0.4 1.38 RF1 ASS1 P00966 Argininosuccinate synthase 0.53 0.31 1.78 FASN P49327 Fatty acid synthase 0.53 0.45 1.7 GGCT O75223 Gamma‐glutamylcyclotransferase 0.55 0.61 2.8 LDHB P07195 L‐lactate dehydrogenase B chain 0.55 0.47 1.53 ARPP19 P56211 cAMP‐regulated phosphoprotein 19 0.56 0.56 1.83 MIF P14174 Macrophage migration inhibitory factor 0.56 0.55 2.92 TXN P10599 Thioredoxin 0.58 0.59 2.44 LDHAL6A Q6ZMR3 L‐lactate dehydrogenase A‐like 6A 0.59 0.65 3 CPNE2 Q96FN4 Copine‐2 0.6 0.67 2.21 EIF5A P63241 Eukaryotic translation initiation factor 5A‐1 0.6 0.61 2.11 PHGDH O43175 D‐3‐phosphoglycerate dehydrogenase 0.6 0.46 1.4 SELENBP1 Q13228 Selenium‐binding protein 1 0.61 0.44 2.21 Chromosome 17 open reading frame 25, C17orf25 D3DTG9 0.62 0.52 1.59 isoform CRA_d EEF2 P13639 Elongation factor 2 0.62 0.66 1.73 UBE2O Q9C0C9 Ubiquitin‐conjugating enzyme E2 O 0.62 0.81 1.88 ARHGDIA P52565 Rho GDP‐dissociation inhibitor 1 0.63 0.52 1.38 Trifunctional purine biosynthetic protein GART P22102 0.63 0.47 2.13 adenosine‐3 S100A13 Q99584 Protein S100‐A13 0.63 0.6 1.03 HSP90AB1 P08238 Heat shock protein HSP 90‐beta 0.64 0.64 1.72 Peptidylprolyl cis‐trans isomerase A‐like PPIAL4A Q9Y536 0.64 0.62 1.67 4A/B/C S100A6 P06703 Protein S100‐A6 0.64 0.65 2.33 UBE2V1 Q13404 Ubiquitin‐conjugating enzyme E2 variant 1 0.64 0.65 1.96 EEF1A1 P68104 Elongation factor 1‐alpha 1 0.65 0.68 1.89 IPO7 O95373 Importin‐7 0.65 0.67 1.75 PDCD6 O75340 Programmed cell death protein 6 0.65 0.64 1.44 SULT1A1 P50225 Sulfotransferase 1A1 0.65 0.49 1.95 AGR2 O95994 Anterior gradient protein 2 homolog 0.66 0.62 0.95 CLIC1 O00299 Chloride intracellular channel protein 1 0.66 0.67 2.39 DNAJA1 P31689 DnaJ homolog subfamily A member 1 0.66 0.61 1.4 DDT P30046 D‐dopachrome decarboxylase 0.66 0.6 1.94 GAPDH P04406 Glyceraldehyde‐3‐phosphate dehydrogenase 0.66 0.68 2.14 PPA1 Q15181 Inorganic pyrophosphatase 0.66 0.66 1.7 TUBA1B P68363 Tubulin alpha‐1B chain 0.66 0.63 2.67 UBA1 P22314 Ubiquitin‐like modifier‐activating enzyme 1 0.66 0.64 1.55 *: Ratio = SKBR3_shVPS4B vs. SKBR3

Table 7. Increased protein expression in SKBR3_shVPS4B cells is mostly the result of increased protein synthesis. Number of Percentage

proteins* (%) Increased synthesis only 21 63.6

Decreased degradation only 3 9.1 Both 3 9.1 Other 6 18.2 *: Number of proteins was calculated from 33 proteins with increased relative abundance (SKBR3_shVPS4B vs. SKBR3 ratio > 1.5) at 24 h.

Table 8. Decreased protein synthesis and increased protein degradation contribute to decreased protein expression in SKBR3_shVPS4B cells.

Number of Percentage proteins* (%) Decreased synthesis only 7 18.4 Increased degradation only 6 15.8 Both 25 65.8 *: Number of proteins was calculated from 38 proteins with decreased relative abundance (SKBR3_shVPS4B vs. SKBR3 ratio < 0.67) at 24 h.

VPS4B down-regulation alters energy metabolism in SKBR3 cells

Proteins involved in energy metabolism are highly abundant in cells. They also

comprise one of the largest protein function categories in this study. Glycolysis and fatty acid β-oxidation are the main sources of acetyl-CoA for the citric acid cycle

(tricarboxylic acid cycle, TCA) to generate adenosine triphosphate (ATP). Most of the major proteins involved in these processes were identified and quantified in our study

(Figure 22, ellipses shaded with colors). More than a 1.5-fold decrease in the relative expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and L-lactate dehydrogenase B chain (LDHB) was observed in SKBR3_shVPS4B cells. Greater than a

1.25-fold decrease in the relative expression of fructose-bisphosphate aldolase A and C

(ALDOA and ALDOC), phosphoglycerate kinase 1 (PGK1), alpha-enolase (ENO1), pyruvate kinase isozymes M2 (PKM2) and L-lactate dehydrogenase A chain (LDHA) was observed in SKBR3_shVPS4B cells. Consistently, most of these proteins had increased degradation and decreased synthesis rates.

In contrast, the relative expression of mitochondrial trifunctional protein alpha- subunit (HADHA), mitochondrial trifunctional protein beta-subunit (HADHB) and mitochondrial hydroxyacyl-coenzyme A dehydrogenase (HADH) was increased more than 1.5-fold in SKBR3_shVPS4B cells. Mitochondrial very long-chain specific acyl-

CoA dehydrogenase (ACADV) and mitochondrial enoyl-CoA hydratase (ECHS1) was increased more than 1.25-fold. Most of these proteins had increased synthesis rates, and their degradation rates were not changed, with the exception of the decreased degradation and increased synthesis rates of HADH. Fatty acid synthase (FASN) expression was

decreased in SKBR3_shVPS4B cells with increased degradation and decreased synthesis rates. Taken together, these results suggest that in SKBR3_shVPS4B cells, fatty acid tends to be oxidized to generate energy rather than being stored, consequently replacing glucose as a main energy source.

The expression of mitochondrial isocitrate dehydrogenase [NAD] subunit alpha

(IDH3A) and beta (IDH3B) was increased more than 1.5-fold. Mitochondrial succinyl-

CoA ligase [ADP-forming] subunit beta (SUCLA2) and mitochondrial malate dehydrogenase (MDH2) expression was increased 1.25-fold. The expression of other proteins related to the TCA cycle, such as mitochondrial citrate synthase (CS), mitochondrial aconitate hydratase (ACO2) and mitochondrial fumarate hydratase (FH), was not changed. These data indicate that increased expression of TCA cycle enzymes could increase ATP production in SKBR3_shVPS4B cells.

Glycolysis Glucose HK1 Fatty acid oxidation HK2 Fatty acid GPI CPT1 PFK CPT2 ALDOA Fatty acid Fatty acid synthesis ALDOC ACSL Fatty acid GAPDH ACADM PGK1 FASN ACADV BPGM ECHS1 Malonyl CoA ENO1 ACC2 PKM2 HADHA Pyruvate HADH ACC1 LDHA PDHX LDHB HADHB Acetyl CoA Lactate Acetyl CoA

FH MDH2 CS ACLY

DHSA Citrate Citrate citrate carrier TCA cycle ACO2 SUCLA2 IDH3A Ratio = SKBR3_shVPS4B vs. SKBR3 KGDH ODO2 IDH3B Ratio 0 0.67 0.80 1.25 1.50 >1.5

Ratio of sythesis rate Catalysis Ratio of degradation rate constant Transportation Protein not identifed Colored ellipse: Protein with the ratio of relative abundance at 24 h

Figure 22. VPS4B down-regulation up-regulates the expression of fatty acid β- oxidation related proteins and down regulates the expression of glycolysis related proteins in SKBR3 cells. The energy metabolism pathway was adapted from the Kyoto Encyclopedia of Genes and Genomes (KEGG) [217]. Colors represent the ratios of relative protein abundance at 24 h (shaded ellipses), protein synthesis rate (shaded upper rectangles), and protein degradation rate constant (shaded lower rectangles) in SKBR3_shVPS4B cells vs. SKBR3 cells.

DISCUSSION

Dysfunction of VPS4B results in altered endosomal trafficking of membrane receptors, such as EGFR, as well as EGFR-associated signaling molecules [66-68][80][96]

[97]. Data from our unpublished phosphoproteomic studies indicate that down-regulation of VPS4B alters EGFR signaling in breast cancer cells. Toward gaining a more thorough understanding of the consequences of altered EGFR signaling caused by VPS4B dysfunction, we developed an iSDMS method to identify changes in the protein synthesis and degradation rates, as well as dynamic protein expression, in SKBR3_shVPS4B cells.

We obtained dynamic profiles for more than 700 proteins in both SKBR3_shVPS4B cells and parental SKBR3 cells. We found that down-regulation of VPS4B results in altered synthesis and degradation rates of proteins involved in the energy metabolism process, which up-regulate fatty acid β-oxidation and down-regulate glycolysis and fatty acid synthesis. The adoption of fatty acid β-oxidation as an alternative energy source could be a distinct feature of breast cancer cells with VPS4B dysfunction, and could be a new drug target for cancer treatment.

Our iSDMS approach has several advantages as compared to other mass spectrometry-based protein synthesis and degradation studies. First, our iSDMS approach can be applied to both steady state and non-steady state conditions. Most methods of analyzing protein turnover use the ratio of labeled peptide to unlabeled peptide [203], or relative isotope abundance (RIA) of labeled and unlabeled peptide

[202][209][210][218][219], to estimate degradation and synthesis rates, respectively.

The estimation is accurate only when steady-state is assumed, in which synthesis and

degradation are balanced and no net protein is produced. However, steady-state conditions cannot be confirmed when cells are under growth, differentiation, or experimental manipulation, which is the case in most cell biology studies. Recently, the introduction of iTRAQ has allowed the incorporation of protein relative abundance changes into RIA calculations, thereby avoiding the need for steady-state assumption

[206]. Instead of using RIA to calculate protein degradation and synthesis, our iSDMS approach normalizes the MS signal from labeled or unlabeled peptide to the MS signal from the internal standard. Changes in the relative abundances of the labeled and unlabeled peptides are directly related to the degradation and synthesis of the peptides.

Thus, steady-state assumption is not needed.

A second advantage of our iSDMS approach is that complete basal stable isotope labeling is not required. In RIA-based methods, cells are required to be completely labeled before they are transferred to standard medium for the determination of the initial

RIA. In iSDMS, the pre-existing unlabeled peptide at the initial time point does not affect the calculation of the degradation of the pre-existing labeled peptide. The interference of the pre-existing unlabeled peptide in the calculation of the peptide’s synthesis rate can be eliminated by subtracting the relative abundance of the pre-existing unlabeled peptide from that of the total unlabeled peptide.

A third advantage of our iSDMS approach is that it can be used to construct complete dynamic proteome profiles: protein synthesis, protein degradation, and protein expression, which enables the discrimination between the different mechanisms that

control protein expression. Gene amplification is often related to elevated oncoprotein

expression; however increasing evidence has shown that impaired degradation signaling

also contributes to increased oncoprotein synthesis [196-200]. Understanding the

specific contributions of the different mechanisms that control protein expression is

important for the development of more effective drug targets for cancer treatments.

Protein degradation rate constants vary among different cell types and tissues. In the same biological system, the degradation rate constants also vary widely among different proteins. In Escherichia coli, protein degradation rate constants range from

0.017 to 0.058 h-1, with a mean value of 0.03 h-1 [205]. In human A549 lung

adenocarcinoma cells, the degradation rate constants range from 2×10-5 to 5.4 h-1, with a mean of 0.081 h-1 [208]. In our study, we found that among more than 700 proteins,

protein degradation rate constants ranged from 0.012 to 0.116 h-1 with a mean of 0.033 h-

1 in SKBR3_shVPS4B cells, and 0.01 to 0.087 h-1 with a mean of 0.028 h-1 in parental

SKBR3 cells (Appendix 3). These degradation rate constants were normally distributed.

Since the shortest chase time period was 2 h in this study, we were not able to obtain the

degradation rate constants for proteins that were rapidly degraded (< 2 h). In future

dynamic global proteome studies, it would be beneficial to include shorter chase times.

Protein expression is controlled by both protein synthesis and degradation.

Generally, higher protein synthesis is related to higher protein expression, and higher protein degradation is related to lower protein expression. These trends are consistent with our results (Figure 20). Increased protein expression could be the result of increased

protein synthesis, decreased protein degradation, or both. Traditionally, increased protein expression has been considered as being mostly attributable to increased gene transcription and translation. A recent study indicates that the influence of protein

translation on protein abundance is 40 % higher than the influence of protein degradation

[205]. However, an accumulating body of evidence has also shown that the abnormally

elevated expression of many important receptors in cancer or drug resistant cancer cells is

caused by decreased or delayed receptor degradation [196-200]. Our results show that only ~ 10 % of proteins with increased expression have decreased degradation, while the increased expression of the majority of these proteins is attributable to changes in protein

synthesis only. These results indicate that in SKBR3 cells, protein synthesis is the main

method of increasing protein expression, whereas altered protein degradation is a more

protein-specific approach to increasing protein expression. On the other hand, decreased

protein expression could be the result of decreased protein synthesis, increased protein

degradation, or both. Our results show that most proteins with decreased expression had

both decreased synthesis and increased degradation, which suggests that cells have

different strategies to increase or decrease protein expression for different proteins.

VPS4B plays an important role in protein degradation, especially in the lysosomal

degradation of membrane receptors [92]. It has been found that loss of function of

VPS4B results in delayed EGFR degradation and prolonged EGFR retention on the

limiting membrane of MVBs [66-68]. We were not able to obtain the dynamic

expression profile for EGFR in this study due to the low abundance of endogenous EGFR

in SKBR3 cells. Overall protein degradation rates were similar in SKBR3_shVPS4B

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cells and parental SKBR3 cells. It is possible that the VPS4B down-regulation-induced delayed lysosomal degradation of endocytosed cargo is protein-specific, which explains why overall protein degradation is not affected. In future studies, it would be interesting to identify the proteins whose degradation rates are selectively affected by VPS4B down- regulation. This information will provide more fundamental knowledge of the VPS4B- related protein degradation pathway.

Glycolysis and fatty acid oxidation are two major metabolic pathways that cells utilize to generate energy. Adaptation to different carbon and energy sources is an important cellular response to environmental or intracellular changes. It has been shown

that growth factors promote fatty acid synthesis by inducing the transcription of the fasn

gene in breast cancer cells [220][221]. The transcription of fasn is controlled by sterol

regulatory element binding protein (SREBP), which is negatively regulated by activated

5' adenosine monophosphate (AMP)-activated protein kinase, AMPK [221][222].

Recently, it has been suggested that growth factor signaling and AMPK activity

negatively regulate each other. For example, activated B-Raf, an event mediated by

extracellular-signal-regulated kinases (ERK1/2) and growth factors, negatively regulates

the AMPK activator, tumor suppressor Serine/threonine kinase 11 (LKB1), to promote

cell proliferation [223][224]. Inhibition of EGF receptor 2 (HER2) by kinase inhibitor

activates AMPK and induces fatty acid oxidation in cardiomyocytes [225]. On the other

hand, activation of AMPK via its agonist 5-aminoimidazole-4-carboxamide-1-b-4-

ribofuranoside (AICAR) preferentially inhibits the growth and lipogenesis of EGFR-

activated glioblastoma cells [226][227]. VPS4B plays roles not only in protein lysosomal

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degradation, but also in the endosomal trafficking of signaling molecules. The impaired

endosomal trafficking induced by VPS4B down-regulation leads to attenuation of growth

signal transduction [97], which may abolish the negative regulation of AMPK and thus

decrease FASN expression and increase fatty acid β-oxidation. Growth factors also

stimulate glycolysis by increasing the expression of related protiens, such as LDHA/B

[228], fructose-2,6-bisphosphatase (PFK) [229], and GADPH [230], via

RAS/PI3K/PTEN/AKT/mTOR pathway [231]. However, we found proteins related to glycolysis had decreased abundance in SKBR3_shVPS4B cells resulting from altered synthesis and degradation. The decreased glycolysis in SKBR3_shVPS4B cells may also result from impaired endosomal trafficking, which attenuates the stimulating signaling from activated EGFR for glycolysis.

The rapid growth of cancer cells requires high energy consumption. It has traditionally been thought that cancer cells rely on the inefficient functioning of the glycolytic pathway to generate ATP [232]. However, recent studies suggest that many types of cancer cells can adapt to fatty acid β-oxidation as an alternative method of energy utilization [233-238]. Increased fatty acid β-oxidation is related to cancer

progression [234] and chemoresistance [239]. Inhibition of fatty acid β-oxidation can

sensitize cancer cells to apoptosis [240-242]. The decreased glycolysis and increased

fatty acid β-oxidation observed in our studies suggest that VPS4B down-regulation shifts

the primary energy source from glucose to fatty acids in SKBR3 cells. Our unpublished

data indicate that VPS4B is down regulated in SKBR3 cells under hypoxic conditions.

The induction of fatty acid β-oxidation in VPS4B down regulated SKBR3 cells may

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contribute to cell survival under such harsh conditions. Further studies are needed in

order to address the exact relationship among hypoxia, VPS4B, energy metabolism and

cell survival. These results will potentially lead to the discovery of new mechanisms of

breast cancer survival and new cancer treatment targets.

Here we have presented a new approach, iSDMS, for the direct measurement of

protein degradation and synthesis, as well as relative protein expression levels in breast

cancer cells with down regulated VPS4B expression. This approach can be feasibly applied to other cell culture systems to determine global protein dynamics under different genetic manipulations or environmental stimulations. As with other mass spectrometry- based approaches, iSDMS is limited by the depth and reproducibility of protein identification, especially when applied to multiple time points and multiple conditions. In the near future, with the advancement of mass spectrometry technology, we believe our iSDMS approach will be a powerful tool to reveal cellular dynamics in cell biology, providing important information for the development of new strategies for disease treatment.

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IV. IsoQuant: A Software Tool for SILAC-Based Mass Spectrometry Quantitation

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ABSTRACT

Accurate protein identification and quantitation are critical when interpreting the

biological relevance of large-scale shotgun proteomics datasets. Although significant technical advances in peptide and protein identification have been made, accurate quantitation of high throughput datasets remains a key challenge in mass spectrometry data analysis and is a labor intensive process for many proteomics laboratories. Here, we

report a new SILAC-based proteomics quantitation software tool, named IsoQuant, which

is used to process high mass accuracy mass spectrometry data. IsoQuant offers a

convenient quantitation framework to calculate peptide/protein relative abundance ratios.

At the same time, it also includes a visualization platform which permits users to validate

the quality of SILAC peptide and protein ratios. The program is written in the C#

programming language under the Microsoft .NET framework version 4.0 and has been

tested to be compatible with both 32-bit and 64-bit Windows 7. It is freely available to

non-commercial users at http://www.proteomeumb.org/MZw.html.

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INTRODUCTION

Mass spectrometry-based proteomics approaches can provide valuable qualitative

and quantitative information regarding not only protein identification, but also protein-

protein interaction, and the dynamics of protein expression levels [243]. Accurate protein quantitation is required in order to make meaningful biological inferences from mass spectrometry data. In proteomics experiments, qualitative data is obtained by identifying candidate proteins with an acceptable false positive identification rate. Several commercially available database search programs, such as SEQUEST [244] and Mascot

[245], and open-source database search programs including X!Tandem [246], Protein

Prospector [247], ProbID [248], OMSSA [249], ProSight [250] and InsPecT [251] are available for this purpose. Although many software programs have been developed for quantitative proteomic analysis, certain challenges pertaining to reliable and accurate quantitation still remain [252].

Several methodologies are currently available for mass spectrometry-based protein quantitation which include either stable isotope labeling (chemical, enzymatic, or

metabolic) or label-free strategies. Stable isotope labeling by amino acids in cell culture

(SILAC) [253][254] is a metabolic labeling method whereby stable isotope-labeled

amino acids (typically Lys and Arg) are incorporated into proteins as they are synthesized,

thereby resulting in robust peptide and protein quantitation ratios. SILAC has significant advantages as compared to chemical labeling strategies, such as isobaric tags for relative and absolute quantitation (iTRAQ) and tandem mass tags (TMT), since the labeling event occurs at the beginning of the sample preparation procedure. Consequently, less

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confounding factors influence the calculation of peptide and protein ratios. Additionally,

SILAC yields more robust quantitation ratios than label-free strategies since the lack of reproducibility in certain label-free strategies can negatively impact the generation of consistent ratio reports. In general, SILAC-based proteomic analyses are limited to only those cells or animals that can metabolically incorporate stable isotope labeled amino acids. Although it has not been widely adopted by most clinical proteomic analyses, recently the SILAC method has been modified and extended to the analysis of clinical biopsy samples that have not traditionally been amenable to this type of approach [255]

[256].

Several methods have been developed for the mass spectrometry-based

determination of SILAC ratios. One method is based on peak area integration of

Extracted Ion Chromatograms (XIC) using software programs such as Xpress [257],

ASAPRatio [258] and MSQuant [259]. In general, XIC peak area is calculated by integrating peak intensity over retention time for a specified m/z value. However, it is often difficult to objectively determine the boundaries of a peak corresponding to a specific peptide, especially when the XIC peak or elution profile of a peptide is not well defined. Another method for determining SILAC-labeled peptide ratios is based on ratios generated from MS1 scans surrounding the MS/MS spectrum of an identified peptide.

The programs Census [260] and RelEx [261] use a linear regression approach to calculate quantitation ratios. MaxQuant [262][263] determines SILAC pairs based on peptide

features and subsequently uses the database search results from Andromeda [264], a novel peptide search engine using a probabilistic scoring model, to calculate ratios. A

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newly developed software program, UNiquant [265], provides a quantitation method

based on overall peak intensity ratio and has been shown to significantly increase the

number of quantified peptides in the analysis of quantitative proteomics data using stable isotope labeling.

A critical issue commonly associated with the processing of quantitative SILAC- based proteomic data is the validation of the accuracy of the large number of SILAC peptide ratios generated from a shotgun proteomics experiment. Since every software program uses a different algorithm and computational method to calculate SILAC peptide ratios, we and others have observed a rather large discrepancy among the SILAC peptide ratios reported by different programs for the same peptides, especially for low abundant peptides. Therefore, the manual validation of SILAC peptide ratios at both the protein and peptide levels is often required for each analysis. Currently, the Qurate [266]

graphical tool can be used to manually validate isotopically labeled peptide quantitation

events. The Rover [267] tool can be used to validate the quantitation reports from several

different packages such as MaxQuant and Census.

In this paper we describe the framework and quantitation accuracy of IsoQuant, a

software tool we developed for SILAC-based mass spectrometry quantitation. IsoQuant

uses unique peak selection and absolute peak intensity integration algorithms combined

with high mass accuracy and dynamic chromatographic retention time filters in order to

maximize the accuracy of peptide and protein quantitation. To improve the efficiency of

manual validation, we have included a graphical user interface in IsoQuant, which allows

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end users to visualize original MS1 spectra and examine how each individual SILAC

peptide ratio was calculated in order to improve the confidence of protein quantitation.

IsoQuant is freely available to non-commercial users at

http://www.proteomeumb.org/MZw.html.

MATERIALS AND METHODS

SILAC labeling and hippocampal slice culture

Hippocampal slice cultures were prepared and maintained as described previously

[268]. Briefly, hippocampi were dissected from 5- to 7-day-old rat pups. Slices were cut

with a tissue chopper at 400 μm and attached to glass coverslips in a chicken plasma clot.

The coverslips and slices were placed in individual sealed test tubes containing semi- synthetic medium and maintained on a roller drum in an incubator for 14 days. On day

15, the cultures were transferred to DMEM-based SILAC medium (Pierce/Thermo Fisher

Scientific) supplemented with D4 L-Lysine-HCl (100 mg/L; purity 96-98%; Cambridge

13 Isotope Laboratories), C6 L-Arginine-HCl (100 mg/L; purity 97-99%; Cambridge

Isotope Laboratories), 10% dialyzed FBS (Invitrogen) and 1% penicillin/streptomycin.

Slices were harvested on the 14th day of being cultured in SILAC DMEM.

Preparation of cell lysates and protein digestion

SILAC-labeled cultured hippocampal slices were homogenized in a buffer

containing 8 M urea, 75 mM NaCl, 50 mM Tris-HCl, pH 8.2, protease inhibitor cocktail

(Roche), 1 mM NaF, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, and 1 mM PMSF using a microtube pellet pestle rod and motor

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(Kontes). Insoluble material was pelleted by centrifugation at 10,000 × g for 10 min at 4

°C. The concentration of the soluble proteins was determined by the BCA protein assay

(Thermo Fisher Scientific/Pierce). Reducing Laemmli sample buffer was added to three aliquots of 25 µg protein prior to 5 min incubation at 95 °C and protein separation via

SDS-PAGE. Gel bands were visualized by Coomassie blue staining. Each gel lane was divided into 15 regions and the corresponding gel regions from each lane were combined and subjected to in-gel tryptic digestion as described previously [269].

The complex cell lysate sample used to evaluate the reproducibility of peptide quantitation by IsoQuant (Figure 4) was derived from SKBR3 cells cultured in SILAC

DMEM supplemented with 10% dialyzed FBS (Invitrogen) and 1% penicillin/streptomycin. Five 15-cm dishes of SKBR3 cells were cultured in

12 supplemented SILAC DMEM to which C6 L-Lysine-HCl (100 mg/L; Thermo

12 Scientific/Pierce) and C6 L-Arginine-HCl (100 mg/L; Thermo Scientific/Pierce) were added; this medium is referred to as SILAC “light”: Lys0, Arg0. Five 15-cm dishes of

13 SKBR3 cells were cultured in supplemented SILAC DMEM to which C6 L-Lysine-HCl

13 (100 mg/L; purity 97-99%; Cambridge Isotope Laboratories) and C6 L-Arginine-HCl

(100 mg/L; purity 97-99%; Cambridge Isotope Laboratories) were added; this medium is referred to as SILAC “heavy”: Lys6, Arg6. Cells were lysed by sonication in the same buffer as described for the hippocampal slices. The concentration of the soluble proteins was determined by the BCA protein assay and 2 mg protein from the SILAC “light” and

“heavy” conditions were combined. Disulfide bonds were reduced by incubation with 5 mM DTT for 25 min at 56 °C followed by alkylation with 15 mM iodoacetamide for 30

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min at room temperature in the dark. Un-reacted iodoacetamide was quenched by addition of DTT to 5 mM final concentration for 30 min at room temperature in the dark.

The protein mixture was diluted at a ratio of 1 to 5 in 25 mM Tris-HCl, pH 8.2 to reduce the concentration of urea to 1.6 M. Proteins were digested with trypsin (2% wt/wt) overnight at 37 °C. The digestion was stopped by acidification with TFA to 1% (vol/vol) followed by desalting with C18 peptide SepPak cartridges (Waters). The de-salted peptides were lyophilized and stored at -80 °C until further analysis.

LC-MS/MS analysis

Peptides were separated by nano-scale reverse-phase liquid chromatography using a nano-flow Xtreme Simple liquid chromatography system (CVC/Micro-Tech). The analytical column was prepared by packing 1.7µm 200Å C18 resin (Prospereon Life

Sciences) into a laser-pulled fused silica capillary (75 µm inner diameter, 10.5 cm length,

10µm tip; Sutter Instruments) using a pressure injection cell (Next Advance). Peptides were injected into the sample loop using an Endurance autosampler (Spark Holland,

Brick, NJ) and were loaded onto the column with 95% solvent A (0.5% acetic acid in water). A 120 min LC gradient method from 5 – 35% B (80% ACN, 0.5% acetic acid) with a post-split flow rate of 0.3 µl/min was used to elute the peptides into the mass spectrometer. The LTQ-Orbitrap mass spectrometer (Thermo Electron) was equipped with a nanospray ionization source. The spray voltage was 1.5 kV and the heated capillary temperature was 180 °C. MS1 data were acquired in the profile mode in the

Orbitrap with a resolution of 60,000 at 400 m/z and the top ten most intense ions in each

MS1 scan were selected for collision-induced dissociation in the linear ion trap.

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Dynamic exclusion was enabled with repeat count 1, repeat duration 30 sec, and exclusion duration 180 sec. Other mass spectrometry data generation parameters were as follows: collision energy 35%, ion selection threshold for MS/MS 500 counts, isolation width 3 m/z, default charge state 3, and charge state screening enabled.

RESULTS AND DISCUSSION

Overview of IsoQuant SILAC-based quantitation

In this report, we have developed a new SILAC-based proteomics quantitation method, named IsoQuant. The program was written in C# programming language under

Microsoft .Net framework version 4.0. The source code can be found in Appendix 4.

Figure 23 illustrates the framework of IsoQuant for calculating SILAC peptide quantitation ratios. Briefly, IsoQuant consists of three major components: .raw file extraction, Peptide/Protein Quantitation, and Data Validation and Visualization. Figure

24A is a screen shot of the three major IsoQuant user modules. To automate and streamline the data processing, IsoQuant takes original .raw files generated from an

Orbitrap mass spectrometer as input and extracts MS1 spectra using the Xcalibur

Developer’s Kit (XDK, Thermo Electron). This .raw file conversion program selects all of the MS1 scans in the .raw binary file and exports them to a single ASCII format text file. The built-in IsoQuant .raw file conversion program is convenient for users since it does not require any third party software tools for mass spectrometry data conversion.

Next, a summary report of peptide quantitation ratios is generated. Since IsoQuant performs its peptide quantitation after the database search, the filtering and selection of peptides based on their false discovery rates can be readily defined by the user. The third

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step is protein identification whereby IsoQuant checks the database search results and groups proteins with homologous peptides into protein clusters. Lastly, IsoQuant calculates protein quantitation ratios and compiles protein quantitation reports based on the peptide quantitation ratios in each protein cluster. The graphical user interface of

IsoQuant’s peptide and protein quantitation module is presented in Figure 24B.

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Figure 23. Framework of IsoQuant

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Figure 24. User interface of IsoQuant. A) IsoQuant consists of three major components: Raw file conversion, Peptide/ Protein Quantitation and Visual Validation. B) Interface of the peptide and protein quantitation module. Currently, IsoQuant supports the following quantitation features: 1. double and triple label SILAC experiments; 2. Single or multiple .raw files; and 3. SEQUEST and Mascot peptide identification results (only SEQUEST search result is shown here).

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IsoQuant minimizes under-sampling problem when a data-dependent method is

used to acquire MS2 spectra

Mass spectrometer data acquisition methods with dynamic exclusion enabled are

commonly used in most proteomic experiments. Mass spectrometers operated in a data-

dependent data acquisition mode have a bias toward selecting the most abundant peaks

for MSn fragmentation. As a result, the use of a dynamic exclusion method often reduces

the number of times a given peptide is selected for MSn fragmentation, thereby

increasing the likelihood that lesser abundant peaks will be selected for fragmentation.

In this study, we found that data-dependent data acquisition methods often result

in the lack of fragmentation of both the heavy- and light-labeled versions of each SILAC

peptide pair in cases where the intensity of one of the peptides is below the threshold

used to trigger MS/MS fragmentation. To compensate for the loss of potentially valuable

quantitation data in cases such as these, IsoQuant only requires either the heavy or light peptide to be selected for MS/MS fragmentation and identified by database search in order for a SILAC peptide ratio to be calculated. Once the peptide is correctly identified, corresponding SILAC-labeled heavy and light peptide MS1 isotopic envelopes will be extracted.

Figure 25 illustrates one of the limitations of data-dependent acquisition instrument methods and indicates that MS1 peaks selected for MS/MS fragmentation do not always have clear isotope distributions for accurate SILAC-pair quantitation. As shown in Figure 25A, an MS1 peak corresponding to 572.77 m/z at retention time (RT)

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32.70 min and RT 32.75 min (indicated by *) was selected for MS/MS fragmentation.

The MS1 scan of RT 32.70 min has a distinct isotopic peak envelope at 572.77 m/z

(heavy SILAC peptide), whereas 569.76 m/z (light SILAC peptide) has an incomplete isotopic peak envelope (Figure 25B). The use of dynamic exclusion in this instrument method precluded the selection of the 572.77 m/z (heavy SILAC peptide) and 569.76 m/z

(light SILAC peptide) peaks for MS/MS fragmentation after RT 32.75 min, as indicated in Figure 25A. On the other hand, at RT 32.84 min, which coincides with the apex of the peak in the extracted ion chromatogram of 572.77 m/z, complete isotopic peak envelopes were obtained for both the light and heavy SILAC peptides (Figure 25C). However, these two peaks at RT 32.84 min were never selected for MS/MS fragmentation since they had already been added to the dynamic exclusion list. To overcome this issue,

IsoQuant uses both high mass accuracy and retention time filters to determine candidate

SILAC peptide pairs for quantitation if either the heavy or light peptide was identified by the database search engine. Therefore, IsoQuant can overcome the limitations of dynamic exclusion-based data-dependent acquisition methods that affect the accuracy of other quantitation programs.

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Figure 25. IsoQuant overcomes the limitation of under-sampling in data-dependent instrument methods, thereby increasing the accuracy of SILAC-based quantitation. A) Extracted ion chromatogram (XIC) of 572.77 m/z (SILAC heavy-labeled peptide). 572.77 m/z was selected for MS2 fragmentation and was identified by SEQUEST in the first two scans (indicated by *; retention time (RT) 32.70 min and 32.75 min). B) MS1 scan at RT 32.70 min (568.0 – 575.0 m/z) demonstrating an incomplete isotope envelope of 569.76 m/z (SILAC light-labeled peptide). C) MS1 spectrum from the most intense peak in A) at RT 32.84 min. Although 572.77 m/z was not selected for MS2 fragmentation due to dynamic exclusion, its complete isotope envelope permitted quantitation by IsoQuant.

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Peptide ratios calculated by weighted average method significantly improve the

reproducibility of peptide quantitation

To ensure the consistency of peptide quantitation ratios calculated by IsoQuant, a

SILAC-labeled cell lysate sample was analyzed twice by LC-MS/MS for two technical replicates. Ratios were considered as outliers if they were beyond 1.5-fold of the interquartile range for each dataset. Peptide ratio outlier removal resulted in an increased correlation between the peptide ratios from the two technical replicates. We used three different methods for calculating peptide ratios based on MS1 pairs: median, average, and weighted average. For weighted average, we used the following formula to calculate ratios:

wi xi ratio  ,wi is intensity of light peptide, xi is ratio of heavy and light pair. wi

In one proof of analysis experiment (Figure 26), we compared the quantitation ratios calculated from both technical replicates using five different methods: average

(R=0.550, Figure 26A), average with outlier removal (R=0.791, Figure 26B), median

(R=0.748, Figure 26C), weighted average (R=0.863, Figure 26D), and weighted average with outlier removal (R=0.893, Figure 26E). Ratio calculation using weighted average with outlier removal resulted in the best peptide ratio correlation (R=0.893, Figure 26E) between the two technical replicates. The median calculation is not affected by outliers; therefore we did not compare the median calculation method with a median with outlier removal method.

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Figure 26. Peptide ratios calculated by weighted average method significantly improves the reproducibility of peptide quantitation ratios. In this test, 105 unique peptides were identified by SEQUEST and quantified by IsoQuant. Each quantified heavy and light SILAC peptide was identified by at least two MS2 spectra. Quantitation calculations were performed using average (A), median (C), and weighted average methods (D). Outlier removal was also used with the average (B) and weighted average methods (E). The weighted average with outlier removal method performed the best with a median standard deviation of 0.0713 and a median CV (coefficient of variance) of 8.9%. R = linear correlation coefficient of the line of best fit.

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Quantitative accuracy of IsoQuant using sPRG2009 SILAC peptide standards and complex biological sample

In order to evaluate the performance and accuracy of IsoQuant, we used the

SILAC-labeled peptide standards provided by the Association of Biomolecular Resource

Facility’s (ABRF) Proteomics Standards Research Group (sPRG) [270]. The natural and

13 15 13 15 synthetic ( C6 N2-lysine and C6 N4-arginine) peptides from five recombinant human proteins (serum albumin, histidyl-tRNA synthetase, ribosyldihydronicotinamide dehydrogenase, peroxiredoxin-1, and ubiquitin) were combined in three defined ratios and quantities. Two technical LC-MS/MS replicates were performed and IsoQuant was used to determine the peptide and protein ratios. The accuracy of the calculated quantitation ratios was assessed using the following criteria. First, the ratios for all occurrences of the same peptide identified in the same run should be consistent. Second, the ratios for all occurrences of the same peptide identified in both runs should be consistent. Lastly, the ratios for all unique peptides identified from the same protein should be consistent.

Table 9 lists the SILAC ratios calculated by IsoQuant as compared to the published consensus ratios from other laboratories who participated anonymously in the

ABRF sPRG09 study. Using IsoQuant, we successfully quantified all 15 peptides and five proteins present in the sample. The average absolute deviation for all protein ratios was 4.99%. Therefore, this study suggests IsoQuant is an accurate method that can be used to reliably determine peptide and protein ratios.

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Table 9. IsoQuant peptide quantitation results from LC-MS/MS analysis of ABRF sPRG09 quantitative proteomics standard.

ABRF % of Absolute Protein UniProt Expected Experimental CV Peptide Sequence Consensus SD Consensus Deviation ID Ratio Ratio (%) Ratio Ratio (%) ALBU_HUMAN LVNEVTEFAK 1 1 1.01 0.02 2.4 100.92 0.92 SLHTLFGDK 1.06 1.09 0.03 2.4 102.37 2.37 AEFAEVSK 1.03 1.07 0.05 5.1 103.77 3.77 NQO2_HUMAN NVAVDELSR 1 0.93 0.92 0.03 3.1 99.28 0.72 PRDX1_HUMAN DISLSDYK 0.1 0.1 0.10 0.01 6.5 103.24 3.24 ADEGISFR 0.11 0.10 0.01 10.1 89.37 10.63 122 GLFIIDDK 0.1 0.11 0.01 4.6 113.56 13.56 PILVQAFQFTDK 0.12 0.11 0.01 4.9 95.36 4.64 SYHC_HUMAN AALEELVK 5 3.73 3.92 0.11 2.9 104.99 4.99 DQGGELLSLR 3.5 3.03 0.40 13.2 86.53 13.47 GLAPEVADR 4.01 4.23 0.09 2.1 105.45 5.45 IFSIVEQR 4.24 4.10 0.48 11.8 96.63 3.37 UBIQ_HUMAN TITLEVEPSDTIENVK 10 8.28 9.09 0.99 10.9 109.74 9.74 TLSDYNIQK 7.81 8.37 0.24 2.9 107.11 7.11 ESTLHLVLR 10.7 11.96 0.82 6.8 111.77 11.77 Median ‐ ‐ ‐ ‐ ‐ ‐ 103.24 4.99

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As mentioned earlier, the accuracy of IsoQuant depends on the correct selection

of MS1 isotopic peaks using IsoQuant’s high mass accuracy and retention time filter.

The correct isotopic pattern of candidate peptides is easily determined when analyzing a

non-complex sample comprised of a small number of peptides, such as ABRF’s SILAC

peptide standards described in Table 9. However, the isotopic envelopes of peptides

derived from complex unknown samples can be difficult to discern. The most common

problem is that the isotopic peak envelope of one peptide sometimes contains isotopic peaks from another co-eluting peptide with a nearly identical m/z. Consequently, two isotopic peak distributions from these co-eluted peptides are overlapped and their m/z

values cannot be easily determined and deconvoluted. Therefore, IsoQuant considers

MS1 peaks with values within the mass accuracy tolerance range (5ppm) as true isotopic

peak pairs to minimize the issue of co-eluting peptides mentioned above. Future updated

versions of IsoQuant will include a more complicated peak selection algorithm which

compares theoretical isotopic peak distributions with observed isotopic patterns to

determine which SILAC-labeled pairs to use for quantitation.

To further demonstrate the ability of IsoQuant to quantify a complex sample, we used IsoQuant to conduct a quantitative analysis of a sample derived from SILAC-labeled rat brain hippocampal slices. A representative table of peptide quantitation results and the overall peptide ratio distribution from this sample are illustrated in Figure 27A. If the quantitation of a specific SILAC peptide pair requires further visual validation, the

IsoQuant peptide quantitation viewer/visualization module will display all corresponding

SILAC MS1 m/z pairs from the original mass spectrometer .raw file that were used to

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calculate that specific peptide ratio. For instance, the highlighted SILAC peptide

MATDPENIIK (Figure 27A, indicated by MS2 scan# 2473) from synaptophysin was quantified with a SILAC ratio of 5.8 ± 0.85. Figure 27B is a representative spectrum of a

MATDPENIIK SILAC peptide pair generated from IsoQuant’s visual validation module.

The “light” MATDPENIIK peptide has an isotope envelope at 574.28 m/z (relative intensity 5%) and the “heavy” MATDEPENIIK peptide has an isotope envelope at

576.30 m/z (relative intensity 30%); heavy/light = 5.88 ± 0.85. Therefore, this visual validation module allows users to quickly confirm the accuracy of SILAC ratios generated by IsoQuant.

The final assembly of peptide ratios into protein ratios is illustrated in Figure 27C.

The heavy/light ratio of the highlighted protein, synaptophysin, is 6.09 ± 0.50, which is in agreement with the heavy/light ratio of the synaptophysin peptides whose MS1 spectra are shown in Figure 27B. In the protein quantitation report, IsoQuant uses a similar weighted average method as described for peptide quantitation. If many peptides belong to a group of proteins, IsoQuant will group these proteins into a common protein group and use a weighted average method to obtain an average protein group ratio. However, if there is a unique peptide from a specific isoform that can be identified within a protein group, IsoQuant will calculate and record the protein ratio of that specific isoform based on the ratio of the quantified unique peptide pair. Therefore, no outlier removal is used during the process of protein ratio calculation in order to maintain the ratios of the unique peptides for each specific protein isoform.

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Figure 27. Interface of IsoQuant visualization module permits users to validate quantitation ratios. A) Screenshot of quantitative SILAC peptide summary report and overall peptide quantitation ratio distribution from a representative double (Lys4, Arg6) SILAC label experiment. B) Screenshot of a representative spectrum from which the peptide ratios were calculated. Users can move a slider at the top of the “MS1 spectrum” view in the “Validate” window to allow the visualization of neighboring MS1 scans used for peptide quantitation. C) Screenshot of protein ratio report.

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CONCLUSIONS

In summary, we have developed a new SILAC-based mass spectrometry

quantitation software tool, named IsoQuant, which provides accurate quantitation ratios

and avoids some of the common quantitation limitations related to data-dependent mass

spectrometry data acquisition methods. The results using SILAC-labeled hippocampal

slice cultures and cell lysate datasets prove that IsoQuant can be used for accurate peptide

and protein SILAC-based quantitation of complex samples. Most importantly, IsoQuant

offers an easy to use graphic interface peptide/protein quantitation report, and it also

includes a visualization platform which permits users to validate the quality of SILAC peptide and protein ratios.

126

V. Conclusion and Future Directions

127

The work outlined here provides the first systems biology evidence demonstrating that VPS4B plays an important role in modulating EGFR signaling in breast cancer.

Figure 28 is our proposed model of the mechanisms underlying altered EGFR signaling caused by VPS4B dysfunction in breast cancer cells. Down-regulation of VPS4B results in impaired endosomal trafficking. Consequently, endocytosed EGFR that is normally targeted to MVBs for subsequent lysosomal degradation accumulates on the limiting endosomal membrane, where EGFR remains active, ligand bound, and associated with signaling adaptors. Accumulated endocytosed EGFR potentiates signaling that is initiated or propagated by endosome localized proteins, such as ERK, which requires endosomal localization for its late stage activation [73-75], and EPS8, which directly interacts with EGFR [271]. Impaired endosomal trafficking affects the signal transduction associated with the trafficking of signaling molecules between cellular compartments. For example, the nuclear translocation of activated ERK is required for the activation of transcription factors ETV3 and ETV6 [158], and the trafficking of Src from late endosomes to focal adhesions is required for STAT3 activation [97].

Phosphorylation of ETV3, ETV6 and STAT3 is attenuated in VPS4B down regulated cells. Signaling related to the transcription, translation or degradation of proteins involved in glycolysis and fatty acid metabolism is also affected. The negative regulation of AMPK by EGFR growth signaling [223][224] could be abolished by impaired endosomal trafficking, resulting in AMPK activation and subsequently decreasing FASN gene transcription [221][222], and increasing fatty acid β-oxidation gene expression. The attenuated EGFR stimulating signaling for glycolysis [228-231] results in the decreased expression of glycolysis-related proteins. By spatially modulating EGFR signal

128

transduction, down-regulation of VPS4B leads to stabilization of cell adhesions and junctions, decreased cell proliferation, and adoption of fatty acids as an alternative energy source, which may support the survival of breast cancer cells under stress conditions.

129

Impaired endosomal trafficking caused by down regulation of VPS4B

EGFR EGFR

EGFR SOS-1 EPS8Ls ABI1 ERK1 Src

SH3BP1 Glycolysis

AMPK RAC ETV6 STAT3 Fatty acid oxidation ETV3 STAT5 SREBP Actin Fatty acid Cell adhesion Cell proliferation sythesis Cell junction FASN

Cell survival under stress conditions

Activation Inhibition Transcriptional activation

Attenuated activation Attenuated inhibition Attenuated transcriptional activation

Figure 28. A model of the mechanisms underlying altered EGFR signaling caused by VPS4B dysfunction. Down-regulation of VPS4B results in impaired endosomal trafficking. Consequently EGFR accumulates within endosomes, which potentiates signaling that is initiated or propagated by endosome localized proteins such as ERK, and EPS8. However, signal transduction associated with the trafficking of signaling molecules between cellular compartments is attenuated, such as activation of transcription factors ETV3 and ETV6 by ERK, and activation of STATs by Src. Affected signaling also includes signaling for the transcription, translation or degradation of proteins involved in glycolysis and fatty acid synthesis and oxidation. The negative regulation of AMPK by EGFR growth signaling could be abolished, resulting in activation of AMPK and consequently decreasing FASN expression by inhibiting its gene transcription factor SREBP, and increasing fatty acid β-oxidation gene expression. The attenuated stimulating signaling for glycolysis results in decreased expression of related proteins. Down-regulation of VPS4B mediated-EGFR signaling results in stabilization of cell adhesions and junctions, decreased cell proliferation, and adoption of fatty acids as an alternative energy source, which may support the survival of breast cancer cells under stress conditions.

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The results from our proteomic studies provide initial data regarding the effects of

VPS4B-mediated endosomal trafficking on EGFR signal transduction in breast cancer.

Additional studies that increase the depth of proteome coverage will likely reveal more comprehensive details of the specific signaling pathways, molecular mechanisms, and cellular functions that are affected by VPS4B-mediated endosomal dysfunction in breast cancer cells. For example, activation of EGFR evokes actin reorganization in many cell types – a process which is normally associated with increased phosphorylation of actin regulatory proteins. In our studies, a large number of cytoskeletal proteins, cell adhesion and cell junction proteins had a lower relative abundance of phosphorylation in VPS4B down regulated SKBR3 cells as compared to parental SKBR3 cells, which suggests that

VPS4B down-regulation stabilizes actin, cell adhesion and junctions. EPS8 is a crucial

part of the EPS8/Sos1/Abi1 complex, which transduces the signals from EGFR or EGFR-

activated RAS to RAC1, a central player in actin reorganization [272]. SH3BP1 has

been shown to inactivate RAC1 and is required for GDP/GTP cycling of RAC1 in the

leading edge of motile cells [119]. Both SH3BP1 and RAC1 exhibited increased

phosphorylation in VPS4B down regulated SKBR3 cells, which indicates they are

involved in the regulation of actin dynamics downstream in the EGFR signal transduction

pathway. Determination of RAC1 activity and its downstream effectors will further

address the specific roles of EPS8 and SH3BP1 in actin organization within the context

of VPS4B dysfunction. Since EPS8 directly interacts with EGFR [271] and forms an

EPS8/Sos1/Abi1 complex [273], further determination of the signal spatiality of EPS8

will address whether the impaired endosomal trafficking caused by VPS4B can affect

EPS8 downstream signal transduction.

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Activation of ERK by EGFR leads to phosphorylation of ETS transcription factors, such as ETV3 and ETV6, and promotes MAPK-related proliferation gene transcription [159][158]. However, we found that phosphorylation of ETV3 and ETV6 was decreased in VPS4B down regulated SKBR3 cells as compared to parental SKBR3 cells, which raises the question of how VPS4B dysfunction affects EGFR-mediated ERK activation and signal transduction in breast cancer cells. EGFR activates ERK through a linear RAF/MEK/ERK cascade. Early stage ERK activation occurs on the plasma membrane following EGFR activation, which is mediated by KSR, the scaffold protein for Raf-1, MEK1/2 and ERK1/2 [274][275]. Late stage ERK activation is mediated by the scaffold protein MP1, which is recruited to late endosomes by the adaptor proteins p14 and p18, where it promotes the assembly and interaction of MEK1 and ERK1 [73-

75]. MP1-mediated late stage ERK1 activation serves as an extension of the initiation of signaling at the cell membrane and maintains signaling at an adequate strength and duration [276]. Our studies indicate that VPS4B down-regulation results in the endosomal accumulation of activated EGFR, causing late stage ERK1 activation, which further activates ERK1 that is recruited to late endosomes by the p14/p18/MP1/MEK scaffold protein complex. The elevated ERK1 activation observed in our studies could also result from increased activation during the early stage of endosomal signaling; however, further studies are needed to clarify this hypothesis.

After activation, more than 60% of ERK molecules translocate from the cytoplasm to the nucleus, where they activate a variety of substrates, including ETS transcription factors. However, the decreased phosphorylation of ETS transcription

132

factors ETV3 and ETV6 in VPS4B down regulated SKBR3 cells suggests that either

VPS4B down-regulation could affect the trafficking of ERK1, or ERK1 could be retained on endosomes due to the impaired endosomal trafficking caused by down-regulation of

VPS4B. In addition to their nuclear localization, ERK molecules translocate to various cellular compartments such as mitochondria, Golgi, and endosomes, where they interact with their specific substrates and scaffold proteins in order to induce the proper signal

[277]. Whether VPS4B down-regulation could also affect these processes is not known.

Further studies are required in order to comprehensively delineate the effects of activated

ERK signaling in VPS4B down regulated cells.

Additional studies are also needed to address the relationship between the altered

EGFR signaling caused by VPS4B-mediated endosomal dysfunction and altered protein synthesis and degradation, especially, proteins related to energy metabolism. Our studies indicate that overall protein synthesis and degradation rates are not significantly changed in VPS4B down regulated breast cancer cells. However, altered EGFR signaling could selectively alter protein synthesis and degradation rates. Protein abundance regulation by modulation of the synthesis and degradation rates of proteins involved in metabolism helps cells to adapt to different carbon and energy sources [205]. In our study, VPS4B down-regulation alters the synthesis and degradation rates of proteins involved in energy metabolism; proteins involved in fatty acid β-oxidation are up regulated, while proteins involved in glycolysis and fatty acid synthesis are down regulated. Recent studies suggest cross talk between fatty acid metabolism and EGFR signaling, in which AMPK has emerged as the main player [223-227]. AMPK is the “fuel gauge” which senses

133

cellular energy demands and coordinates energy metabolism in response to environmental and intracellular microenvironmental changes. Activation of AMPK

switches off ATP-consuming pathways, such as fatty acid synthesis, and switches on

alternative pathways for ATP generation, such as fatty acid β-oxidation [278]. AMPK

can down regulate fatty acid synthesis by blocking the nuclear translocation of the fasn

gene transcription activator, SREBP, consequently decreasing FASN expression [222].

VPS4B down-regulation results in decreased FASN expression. Whether decreased

FASN expression results from activated AMPK requires further study.

Growth signaling mediated by EGFR or growth factors has been shown to inhibit

AMPK activation [223][224], while activation of AMPK decreases EGFR-mediated cell

growth [226][227]. Given that VPS4B dysfunction-mediated impaired endosomal

trafficking alters EGFR signal transduction, it is possible that the inhibitory signal

transduction from activated EGFR to AMPK results in activation of AMPK and therefore down-regulation of FASN. Analysis of AMPK activity and signaling intermediates in response to EGFR activation in VPS4B down regulated cells will address this possibility.

In addition, growth factors also stimulate glycolysis by increasing the expression of related proteins, such as LDHA/B [228], fructose-2,6-bisphosphatase (PFK) [229],

GADPH [230], via RAS/PI3K/PTEN/AKT/mTOR pathway [231]. However, we found that the relative abundance of proteins related to glycolysis was decreased in VPS4B down regulated cells as a result of altered synthesis and degradation. Whether the decrease in the relative abundance of glycolysis related proteins in VPS4B down

134

regulated cells also results from the impaired endosomal trafficking, which attenuates the stimulating signaling, necessitates further study.

The last aspect of our studies to be addressed is how VPS4B down-regulation-

mediated alteration of EGFR signal transduction and protein synthesis and degradation affect breast cancer cellular function under hypoxic conditions. Our studies suggest that

VPS4B down-regulation could result in: 1) stabilization of actin, cell adhesions and cell

junctions; 2) alteration of chromatin structure, gene transcription and protein translation;

and 3) adoption of fatty acid as an alternative energy resource. VPS4B is down regulated

in SKBR3 cells under hypoxia, which indicates that VPS4B is involved in the cell stress

response. Studies on the role of VPS4B dysfunction mediated EGFR signal transduction

under hypoxic conditions will further reveal the role of VPS4B in breast cancer survival.

In summary, our proteomics-based systems biology studies provide a valuable

framework for the future design of specific hypothesis-driven studies to understand the

role of VPS4B dysfunction in EGFR signal transduction. These studies will further

reveal the survival mechanism of breast cancer, which will shed new light on the

development of new drug targets or new breast cancer treatment strategies.

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Appendix 1. Quantified phosphopeptides.

Ratio = Gene UniProt Delta Phosphopeptide Sequence* Position Known Xcorr SKBR3_shVPS4 Name Accession Cn B vs. SKBR3 A6NFI3 A6NFI3 GGDAKS@PVLQEK S112 No 3.82 0.56 0.77 ± 0.13 AAK1 Q2M2I8 ILS@DVTHSAVFGVPASK S637 Yes 3.31 0.16 0.79 ± 0.18 ABCC5 O15440 NATLAWDSSHSSIQNS@PK S509 Yes 5.16 0.3 0.73 ± 0.26 ABI1 Q8IZP0 LGSQHS@PGR S225 Yes 2.88 0.17 0.91 ± 0.09 ABI1 Q8IZP0 TNPPTQKPPS@PPMSGR S183 Yes 2.67 0.28 1.30 ± 0.33 ACIN1 Q9UKV3 SSSISEEKGDS@DDEKPR S216 Yes 3.1 0.3 0.91 ± 0.10 ACIN1 Q9UKV3 RLS@QPESAEK S710 Yes 4.22 0.49 1.02 ± 0.11 ACIN1 Q9UKV3 ASLVALPEQTAS@EEETPPPLLTK S410 Yes 4.27 0.16 1.27 ± 0.65 ACIN1 Q9UKV3 TAQVPS@PPR S1004 Yes 2.53 0.41 1.30 ± 0.12 ACLY P53396 TAS@FSESR S455 Yes 2.69 0.14 1.19 ± 0.09 ACLY P53396 AKPAMPQDSVPS@PR S481 Yes 3.35 0.39 1.26 ± 0.15 ADAR P55265 TAESQT@PTPSATSFFSGK T601 Yes 3.84 0.13 0.83 ± 0.14 ADD1 P35611 AAVVTS@PPPTTAPHK S12 Yes 2.64 0.11 0.66 ± 0.07 T150, No AFG3L2 Q9Y4W6 MFFLWT@ALFWGGVMFY@LLLKR 3.28 0.13 1.04 ± 0.08 Y160 No AGFG1 P52594 GTPSQS@PVVGR S181 Yes 3.14 0.19 0.91 ± 0.15 AHNAK Q09666 FGTFGGLGS@K S5830 Yes 3.06 0.38 0.66 ± 0.09 AHNAK Q09666 VDVKS@PK S5077 Yes 2.64 0.25 0.92 ± 0.06 AHNAK Q09666 LPSGS@GAASPTGSAVDIR S212 Yes 2.7 0.3 0.94 ± 0.13 AHNAK Q09666 FGFGAKS@PK S4986 Yes 3.14 0.37 1.02 ± 0.11 AHNAK Q09666 ISMPDLDLHLKS@PK S2397 Yes 3.07 0.5 1.02 ± 0.18 AHNAK Q09666 IS@MSEVDLNVAAPK S570 Yes 4.47 0.12 1.02 ± 0.10 AHNAK Q09666 FKAEAPLPS@PK S5110 Yes 3.33 0.43 1.10 ± 0.10 AHNAK Q09666 LKS@EDGVEGDLGETQSR S135 Yes 4.13 0.64 1.14 ± 0.25 AHNAK Q09666 GGVTGS@PEASISGSK S5731 Yes 3.83 0.15 1.16 ± 0.10 AHNAK Q09666 DIDISS@PEFK S177 No 2.79 0.4 1.17 ± 0.21 AHNAK Q09666 IS@APNVDFNLEGPK S5448 Yes 3.39 0.59 1.18 ± 0.21 AHNAK Q09666 ASLGSLEGEAEAEASS@PK S5763 Yes 4.14 0.17 1.22 ± 0.26 AHSG P02765 CDSSPDS@AEDVRK S138 Yes 3.98 0.2 1.23 ± 0.23 AKAP17A Q02040 VVPEDGS@PEKR S537 Yes 2.97 0.42 0.98 ± 0.20 AKT1S1 Q96B36 S@LPVSVPVWGFK S183 Yes 3.47 0.46 0.97 ± 0.19 AKT1S1 Q96B36 LNT@SDFQK T246 Yes 2.9 0.13 1.08 ± 0.06 ALDOA P04075 GILAADESTGS@IAKR S39 Yes 3.27 0.11 1.10 ± 0.26 ALMS1 Q8TCU4 PDIFY@QKDLPDR Y2151 No 2.52 0.34 1.05 ± 0.24 ANK3 Q12955 RQS@FASLALR S1459 Yes 3.08 0.29 0.55 ± 0.19 ANK3 Q12955 LS@LHEEEGSSGSEQK S4342 No 2.52 0.48 0.88 ± 0.25 ANLN B4DSL6 AAS@PPKPLLSNASATPVGR S59 No 3.86 0.17 1.35 ± 0.36 ANLN Q9NQW6 TQS@LPVTEK S485 Yes 2.6 0.35 0.66 ± 0.19 ANLN Q9NQW6 TPIS@PLK S323 Yes 2.8 0.35 1.23 ± 0.15 ANP32A P39687 NRT@PSDVK T15 Yes 2.55 0.24 0.83 ± 0.17 AP3B1 O00203 NFYES@DDDQKEK S276 Yes 3.15 0.31 1.06 ± 0.29 ARFGAP2 Q8N6H7 YKDNPFSLGES@FGSR S368 Yes 3.75 0.14 0.86 ± 0.26 ARFGEF2 Q9Y6D5 ELEKPIQSKPQSPVIQAAAVS@PK S227 Yes 4.64 0.41 1.15 ± 0.24 ARFIP1 P53367 KWS@LNTYK S132 Yes 2.79 0.32 0.69 ± 0.16 ARHGAP1 AESSSGGGTVPSSAGILEQGPSPGDGS@P Q68EM7 S575 Yes 3.85 0.23 1.54 ± 0.20 7 PKPK ARHGAP3 A7KAX9 DATNRS@PTQIVK S952 No 2.66 0.13 1.15 ± 0.19 2 ARHGEF16 Q5VV41 HQS@FGAAVLSR S107 Yes 3.87 0.49 0.95 ± 0.43 ARHGEF16 Q5VV41 GLGKPGGQGDAIQLS@PK S174 Yes 3.17 0.57 1.28 ± 0.27 ARHGEF6 Q15052 MS@GFIYQGK S488 Yes 3.17 0.52 0.76 ± 0.11 ARHGEF6 Q15052 KS@TAALEEDAQILK S649 Yes 4.21 0.1 0.81 ± 0.29 ARHGEF7 Q14155 KPS@DEEFASR S694 Yes 2.91 0.34 0.96 ± 0.22 ARL6IP4 Q66PJ3 S@AGEEEDGPVLTDEQK S271 Yes 4.68 0.51 0.85 ± 0.08 ATAD2 Q6PL18 LRS@AGAAQK S41 Yes 3.06 0.33 1.11 ± 0.09 ATAD2 Q6PL18 S@QVEQQQLITVEK S1302 Yes 5.09 0.62 1.17 ± 0.27 ATAD2 Q6PL18 LSSAGPRS@PYCK S342 Yes 3.04 0.17 1.18 ± 0.30

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Appendix 1. Quantified phosphopeptides (continued).

ATXN2L Q8WWM7 EKEVDGLLTSEPMGS@PVSSK S594 Yes 3.89 0.11 0.85 ± 0.07 ATXN2L Q8WWM7 GPPQS@PVFEGVYNNSR S111 Yes 4.85 0.37 1.08 ± 0.33 ATXN2L Q8WWM7 STSTPTS@PGPR S684 Yes 2.51 0.16 1.09 ± 0.08 AUP1 Q9Y679 LRPQSAQSSFPPS@PGPSPDVQLATLAQR S354 Yes 6.71 0.27 1.16 ± 0.21 BAIAP2 Q9UQB8 NSYATTENKT@LPR T360 Yes 2.86 0.21 1.53 ± 0.13 BAT2 P48634 EGTLTQVPLAPPPPGAPPS@PAPAR S1147 Yes 3.38 0.55 1.61 ± 0.88 BAT2L1 Q5JSZ5 QAEKEVPWSPS@AEK S558 No 2.66 0.26 1.37 ± 0.35 BAT2L2 Q9Y520 LPDLS@PVENK S2105 Yes 4.12 0.52 0.94 ± 0.15 BAT3 P46379 VGDPPQPLPEEPMEVQGAERAS@PEPQR S964 Yes 4.06 0.65 1.58 ± 0.25 BCAS1 O75363 TLVS@PNKAETK S296 No 2.66 0.28 1.21 ± 0.17 BCLAF1 Q9NYF8 AEGEPQEES@PLKSK S177 Yes 3.3 0.3 0.98 ± 0.29 BCLAF1 Q9NYF8 IDISPST@LR T661 Yes 2.58 0.47 1.01 ± 0.11 BCLAF1 Q9NYF8 IDISPS@TLR S660 Yes 2.6 0.12 1.01 ± 0.06 BCLAF1 Q9NYF8 KETQS@PEQVK S496 Yes 3.58 0.15 1.04 ± 0.11 BCLAF1 Q9NYF8 IDIS@PSTLR S658 Yes 2.82 0.13 1.04 ± 0.10 BCLAF1 Q9NYF8 DLFDYS@PPLHK S512 Yes 3.72 0.11 1.08 ± 0.17 BET1 O15155 SLS@IEIGHEVK S50 Yes 3.47 0.16 0.60 ± 0.14 BNIP2 Q12982 KGS@ITEYTAAEEK S114 Yes 4.31 0.1 0.48 ± 0.10 BRD3 Q15059 SES@PPPLSDPK S263 Yes 2.73 0.12 0.85 ± 0.12 BRD4 O60885 IHS@PIIR S1117 Yes 2.55 0.56 0.75 ± 0.14 BTG4 Q9NY30 VS@NPKSIYQVENLK S163 No 2.5 0.22 0.62 ± 0.10 C11orf52 Q96A22 VLQQQGS@QER S62 No 3.04 0.5 0.55 ± 0.06 C14orf4 Q9H1B7 RPGS@VSSTDQER S334 Yes 3.12 0.26 0.94 ± 0.12 C14orf4 Q9H1B7 KAS@PEPPDSAEGALK S547 Yes 3.65 0.37 1.00 ± 0.10 C14orf43 Q6PJG2 RAS@QEANLLTLAQK S461 Yes 3.93 0.37 0.69 ± 0.09 C19orf62 Q9NWV8 TRS@NPEGAEDR S29 Yes 3.53 0.14 0.87 ± 0.25 C1orf226 A1L170 ASS@PSLIER S223 Yes 2.76 0.19 0.33 ± 0.00 C7orf47 Q8TAP8 APVPEPGLDLSLSPRPDS@PQPR S52 Yes 3.23 0.24 0.79 ± 0.00 C9orf142 Q9BUH6 LAAAEETAVS@PR S148 Yes 3.36 0.31 2.80 ± 0.38 C9orf78 Q9NZ63 VGDTEKPEPERS@PPNR S261 Yes 3.24 0.12 0.91 ± 0.14 CAD P27708 IHRAS@DPGLPAEEPK S1859 Yes 3.58 0.47 0.68 ± 0.00 CAMK2B Q13555 QET@VECLR T287 Yes 2.69 0.41 0.56 ± 0.04 CANX P27824 AEEDEILNRS@PR S583 Yes 2.7 0.21 0.93 ± 0.16 CARHSP1 Q9Y2V2 GNVVPS@PLPTR S41 Yes 2.96 0.14 1.07 ± 0.09 CARHSP1 Q9Y2V2 GNVVPS@PLPTRR S41 Yes 2.73 0.25 1.07 ± 0.09 CBX3 Q13185 S@LSDSESDDSK S93 Yes 3.23 0.22 0.84 ± 0.04 CBX3 Q13185 SLS@DSESDDSK S95 Yes 4.32 0.18 0.84 ± 0.06 CCDC137 Q6PK04 VQAGPGS@PR S19 Yes 2.65 0.45 1.07 ± 0.04 CCDC6 Q16204 SNS@PDKFK S419 Yes 2.62 0.24 0.92 ± 0.06 CCDC6 Q16204 LDQPVSAPPS@PR S244 Yes 3.12 0.36 1.48 ± 0.14 CCDC86 Q9H6F5 LGGLRPES@PESLTSVSR S18 Yes 3.11 0.15 1.14 ± 0.26 CCDC86 Q9H6F5 ALVEFESNPEETREPGS@PPSVQR S47 Yes 3.65 0.25 1.26 ± 0.26 CCNK O75909 AVVVS@PKEENK S340 Yes 2.81 0.53 0.99 ± 0.19 CCNL2 Q96S94 AKADS@PVNGLPK S369 Yes 3.1 0.51 1.55 ± 0.17 CD97 P48960 ALRAS@ESGI S831 Yes 2.6 0.13 1.10 ± 0.09 NGAAGPHS@PDPLLDEQAFGDLTDLPVV CDC42EP4 Q9H3Q1 S174 Yes 4.12 0.41 1.57 ± 0.32 PK CDK1 P06493 IGEGTY@GVVYK Y15 Yes 3.1 0.13 1.06 ± 0.21 CDK12 Q9NYV4 ESKGS@PVFLPR S423 Yes 2.62 0.37 0.84 ± 0.23 CDK12 Q9NYV4 HSSIS@PVRLPLNSSLGAELSR S385 Yes 3.61 0.1 0.87 ± 0.04 CDK12 Q9NYV4 AIT@PPQQPYK T692 Yes 3.01 0.5 0.87 ± 0.04 CDK7 P50613 AYT@HQVVTR T170 Yes 2.99 0.12 0.46 ± 0.19 CDYL Q9Y232 ILVPKS@PVK S201 Yes 3.06 0.56 0.94 ± 0.08 CGN Q9P2M7 KVS@LVLEK S332 Yes 3.52 0.46 0.32 ± 0.03 CGN Q9P2M7 SHS@QASLAGPGPVDPSNR S131 Yes 4.07 0.11 0.39 ± 0.13 CGN Q9P2M7 QPAQSQNLS@PLSGFSR S252 Yes 2.93 0.24 0.45 ± 0.07 CGN Q9P2M7 VAS@PGSTIDTAPLSSVDSLINK S159 Yes 5.2 0.14 0.77 ± 0.00 CHD4 Q14839 MSQPGS@PSPK S1535 Yes 2.59 0.46 0.99 ± 0.05 CHMP7 Q8WUX9 IS@DAELEAELEK S417 Yes 2.77 0.52 1.31 ± 0.09 CLASP1 Q7Z460 SRS@DIDVNAAASAK S600 Yes 4.86 0.14 0.70 ± 0.35 CLCC1 Q96S66 FQTGNKS@PEVLR S438 Yes 3.74 0.32 1.23 ± 0.26

137

Appendix 1. Quantified phosphopeptides (continued).

CLDN7 O95471 SNS@SKEYV S206 Yes 2.67 0.2 0.68 ± 0.07 CLN6 Q9NWW5 HGS@VSADEAAR S31 Yes 4.09 0.25 1.08 ± 0.11 CLTA P09496 LQS@EPESIR S105 Yes 2.57 0.11 0.38 ± 0.03 CNNM4 Q6P4Q7 SAS@LSYPDR S664 Yes 3.03 0.29 1.02 ± 0.10 COPB2 P35606 STAQQELDGKPAS@PTPVIVASHTANK S859 Yes 4.85 0.15 1.04 ± 0.22 CTNND1 O60716 VGGS@SVDLHR S268 Yes 2.97 0.16 0.50 ± 0.18 CTTN Q14247 AKTQT@PPVSPAPQPTEER T401 Yes 3.1 0.1 0.99 ± 0.23 CUL4A Q13619 KGS@FSALVGR S10 Yes 3.47 0.25 0.82 ± 0.21 DAPP1 Q9UN19 SRS@FIFK S276 No 2.64 0.2 0.51 ± 0.12 DBN1 Q16643 LSS@PVLHR S142 Yes 2.67 0.21 1.11 ± 0.10 DBNL Q9UJU6 LRS@PFLQK S283 Yes 2.63 0.14 0.80 ± 0.07 DDX21 Q9NR30 KKEEPSQNDIS@PK S89 Yes 4.55 0.36 1.11 ± 0.10 DDX21 Q9NR30 NEEPS@EEEIDAPKPK S121 Yes 3.7 0.36 1.35 ± 0.21 DENND4C Q5VZ89 RSS@LYGIAK S948 Yes 2.78 0.2 0.56 ± 0.03 DENND4C Q5VZ89 STS@LSALVR S1089 Yes 2.91 0.16 0.66 ± 0.14 DKC1 O60832 AKEVELVS@E S513 Yes 2.78 0.44 0.78 ± 0.09 DKC1 O60832 AGLESGAEPGDGDS@DTTKK S494 Yes 3.02 0.1 0.86 ± 0.21 DLGAP4 Q9Y2H0 QNS@ATESADSIEIYVPEAQTR S970 Yes 4.52 0.13 0.97 ± 0.13 DLGAP4 Q9Y2H0 SEQGTLTSSES@HPEAAPK S654 No 2.68 0.16 1.11 ± 0.12 DNAJC5 Q9H3Z4 SLS@TSGESLYHVLGLDK S10 Yes 3.37 0.14 0.70 ± 0.19 DNAJC9 Q8WXX5 KIS@LEDIQAFEK S109 Yes 3.46 0.4 1.21 ± 0.00 DNM1L O00429 SKPIPIMPAS@PQKGHAVNLLDVPVPVAR S616 Yes 3.57 0.23 0.94 ± 0.23 DSP P15924 AES@GPDLR S22 Yes 3.1 0.48 0.90 ± 0.07 DSP P15924 SMS@FQGIR S2209 Yes 2.75 0.27 1.14 ± 0.17 DYRK1A Q13627 IYQY@IQSR Y321 Yes 2.97 0.31 0.85 ± 0.14 EBAG9 O00559 KLS@GDQITLPTTVDYSSVPK S36 Yes 5.04 0.38 0.80 ± 0.28 ECT2 Q9H8V3 ANT@PELKK T328 Yes 2.7 0.34 0.90 ± 0.15 EEF1D P29692 ATAPQTQHVS@PMR S133 Yes 3.36 0.27 1.45 ± 0.27 EIF3C Q99613 QPLLLS@EDEEDTKR S39 Yes 2.75 0.5 0.95 ± 0.11 EIF4B P23588 SQS@SDTEQQSPTSGGGK S497 Yes 5.03 0.12 0.99 ± 0.09 EIF4B P23588 SQSS@DTEQQSPTSGGGK S498 Yes 2.71 0.2 0.99 ± 0.09 EIF4B P23588 ARPATDS@FDDYPPR S207 Yes 2.89 0.21 1.54 ± 0.53 EIF4G1 Q04637 ITKPGS@IDSNNQLFAPGGR S1077 Yes 4.56 0.17 0.57 ± 0.05 EIF4G1 Q04637 SFS@KEVEER S1187 Yes 2.92 0.14 0.78 ± 0.07 EIF4G1 Q04637 EAALPPVS@PLK S1231 Yes 2.97 0.54 1.19 ± 0.14 EIF4G2 P78344 FSASSGGGGS@R S22 No 2.74 0.36 0.73 ± 0.11 EIF4G3 O43432 RS@PVPAQIAITVPK S495 Yes 3.57 0.58 0.85 ± 0.09 EIF5B O60841 NKPGPNIES@GNEDDDASFK S214 Yes 5.35 0.48 1.14 ± 0.12 EMD P50402 LS@PPSSSAASSYSFSDLNSTR S49 Yes 5.64 0.12 1.04 ± 0.28 ENAH Q8N8S7 QNS@QLPAQVQNGPSQEELEIQR S125 Yes 4.79 0.56 0.45 ± 0.09 ENSA O43768 KSS@LVTSK S109 Yes 3.05 0.17 0.86 ± 0.04 EPB41L1 Q9H4G0 HQAS@INELK S510 Yes 3.08 0.22 0.44 ± 0.08 EPB41L1 Q9H4G0 SLS@PIIGK S784 Yes 2.51 0.35 0.64 ± 0.07 EPB41L1 Q9H4G0 SEAEEGEVRT@PTK T475 Yes 3.07 0.17 0.75 ± 0.05 EPB41L1 Q9H4G0 LPSSPAS@PSPK S544 Yes 2.67 0.11 0.81 ± 0.12 EPS8L1 Q8TE68 DRSPAAET@PPLQR T187 Yes 3.89 0.26 1.09 ± 0.21 EPS8L1 Q8TE68 DRS@PAAETPPLQR S182 Yes 3.77 0.5 1.62 ± 0.22 EPS8L2 Q9H6S3 YWGPAS@PTHK S570 Yes 2.71 0.1 0.87 ± 0.12 EPS8L2 Q9H6S3 YWGPASPT@HK T572 No 2.68 0.14 0.96 ± 0.13 EPS8L2 Q9H6S3 HSPTSEPTPPGDALPPVSSPHT@HR T483 No 3.76 0.1 1.28 ± 0.51 EPS8L3 Q8TE67 MQVLY@EFEARNPR Y459 No 2.55 0.39 2.29 ± 0.71 ERBB2 P04626 GLQSLPTHDPS@PLQR S1107 Yes 3.37 0.11 0.97 ± 0.19 ERBB2 P04626 LLQETELVEPLT@PSGAMPNQAQMR T701 Yes 3.81 0.18 0.98 ± 0.10 ERBB2 P04626 TLS@PGKNGVVK S1174 No 2.56 0.25 1.12 ± 0.15 ERBB2 P04626 GAPPSTFKGT@PTAENPEYLGLDVPV T1240 No 2.69 0.13 1.18 ± 0.14 ERBB2 P04626 S@GGGDLTLGLEPSEEEAPR S1054 Yes 5.08 0.13 1.31 ± 0.19 ERBB2 P04626 PAGAT@LERPK T1166 Yes 3.64 0.6 1.41 ± 0.22 ERCC5 P28715 NAPAAVDEGSIS@PR S384 Yes 4.63 0.18 0.86 ± 0.14 ESF1 Q9H501 FKIDSNIS@PK S153 Yes 2.87 0.16 1.13 ± 0.22 ESF1 Q9H501 TLDSGTSEIVKS@PR S198 Yes 3.42 0.19 1.13 ± 0.17 ETV3 P41162 SSGVVPQS@APPVPTASSR S139 Yes 4.18 0.27 0.49 ± 0.00

138

Appendix 1. Quantified phosphopeptides (continued).

ETV6 P41212 RLS@PAER S213 Yes 2.8 0.36 0.56 ± 0.17 F11R Q9Y624 KVIYSQPS@AR S284 Yes 2.81 0.21 0.94 ± 0.11 PVAPQVPS@PGGAPGQGPYPYSLSEPAPL FAM120A Q5T035 S32 No 3.44 0.42 0.87 ± 0.18 TLDTSGK GLLAQGLRPES@PPPAGPLLNGAPAGESP FAM129B Q96TA1 S652 Yes 7.12 0.65 1.21 ± 0.19 QPK FAM164A Q96GY0 LQTLS@PSHK S223 Yes 2.55 0.15 0.75 ± 0.18 FAM164A Q96GY0 NST@PPSLAR T244 Yes 2.68 0.23 0.76 ± 0.13 FAM164A Q96GY0 GIEGHS@PGNLPK S292 Yes 2.96 0.36 1.15 ± 0.14 FAM21A Q641Q2 SRPTS@FADELAAR S288 No 3.48 0.12 0.93 ± 0.24 FAM53B Q14153 SSS@FSLPSR S167 Yes 2.67 0.32 0.93 ± 0.27 FAM83H Q6ZRV2 SDS@LGTQGR S805 No 2.93 0.29 0.75 ± 0.07 FAM83H Q6ZRV2 RGS@PTTGFIEQK S599 No 3.42 0.18 0.97 ± 0.15 FAM83H Q6ZRV2 KGS@PTPGFSTR S588 No 2.9 0.26 0.97 ± 0.12 FAM83H Q6ZRV2 HGS@DPAFAPGPR S230 No 3.59 0.72 1.15 ± 0.17 FAM83H Q6ZRV2 RGS@PVPPVPER S621 No 3.14 0.48 1.21 ± 0.16 FAM83H Q6ZRV2 RSS@PVPPVPER S632 No 2.52 0.17 1.34 ± 0.26 FARP1 Q9Y4F1 LGAPENSGIS@TLER S23 Yes 3.41 0.15 0.81 ± 0.19 FASN P49327 ADEASELACPT@PK T2204 Yes 3.86 0.53 1.52 ± 0.17 FIP1L1 Q6UN15 DHS@PTPSVFNSDEER S492 Yes 3.38 0.2 1.05 ± 0.33 FKBP15 Q5T1M5 RPS@QEQSASASSGQPQAPLNR S956 Yes 4.49 0.36 0.94 ± 0.26 FLII Q13045 NAEAVLQS@PGLSGK S856 Yes 3.95 0.28 1.10 ± 0.15 FLNB B2ZZ83 ADDTDSQSWRS@PLK S1074 No 3.08 0.2 1.35 ± 0.07 FLNB O75369 LVS@PGSANETSSILVESVTR S2478 Yes 5.49 0.2 0.80 ± 0.12 FLNB O75369 YADEEIPRS@PFK S1505 Yes 3.84 0.41 0.92 ± 0.00 FLNB O75369 APS@VATVGSICDLNLK S2107 Yes 4.99 0.24 0.97 ± 0.14 FNBP1L Q5T0N5 TIS@DGTISASK S295 Yes 3.41 0.17 0.68 ± 0.13 TGQLEGAPAPGPAAS@PQTLDHSGATAT FOXA1 P55317 S332 Yes 3.86 0.18 1.27 ± 0.32 GGASELK FOXK1 P85037 EGS@PIPHDPEFGSK S445 Yes 2.5 0.37 1.09 ± 0.13 FOXK1 P85037 EEAPAS@PLRPLYPQISPLK S213 Yes 3.33 0.39 1.31 ± 0.37 FOXK1 P85037 S@APASPTHPGLMSPR S416 Yes 3.39 0.1 1.55 ± 0.19 FOXK1 P85037 SAPAS@PTHPGLMSPR S420 Yes 3.02 0.16 1.55 ± 0.19 FOXK2 Q01167 EGS@PAPLEPEPGAAQPK S398 Yes 3.61 0.58 1.28 ± 0.35 FRYL O94915 TRS@LSSLR S1978 Yes 2.74 0.18 0.78 ± 0.22 FTH1 P02794 HTLGDS@DNES S179 Yes 2.67 0.26 1.00 ± 0.00 FXYD3 Q14802 SGHHPGET@PPLITPGSAQS T76 No 3.06 0.17 1.00 ± 0.00 GAPDH P04406 LVINGNPITIFQERDPS@KIK S83 Yes 3.07 0.24 1.12 ± 0.30 GAPVD1 Q14C86 SRS@SDIVSSVR S902 No 3.29 0.22 0.95 ± 0.16 GFPT1 Q06210 VDS@TTCLFPVEEK S261 Yes 4.54 0.11 0.89 ± 0.11 GLYR1 Q49A26 KLS@LSEGK S130 Yes 2.73 0.28 0.81 ± 0.10 GNAS Q5JWF2 ISTAS@GDGR S995 No 2.53 0.23 0.90 ± 0.11 GOSR1 B4DQA8 RDS@SDTTPLLNGSSQDR S49 No 2.61 0.16 1.20 ± 0.32 GPN1 Q9HCN4 DSLS@PVLHPSDLILTR S314 Yes 3.06 0.11 0.92 ± 0.25 GRB7 Q14451 HLHPSCLGS@PPLR S361 Yes 2.65 0.14 0.84 ± 0.16 GRLF1 Q9NRY4 NQKNS@LSDPNIDR S589 Yes 4.75 0.11 1.00 ± 0.18 GSK3A P49840 GEPNVSY@ICSR Y279 Yes 2.61 0.13 1.12 ± 0.24 GTF2I P78347 ESTSSKS@PPR S823 Yes 2.79 0.14 0.80 ± 0.03 GTF2I P78347 RPS@TYGIPR S412 Yes 2.67 0.18 1.10 ± 0.14 H1FX Q92522 AGGSAALS@PSK S31 Yes 3.61 0.11 1.61 ± 0.15 HARS2 P49590 DLS@PQHMVVR S67 Yes 3.11 0.54 0.82 ± 0.12 hCG_1795 P05114 KVS@SAEGAAK S7 Yes 2.91 0.23 0.95 ± 0.05 5 HDGF P51858 AGDLLEDS@PK S165 Yes 3.13 0.54 0.80 ± 0.08 HDGF P51858 NSTPS@EPGSGR S202 Yes 2.58 0.21 0.80 ± 0.11 HDGF P51858 NST@PSEPGSGR T200 Yes 2.52 0.34 0.87 ± 0.18 HDGF2 Q7Z4V5 ARGDS@EALDEES S664 Yes 3.09 0.73 1.02 ± 0.07 HIRIP3 Q9BW71 NGVAAEVS@PAKEENPR S125 Yes 4.53 0.53 1.03 ± 0.20 HIST1H1E P16403 KAS@GPPVSELITK S36 Yes 4.87 0.36 2.04 ± 0.29 HMG20A Q9NP66 DSNAPKS@PLTGYVR S105 Yes 3.51 0.18 1.07 ± 0.21 HMGA1 P17096 KQPPVS@PGTALVGSQK S36 Yes 4.27 0.16 0.82 ± 0.09

139

Appendix 1. Quantified phosphopeptides (continued).

HMGXB3 Q12766 QIHDIVQS@CQPGEVVIR S1500 No 2.52 0.23 0.97 ± 0.13 HN1 Q9UK76 RNS@SEASSGDFLDLK S87 Yes 3.4 0.11 0.67 ± 0.20 HN1L Q9H910 TSDIFGS@PVTATSR S97 Yes 4.9 0.14 1.03 ± 0.10 HNRNPA1 P0C7M2 SES@PKEPEQLR S6 No 3.04 0.16 0.91 ± 0.10 HNRNPC P07910 NDKS@EEEQSSSSVK S233 Yes 3.82 0.35 0.84 ± 0.12 HNRNPH1 P31943 HTGPNS@PDTANDGFVR S104 Yes 4.02 0.18 1.04 ± 0.13 HNRNPK P61978 DYDDMS@PR S284 Yes 2.52 0.67 0.91 ± 0.12 HNRNPUL Q1KMD3 REEDEPEERS@GDETPGSEVPGDK S161 Yes 3.32 0.24 0.80 ± 0.12 2 HSP90AA1 P07900 ESEDKPEIEDVGS@DEEEEKK S263 Yes 3.07 0.42 1.08 ± 0.25 HSP90AB1 Q58FF8 IEDVGS@DEEDDSGK S218 No 4.48 0.26 1.13 ± 0.12 HSPB1 P04792 QLSS@GVSEIR S83 Yes 2.79 0.18 0.38 ± 0.06 HSPB1 P04792 QLS@SGVSEIR S82 Yes 2.78 0.19 0.42 ± 0.04 HSPB1 P04792 GPS@WDPFR S15 Yes 3.09 0.36 0.43 ± 0.06 HSPH1 Q92598 IES@PKLER S809 Yes 2.54 0.4 0.90 ± 0.03 HTATSF1 O43719 SDS@VSASER S387 Yes 2.95 0.23 0.73 ± 0.05 HUWE1 Q7Z6Z7 GSGTAS@DDEFENLR S1907 Yes 4.19 0.22 0.83 ± 0.15 IGF2R P11717 ALSSLHGDDQDS@EDEVLTIPEVK S2409 Yes 4.12 0.13 0.82 ± 0.11 ILF3 Q12906 RPMEEDGEEKS@PSK S382 Yes 3.15 0.1 1.18 ± 0.21 INTS12 Q96CB8 SVS@CDNVSK S378 No 2.6 0.37 0.84 ± 0.18 IQGAP1 P46940 ALQS@PALGLR S330 Yes 2.83 0.51 1.00 ± 0.07 IQGAP3 Q86VI3 S@LTAHSLLPLAEK S1424 Yes 2.59 0.13 0.97 ± 0.58 IRF2BP1 Q8IU81 AGGAS@PAASSTAQPPTQHR S453 Yes 3.22 0.39 0.89 ± 0.34 IRF2BP2 Q7Z5L9 KPS@PEPEGEVGPPK S360 Yes 2.82 0.6 0.72 ± 0.12 IRF2BP2 Q7Z5L9 LEEPPELNRQS@PNPR S10 No 4.08 0.5 0.75 ± 0.07 ITPR3 Q14573 VAS@FSIPGSSSR S1832 Yes 3.5 0.26 0.54 ± 0.14 JUND P17535 LAS@PELER S100 Yes 2.79 0.53 0.89 ± 0.07 KIAA1217 Q5T5P2 ISSLPVSRPIS@PSPSAILER S361 Yes 3.52 0.14 0.89 ± 0.11 KIAA1244 Q5TH69 AGGGDLLLPPS@PK S1991 Yes 3.4 0.46 0.92 ± 0.24 KIAA1429 Q69YN4 SFLSEPS@SPGR S1578 Yes 2.71 0.32 1.15 ± 0.20 KIAA1429 Q69YN4 SFLSEPSS@PGR S1579 Yes 2.96 0.4 1.19 ± 0.17 KIAA1522 Q9P206 GLAGPPAS@PGK S545 Yes 2.52 0.53 1.02 ± 0.29 KIAA1543 Q9P1Y5 KFS@PSQVPVQTR S814 Yes 2.75 0.23 1.20 ± 0.11 KIAA1543 Q9P1Y5 DLPDGHAAS@PR S334 Yes 2.5 0.41 1.21 ± 0.15 KIAA1543 Q9P1Y5 HPLLSSGGPQS@PLR S368 Yes 3.63 0.25 1.30 ± 0.24 KIAA1598 A0MZ66 S@MPVLGSVSSVTK S506 Yes 4.32 0.55 0.94 ± 0.15 KLC1 Q7RTP8 AS@SLNVLNVGGK S595 No 2.69 0.6 0.67 ± 0.07 KLC2 Q9H0B6 ASS@LNFLNK S582 Yes 2.75 0.27 0.90 ± 0.13 KLC4 Q9NSK0 AAS@LNYLNQPSAAPLQVSR S590 Yes 5.52 0.18 0.82 ± 0.10 KRT18 P05783 PVSSAASVY@AGAGGSGSR Y36 Yes 5.05 0.17 0.83 ± 0.07 KRT18 P05783 PVSS@AASVYAGAGGSGSR S31 No 3.26 0.2 0.84 ± 0.54 KRT18 P05783 S@TFSTNYR S7 Yes 2.66 0.14 0.86 ± 0.06 KRT18 P05783 PVS@SAASVYAGAGGSGSR S30 No 3.27 0.32 0.89 ± 0.00 KRT18 P05783 PVSSAAS@VYAGAGGSGSR S34 Yes 4.35 0.14 0.95 ± 0.29 KRT18 P05783 SLGS@VQAPSYGAR S18 Yes 3.65 0.13 1.07 ± 0.16 KRT19 P08727 QSSATS@SFGGLGGGSVR S13 Yes 3.12 0.16 0.80 ± 0.16 KRT19 P08727 QSSAT@SSFGGLGGGSVR T12 No 3.32 0.2 0.85 ± 0.04 KRT19 P08727 QSS@ATSSFGGLGGGSVR S10 No 3.81 0.19 0.85 ± 0.06 KRT19 P08727 QSSATSSFGGLGGGS@VR S22 No 5.32 0.28 0.86 ± 0.08 KRT19 P08727 QSSATSS@FGGLGGGSVR S14 No 3.32 0.22 0.86 ± 0.16 KRT19 P08727 GVS@VSSAR S46 Yes 2.81 0.19 0.97 ± 0.09 KRT19 P08727 APS@IHGGSGGR S35 Yes 2.6 0.59 1.15 ± 0.08 KRT4 P19013 GFS@CGSAIVGGGK S16 No 3.57 0.47 0.99 ± 0.18 KRT4 P19013 GFSCGS@AIVGGGK S19 Yes 3.74 0.23 1.05 ± 0.11 KRT7 P08729 PGGLGSSSLYGLGAS@RPR S45 Yes 3.38 0.11 0.88 ± 0.17 KRT7 P08729 PGGLGS@SSLYGLGASR S36 Yes 3.58 0.14 1.15 ± 0.16 KRT8 P05787 VGS@SNFR S43 Yes 2.59 0.13 0.81 ± 0.08 KRT8 P05787 S@YTSGPGSR S24 Yes 2.54 0.31 1.13 ± 0.02 LAD1 O00515 NLSS@TTDDEAPR S39 No 4.27 0.11 0.86 ± 0.23 LAD1 O00515 IPSKEEEADMSS@PTQR S356 Yes 3.55 0.13 1.07 ± 0.15 LAD1 O00515 NSLS@PVQATQKPLVSK S123 No 4.02 0.11 1.12 ± 0.20

140

Appendix 1. Quantified phosphopeptides (continued).

LAD1 O00515 GVPEKS@PVLEK S177 Yes 2.72 0.33 1.24 ± 0.17 LARP1 Q6PKG0 AVT@PVPTKTEEVSNLK T526 Yes 3.39 0.34 0.95 ± 0.10 LARP1 Q6PKG0 S@LPTTVPESPNYR S766 Yes 2.7 0.24 1.47 ± 0.24 LARP4 Q71RC2 DGLNQTTIPVS@PPSTTKPSR S582 No 3.39 0.2 0.86 ± 0.12 LARP4B Q92615 KNS@FGYR S498 Yes 2.67 0.38 0.77 ± 0.07 LIG1 P18858 VLGS@EGEEEDEALSPAK S66 Yes 4.85 0.53 1.79 ± 0.40 LIMA1 Q9UHB6 ASSLSES@SPPK S373 Yes 2.64 0.19 0.54 ± 0.05 LIMA1 Q9UHB6 ASS@LSESSPPK S369 Yes 3.08 0.19 0.56 ± 0.06 LIMA1 Q9UHB6 KGWS@MSEQSEESVGGR S617 Yes 3.66 0.21 0.64 ± 0.11 LIMA1 Q9UHB6 LRS@PPEALVQGR S132 Yes 3.01 0.61 0.77 ± 0.08 LIMA1 Q9UHB6 ETPHS@PGVEDAPIAK S490 Yes 3.01 0.28 0.83 ± 0.15 LIMA1 Q9UHB6 SEVQQPVHPKPLS@PDSR S362 Yes 3.56 0.26 0.91 ± 0.10 LIMCH1 Q9UPQ0 SINHQIES@PSER S973 No 3.49 0.2 0.59 ± 0.12 LIMCH1 Q9UPQ0 QTPS@PDVVLR S217 No 2.66 0.32 0.74 ± 0.15 LIMD1 Q9UGP4 VSPGLPS@PNLENGAPAVGPVQPR S277 Yes 4.26 0.15 1.24 ± 0.31 LMNA P02545 ASSHSSQTQGGGS@VTK S414 Yes 4.42 0.21 0.83 ± 0.05 LMNA P02545 SGAQASST@PLSPTR T19 Yes 2.59 0.31 1.00 ± 0.15 LMNA P02545 SGAQASSTPLSPT@R T24 No 3.56 0.21 1.01 ± 0.10 LMNA P02545 SGAQASSTPLS@PTR S22 Yes 4.37 0.1 1.02 ± 0.12 LMNA P02545 LRLS@PSPTSQR S390 Yes 2.81 0.13 1.26 ± 0.05 LRRC16A Q5VZK9 RSS@GFISELPSEEGK S968 Yes 4.61 0.11 0.65 ± 0.19 LSM14A Q8ND56 S@PTMEQAVQTASAHLPAPAAVGR S192 Yes 3.54 0.15 0.77 ± 0.25 LSM14A Q8ND56 S@PVSTRPLPSASQK S216 Yes 2.9 0.1 0.92 ± 0.06 LSR Q86X29 NLALSRES@LVV S646 Yes 2.86 0.2 1.49 ± 0.07 LUZP1 Q86V48 DLKS@LEDPPTR S932 No 2.51 0.27 1.05 ± 0.53 MAF1 Q9H063 LSKS@QGGEEEGPLSDK S75 Yes 4.32 0.1 1.06 ± 0.25 MAP2K2 P36507 LNQPGT@PTR T394 Yes 2.65 0.16 1.08 ± 0.10 MAP4 P27816 VGS@LDNVGHLPAGGAVK S1073 Yes 3.34 0.56 0.82 ± 0.16 MAP4 P27816 DMES@PTKLDVTLAK S280 Yes 3.2 0.12 0.91 ± 0.15 MAP4 P27816 KCS@LPAEEDSVLEK S636 Yes 3.62 0.33 0.96 ± 0.16 MAPK1 P28482 VADPDHDHTGFLTEY@VATR Y187 Yes 3.45 0.1 1.29 ± 0.33 MAPK3 P27361 IADPEHDHTGFLTEY@VATR Y204 Yes 4.34 0.13 1.95 ± 0.34 MARCKS P29966 AEDGATPSPSNET@PKK T150 Yes 3.72 0.22 0.61 ± 0.03 MARCKSL P49006 AAAT@PESQEPQAK T148 Yes 3.96 0.23 0.44 ± 0.14 1 MARCKSL P49006 GEVPPKET@PK T85 Yes 2.68 0.49 0.45 ± 0.03 1 MARK2 Q7KZI7 SRNS@PLLER S486 Yes 2.53 0.22 0.88 ± 0.10 MARK2 Q7KZI7 VPAS@PLPGLER S456 Yes 3.03 0.56 1.04 ± 0.21 MCM2 P49736 GNDPLTSS@PGR S27 Yes 2.71 0.12 1.48 ± 0.11 MCM2 Q9UK58 AEEKS@PISINVK S352 Yes 3.46 0.12 0.79 ± 0.09 MDC1 Q14676 AAESLTAIPEPAS@PQLLETPIHASQIQK S1775 Yes 3.41 0.37 1.42 ± 0.31 MEPCE Q7L2J0 NS@CNVGGGGGGFK S152 Yes 2.6 0.34 0.87 ± 0.02 METTL3 Q86U44 NPEAALS@PTFR S43 Yes 3.78 0.16 1.12 ± 0.15 MKL2 Q9ULH7 EQLVDQGIMPPLKS@PAAFHEQIK S66 Yes 3.94 0.47 0.88 ± 0.28 MLF2 Q15773 LAIQGPEDS@PSR S238 Yes 2.78 0.24 1.00 ± 0.18 MLPH Q6UWC1 ASSES@QGLGAGVR S223 No 2.9 0.61 0.97 ± 0.06 MLPH Q9BV36 KFS@NSLK S552 No 2.82 0.13 0.98 ± 0.00 MTMR1 Q13613 GSS@PSHSATSVHTSV S653 Yes 3.53 0.12 1.00 ± 0.00 MTMR12 Q9C0I1 QLS@LPLTQSK S564 No 2.66 0.37 1.07 ± 0.12 MTOR P42345 T@LDQSPELR T1162 Yes 2.62 0.2 0.38 ± 0.03 MTSS1L Q765P7 GLS@LEHQK S456 Yes 2.54 0.56 1.01 ± 0.16 MYCBP2 O75592 S@LSPNHNTLQTLK S2749 Yes 2.93 0.14 0.97 ± 0.15 MYEF2 Q9P2K5 AEVPGATGGDS@PHLQPAEPPGEPR S17 Yes 3.74 0.21 0.64 ± 0.04 LEEGVAS@DEEAEEAQPGSGPSPEPEGSP MYH7B Q7Z406 S1969 No 5.17 0.54 1.00 ± 0.00 PAHPQ MYH9 P35579 GAGDGS@DEEVDGK S1943 Yes 3.82 0.73 0.60 ± 0.08 MYO18A Q92614 SLAPDRS@DDEHDPLDNTSRPR S2020 Yes 3.93 0.18 1.20 ± 0.50 MYO19 Q96H55 FKAS@LEQLLQVLHSTTPHYIR S3 No 3.46 0.16 1.86 ± 0.29 MYO9B Q13459 RTS@FSTSDVSK S1354 Yes 3.58 0.12 0.69 ± 0.10 MYO9B Q13459 VQEKPDS@PGGSTQIQR S1290 Yes 4.38 0.19 0.88 ± 0.10

141

Appendix 1. Quantified phosphopeptides (continued).

NAV1 Q8NEY1 TPPVAVT@SPITHTAQSALK T540 No 3.21 0.12 0.59 ± 0.05 NAV1 Q8NEY1 RNS@TIVLR S452 Yes 3.02 0.15 0.81 ± 0.08 NAV1 Q8NEY1 VNSNS@LDLPSSSDTTHASK S808 Yes 4.86 0.1 1.03 ± 0.08 NBEAL2 Q6ZNJ1 DNLGEVPLT@PTEEASLPLAVTK T1873 No 3.79 0.13 1.13 ± 0.15 NBEAL2 Q6ZNJ1 RIS@QVSSGETEYNPTEAR S2745 No 4.36 0.38 1.15 ± 0.30 IKTLHCEGT@EINSDDEQES@KEVEETAT T670, NCAPG Q9BPX3 Yes, No 3.05 0.12 1.67 ± 0.37 AK S680 NCL P19338 ALVAT@PGKK T121 Yes 2.97 0.54 1.04 ± 0.12 NCL P19338 KVVVS@PTK S67 Yes 2.58 0.2 1.46 ± 0.10 NCL P19338 LELQGPRGS@PNAR S563 Yes 2.61 0.38 1.57 ± 0.29 NCOR2 Q9Y618 ANAS@PQKPLDLK S956 Yes 2.74 0.1 0.84 ± 0.17 NDE1 Q9NXR1 RPS@STSVPLGDK S306 Yes 2.67 0.12 0.94 ± 0.17 NDRG3 Q9UGV2 THS@TSSSLGSGESPFSR S331 Yes 4.59 0.1 1.39 ± 0.20 NEBL O76041 SMQHS@PNLR S953 No 2.74 0.44 0.72 ± 0.07 NFATC2IP Q8NCF5 KKTEFLDLDNSPLSPPS@PR S204 Yes 3.28 0.26 0.95 ± 0.00 NIPBL Q6KC79 AITSLLGGGS@PK S2658 Yes 3.06 0.4 0.83 ± 0.13 NKAP Q8N5F7 IGELGAPEVWGLS@PK S149 Yes 3.95 0.33 1.00 ± 0.19 NOP2 P46087 GPQPPTVS@PIR S786 Yes 2.69 0.2 1.42 ± 0.20 NPDC1 Q9NQX5 APGS@PAAPR S229 Yes 2.55 0.55 1.03 ± 0.19 NPM1 P06748 SIRDT@PAK T199 Yes 2.63 0.23 1.05 ± 0.04 CGSGPVHISGQHLVAVEEDAES@EDEEEE NPM1 P06748 S125 Yes 6.17 0.55 1.06 ± 0.26 DVK NRD1 O43847 RGS@LSNAGDPEIVK S94 Yes 3.86 0.22 0.80 ± 0.12 NSFL1C Q9UNZ2 KKS@PNELVDDLFK S114 Yes 3.82 0.25 0.68 ± 0.17 AGEPNS@PDAEEANSPDVTAGCDPAGVH NSUN2 Q08J23 S743 No 4.17 0.2 1.55 ± 0.05 PPR NUCKS1 Q9H1E3 EEDEEPES@PPEKK S214 Yes 3.55 0.37 0.95 ± 0.26 NUCKS1 Q9H1E3 ATVTPS@PVKGK S181 Yes 2.62 0.24 1.10 ± 0.08 NUCKS1 Q9H1E3 NSQEDS@EDSEDKDVK S58 Yes 5.23 0.15 1.13 ± 0.08 NUF2 Q9BZD4 IVDS@PEKLK S247 Yes 3.05 0.49 1.09 ± 0.08 NUFIP2 Q7Z417 DYEIESQNPLAS@PTNTLLGSAK S629 Yes 5.43 0.12 0.88 ± 0.30 NUMA1 Q14980 VSLEPHQGPGT@PESK T2000 Yes 3.53 0.23 0.81 ± 0.20 NUMA1 Q14980 TQPDGTSVPGEPASPIS@QR S1760 Yes 4.51 0.17 1.24 ± 0.20 NUMA1 Q14980 APVPSTCSSTFPEELS@PPSHQAK S169 Yes 3.9 0.24 1.44 ± 0.46 NUP153 P49790 EGSVLDILKS@PGFASPK S614 Yes 3.21 0.15 1.03 ± 0.20 NUP214 P35658 ITPPAAKPGS@PQAK S678 Yes 2.58 0.47 1.26 ± 0.22 NUP98 P52948 NLNNSNLFS@PVNR S612 Yes 5.26 0.2 0.90 ± 0.08 OGFR Q9NZT2 SQGDEAGGHGEDRPEPLS@PK S378 Yes 3.67 0.58 0.91 ± 0.19 OSBPL3 Q9H4L5 LHSS@NPNLSTLDFGEEK S304 Yes 5.46 0.1 1.31 ± 0.21 P58107 P58107 QVS@ASELHTSGILGPETLR S2691 No 3.52 0.12 0.99 ± 0.20 PAICS P22234 TKEVYELLDS@PGK S27 Yes 4.97 0.47 0.97 ± 0.37 PAK2 Q13177 S@VIDPVPAPVGDSHVDGAAK S197 Yes 3.61 0.38 0.70 ± 0.15 PARD6B Q9BYG5 HGAGS@GCLGTMEVK S11 Yes 4.35 0.29 0.81 ± 0.09 PCBP1 Q15365 VMTIPYQPMPASS@PVICAGGQDR S190 Yes 4.42 0.11 0.94 ± 0.17 PDAP1 Q13442 MQS@LSLNK S176 Yes 2.93 0.17 0.96 ± 0.20 PDCD4 Q53EL6 DSGRGDS@VSDSGSDALR S76 Yes 4.05 0.1 1.05 ± 0.24 PDHA1 P08559 YGMGTS@VER S232 Yes 2.98 0.13 1.13 ± 0.13 PDHA1 P08559 YGMGT@SVER T231 Yes 2.74 0.6 1.15 ± 0.10 PDHA2 P29803 YHGHS@MSDPGVSYR S291 Yes 3.59 0.11 1.26 ± 0.20 PDLIM5 Q96HC4 APRPFGSVSS@PK S137 Yes 2.67 0.13 0.91 ± 0.13 PDPK1 O15530 ANS@FVGTAQYVSPELLTEK S241 Yes 4.88 0.27 0.95 ± 0.13 PDZD2 O15018 ANS@LVTLGSHR S920 No 3.24 0.38 0.78 ± 0.42 PEA15 Q15121 DIIRQPS@EEEIIK S116 Yes 3.74 0.41 0.60 ± 0.13 PFKFB2 O60825 RNS@FTPLSSSNTIR S466 Yes 3.2 0.25 1.10 ± 0.22 PGK1 P00558 ALES@PERPFLAILGGAK S203 Yes 3.6 0.47 1.12 ± 0.19 PGM1 P36871 AIGGIILTAS@HNPGGPNGDFGIK S117 Yes 3.26 0.13 1.15 ± 0.31 PGRMC1 O00264 EGEEPTVYS@DEEEPKDESAR S181 Yes 3.69 0.16 0.76 ± 0.10 PHRF1 Q9P1Y6 KENPS@PLFSIK S814 Yes 3.6 0.21 0.98 ± 0.61 PIK3C2A O00443 VSNLQVS@PK S259 Yes 2.97 0.54 0.84 ± 0.14 PKP3 Q9Y446 ADYDTLS@LR S180 Yes 3.12 0.11 0.69 ± 0.11 PKP3 Q9Y446 S@AVDLSCSR S123 No 2.64 0.25 0.72 ± 0.13

142

Appendix 1. Quantified phosphopeptides (continued).

PKP3 Q9Y446 SLS@LSLADSGHLPDVHGFNSYGSHR S285 No 3.9 0.1 0.85 ± 0.27 PLEC Q15149 KQIT@MEELVR T4030 Yes 3.96 0.61 0.86 ± 0.17 PLEC Q15149 RAS@FAEK S1721 Yes 2.63 0.17 1.04 ± 0.14 PLEKHA6 Q9Y2H5 S@LQLPASPAPDPSPR S842 No 3.66 0.47 0.80 ± 0.09 PLEKHF1 Q96S99 QEEAEEQGAGS@PGQPAHLAR S227 No 2.65 0.48 1.01 ± 0.23 POGZ Q7Z3K3 SLDSEPSVPSAAKPPS@PEK S425 Yes 4.25 0.4 0.97 ± 0.14 PPHLN1 Q8NEY8 DNTFFRES@PVGR S133 Yes 2.52 0.11 0.68 ± 0.07 PPHLN1 Q8NEY8 DTS@PSSGSAVSSSK S205 Yes 3.82 0.11 0.86 ± 0.08 PPIG Q13427 ADRDQS@PFSK S687 Yes 2.57 0.18 1.01 ± 0.08 PPL O60437 GKYS@PTVQTR S14 No 2.81 0.12 0.59 ± 0.12 PPP1R10 Q96QC0 VLS@PTAAKPSPFEGK S313 Yes 2.85 0.18 0.92 ± 0.12 PPP1R12A O14974 LAS@TSDIEEKENR S507 Yes 2.72 0.11 0.69 ± 0.14 PPP1R12C Q9BZL4 IPEPES@PAKPNVPTASTAPPADSR S509 Yes 4.09 0.28 1.15 ± 0.27 PPP1R13L Q8WUF5 SESAPTLHPYSPLS@PK S113 Yes 3.89 0.36 1.14 ± 0.06 PPP4R2 Q9NY27 NHSDSSTSESEVSSVS@PLK S226 Yes 6.5 0.14 1.04 ± 0.31 PRKAR1A P10644 TDSREDEIS@PPPPNPVVK S83 Yes 3.52 0.22 0.82 ± 0.11 PRKCD Q05655 RSDS@ASSEPVGIYQGFEK S304 Yes 4.4 0.15 0.90 ± 0.26 PRPF38A Q8NAV1 VPS@PDHR S209 Yes 2.58 0.35 1.21 ± 0.10 PRPF4B Q13523 LCDFGSASHVADNDITPY@LVSR Y849 Yes 6.06 0.13 0.93 ± 0.41 PRPF4B Q13523 KKS@PIINESR S277 Yes 2.68 0.5 0.94 ± 0.09 PSMA3 P25788 ESLKEEDES@DDDNM S250 Yes 5.53 0.33 0.86 ± 0.10 PSMD2 Q13200 APVQPQQS@PAAAPGGTDEKPSGK S16 Yes 3.63 0.36 1.64 ± 0.45 PTDSS2 Q9BVG9 DAGGPRPES@PVPAGR S16 Yes 2.66 0.5 1.64 ± 0.15 PTPN3 P26045 SLS@VEHLETK S359 Yes 3.49 0.16 0.96 ± 0.26 PUM1 Q14671 RDS@LTGSSDLYK S709 Yes 4.09 0.25 0.68 ± 0.10 PUM2 Q8TB72 QAS@PTEVVER S182 Yes 2.62 0.31 0.85 ± 0.28 PURB Q96QR8 RGGGSGGGEES@EGEEVDED S304 Yes 5.19 0.28 0.89 ± 0.14 PWWP2A Q96N64 S@PEAVGPELEAEEK S81 Yes 3.78 0.57 0.86 ± 0.07 PYGB P11216 KQIS@VR S15 Yes 2.53 0.1 1.06 ± 0.10 PYGO2 Q9BRQ0 GGGT@PDANSLAPPGK T302 Yes 2.84 0.23 0.83 ± 0.10 RAB12 Q6IQ22 AGGGGGLGAGS@PALSGGQGR S21 Yes 4.89 0.24 0.85 ± 0.13 RAB3GAP Q9H2M9 ADFS@PFGNSQGPSR S450 Yes 3.84 0.27 0.93 ± 0.19 2 RACGAP1 Q9H0H5 S@IGSAVDQGNESIVAK S203 Yes 4.59 0.11 1.16 ± 0.28 RALGAPA1 Q6GYQ0 QKT@VDIDDAQILPR T754 Yes 3.17 0.37 0.84 ± 0.21 RALY Q9UKM9 GRLS@PVPVPR S135 Yes 2.8 0.65 1.10 ± 0.06 RANBP2 P49792 HSTPS@PTR S781 Yes 2.8 0.11 1.13 ± 0.08 RANBP2 P49792 AVVS@PPKFVFGSESVK S2510 Yes 2.77 0.43 1.57 ± 0.37 RANBP3 Q9H6Z4 VLS@PPKLNEVSSDANR S333 Yes 2.66 0.44 0.94 ± 0.17 RAP1GAP P47736 AAGISLIVPGKS@PTR S484 Yes 3.69 0.22 0.99 ± 0.20 RBM15 Q96T37 SLS@PGGAALGYR S294 Yes 3.09 0.18 0.89 ± 0.15 RBM15 Q96T37 DRT@PPLLYR T568 Yes 2.91 0.36 1.07 ± 0.33 RBM25 P49756 LGASNS@PGQPNSVK S618 No 4.15 0.24 1.03 ± 0.21 RBM26 Q5T8P6 LNHS@PPQSSSR S127 Yes 3.61 0.27 1.26 ± 0.18 RBM33 Q96EV2 VKPAS@PVAQPK S775 No 2.81 0.5 1.15 ± 0.27 RBM39 Q14498 YRS@PYSGPK S97 Yes 3.28 0.16 1.08 ± 0.10 RBM39 Q14498 DKS@PVREPIDNLTPEER S136 Yes 3.27 0.43 1.27 ± 0.20 RBMXL1 P38159 DVYLS@PR S208 Yes 2.59 0.29 1.00 ± 0.08 RCC1 P18754 RS@PPADAIPK S11 Yes 2.57 0.24 0.92 ± 0.14 REPS2 Q8NFH8 HQAS@LIR S254 Yes 2.55 0.51 0.39 ± 0.09 RIF1 Q5UIP0 RDS@FDNCSLGESSK S1688 Yes 4.4 0.29 1.16 ± 0.19 RNF31 Q96EP0 RLS@APLPSSCGDPEK S466 Yes 3.62 0.41 0.70 ± 0.04 RPL14 P50914 AALLKAS@PK S139 Yes 2.84 0.63 1.37 ± 0.18 RPL23A P62750 IRTS@PTFR S43 Yes 2.51 0.11 0.85 ± 0.07 RPL4 P36578 ILKS@PEIQR S295 Yes 2.69 0.39 1.26 ± 0.19 RPRD1B Q9NQG5 LSMEDSKS@PPPK S134 Yes 3.04 0.17 1.18 ± 0.20 RPRD2 Q5VT52 RMS@GEPIQTVESIR S1099 Yes 3.8 0.45 0.93 ± 0.17 RPS28 P62857 TGS@QGQCTQVR S23 Yes 3.3 0.44 0.52 ± 0.07 RPS3 P23396 DEILPTT@PISEQK T221 Yes 3.25 0.12 1.13 ± 0.14 RPS6KA1 Q15418 KLPST@TL T733 Yes 2.84 0.15 1.27 ± 0.16 RRBP1 Q9P2E9 NTDVAQS@PEAPKQEAPAK S615 Yes 4.88 0.35 0.99 ± 0.21

143

Appendix 1. Quantified phosphopeptides (continued).

RRM2 P31350 VPLAPITDPQQLQLS@PLK S20 Yes 5.69 0.45 1.54 ± 0.14 RSL1D1 O76021 AAESETPGKS@PEK S427 Yes 3.03 0.17 0.93 ± 0.10 RSL1D1 O76021 ATNES@EDEIPQLVPIGK S361 Yes 2.67 0.18 1.10 ± 0.23 RTKN Q9BST9 TFS@LDAVPPDHSPR S520 Yes 2.78 0.13 1.05 ± 0.19 SAFB Q15424 SVVS@FDKVK S604 Yes 3.08 0.43 1.03 ± 0.17 SCAMP3 O14828 KLS@PTEPK S76 Yes 2.98 0.18 0.67 ± 0.11 SCRIB Q14160 MKS@LEQDALR S1508 Yes 3.03 0.31 0.82 ± 0.08 SEC16A O15027 FANLT@PSR T1876 No 2.68 0.19 1.12 ± 0.62 SEC16A O15027 LLPSAPQTLPDGPLAS@PAR S1786 No 5.75 0.49 1.67 ± 0.20 SEPT2 Q15019 IYHLPDAES@DEDEDFKEQTR S218 Yes 3.65 0.34 1.00 ± 0.19 SERBP1 Q8NC51 DELTES@PKYIQK S234 Yes 3.69 0.13 1.02 ± 0.09 SET Q01105 RQS@PLPPQK S7 Yes 2.69 0.41 1.07 ± 0.13 SETPt9 Q9UHD8 LGDS@SGPALKR S22 No 2.51 0.14 0.69 ± 0.13 SETPt9 Q9UHD8 HVDSLSQRS@PK S85 Yes 3.39 0.1 1.11 ± 0.24 SETPt9 Q9UHD8 S@FEVEEVETPNSTPPR S30 Yes 5.4 0.54 1.17 ± 0.18 SGPP1 Q9BX95 RNS@LTGEEGQLAR S112 Yes 3.74 0.24 0.62 ± 0.06 SGTA O43765 SRTPS@ASNDDQQE S305 Yes 4.22 0.14 1.43 ± 0.11 SH3BP1 Q9Y3L3 TESEVPPRPAS@PK S544 Yes 2.98 0.45 2.06 ± 0.08 SH3BP2 P78314 SFS@FEKPR S427 No 2.7 0.3 0.91 ± 0.08 SH3BP4 Q9P0V3 SYS@LSELSVLQAK S246 Yes 5.19 0.13 1.01 ± 0.15 SH3GL1 Q99961 IAASSS@FR S288 Yes 2.6 0.2 0.66 ± 0.07 SH3GL1 Q99961 IAASS@SFR S287 Yes 2.67 0.1 0.68 ± 0.08 SHROOM3 Q8TF72 HSS@LELGR S676 No 3.1 0.2 0.64 ± 0.13 SHROOM3 Q8TF72 EDS@LPEESSAPDFANLK S1440 No 4.06 0.24 0.66 ± 0.30 SLC38A1 Q9H2H9 S@LTNSHLEK S52 Yes 2.83 0.17 0.94 ± 0.10 SLC4A1AP Q9BWU0 KPALPVS@PAAR S82 Yes 2.68 0.6 1.26 ± 0.39 SLC9A1 P19634 IGS@DPLAYEPK S703 Yes 4.39 0.42 0.94 ± 0.12 SLC9A3R1 O14745 EALAEAALES@PRPALVR S280 Yes 3.06 0.49 1.18 ± 0.24 VTPTEEHVEGPLPS@PVTNGTSPAQLNGG SLC9A3R2 Q15599 S254 Yes 4.93 0.11 1.08 ± 0.24 SACSSR SLTM Q9NWH9 DGQDAIAQS@PEKESK S289 Yes 4.16 0.36 0.93 ± 0.10 SLU7 O95391 LVEQANS@PK S215 Yes 2.71 0.3 0.86 ± 0.08 SMARCA4 P51532 GRPPAEKLS@PNPPNLTK S1452 Yes 3.74 0.32 1.12 ± 0.20 SMARCC2 Q8TAQ2 TLTDEVNS@PDSDRR S283 Yes 3.4 0.14 0.96 ± 0.13 SNRNP70 P08621 YDERPGPS@PLPHR S226 Yes 3.85 0.52 1.26 ± 0.13 SNTB2 Q13425 GLGPPS@PPAPPR S95 Yes 2.55 0.55 1.48 ± 0.00 SON P18583 ESDQTLAALLS@PK S1697 Yes 3.52 0.48 1.01 ± 0.23 SON P18583 SVESTS@PEPSK S283 Yes 2.85 0.1 1.14 ± 0.14 SPEN Q96T58 SQS@PVHLR S727 Yes 2.6 0.23 1.24 ± 0.09 SPTBN1 Q01082 RPPS@PEPSTK S2102 Yes 3.14 0.28 1.05 ± 0.07 SQSTM1 Q13501 SRLT@PVSPESSSTEEK T269 Yes 2.79 0.14 0.42 ± 0.07 SRA1 B3KNT3 S@PPVGSGPASGVEPTSFPVESEAR S87 No 3.64 0.12 1.11 ± 0.30 SRRM1 Q8IYB3 KETES@EAEDNLDDLEK S874 Yes 3.86 0.15 0.88 ± 0.10 SRRM1 Q8IYB3 VSHS@PPPK S638 Yes 2.58 0.43 0.95 ± 0.08 SRRM1 Q8IYB3 RS@PSLSSK S653 Yes 2.63 0.16 1.04 ± 0.08 SRRM1 Q8IYB3 KAAS@PSPQSVR S738 Yes 3.85 0.12 1.04 ± 0.05 SRRM1 Q8IYB3 RYS@PPIQR S597 Yes 2.58 0.26 1.14 ± 0.09 SRRM1 Q8IYB3 EKT@PELPEPSVK T220 Yes 2.76 0.35 1.22 ± 0.08 SRRM1 Q8IYB3 APQTS@SSPPPVR S694 Yes 2.56 0.37 1.29 ± 0.09 SRRM1 Q8IYB3 APQTSS@SPPPVR S695 Yes 3.22 0.23 1.30 ± 0.12 SRRM1 Q8IYB3 RES@PSPAPKPR S450 Yes 3.61 0.18 1.34 ± 0.07 SRRM1 Q8IYB3 TAS@PPPPPK S616 Yes 3.29 0.24 1.50 ± 0.21 SRRM1 Q8IYB3 VPKPEPIPEPKEPS@PEK S260 Yes 3.54 0.43 1.59 ± 0.22 SRRM2 Q9UQ35 ELSNS@PLRENSFGSPLEFR S1320 Yes 4.01 0.14 0.88 ± 0.14 SRRM2 Q9UQ35 SSGHSSSELS@PDAVEK S1387 Yes 5.2 0.12 0.93 ± 0.16 SRRM2 Q9UQ35 SGMS@PEQSR S1132 Yes 2.59 0.29 0.95 ± 0.06 SRRM2 Q9UQ35 TPAAAAAMNLAS@PR S2272 Yes 4.63 0.56 1.04 ± 0.14 SRRM2 Q9UQ35 SRTS@PITR S1975 Yes 2.51 0.21 1.05 ± 0.08 SRRM2 Q9UQ35 QSHSES@PSLQSK S1083 Yes 2.65 0.14 1.07 ± 0.07 SRRM2 Q9UQ35 GEFSAS@PMLK S1124 Yes 2.72 0.32 1.08 ± 0.19 SRRM2 Q9UQ35 SDTSS@PEVR S1073 Yes 2.55 0.14 1.09 ± 0.20

144

Appendix 1. Quantified phosphopeptides (continued).

SRRM2 Q9UQ35 SRAS@PVSR S1916 Yes 2.85 0.36 1.12 ± 0.07 SRRM2 Q9UQ35 TVART@PLGQR T1531 Yes 3.1 0.3 1.13 ± 0.06 SRRM2 Q9UQ35 SCFESS@PDPELK S876 Yes 4.26 0.11 1.13 ± 0.17 SRRM2 Q9UQ35 SRTS@PVSR S1893 Yes 2.64 0.13 1.18 ± 0.05 SRRM2 Q9UQ35 SRTS@PVTR S1987 Yes 2.67 0.14 1.20 ± 0.08 SRRM2 Q9UQ35 QGSITSPQANEQSVT@PQRR T866 Yes 2.58 0.12 1.20 ± 0.10 SRRM2 Q9UQ35 VSGRTS@PPLLDR S2398 Yes 3.35 0.12 1.22 ± 0.14 SRRM2 Q9UQ35 SRT@PPSAPSQSR T2409 Yes 2.83 0.11 1.22 ± 0.15 SRRM2 Q9UQ35 RVPS@PTPAPK S2581 Yes 2.7 0.16 1.32 ± 0.13 SRRM2 Q9UQ35 CRS@PGMLEPLGSSR S2132 Yes 2.79 0.53 1.32 ± 0.37 SRRM2 Q9UQ35 ALPQT@PRPR T1492 Yes 2.76 0.46 1.47 ± 0.31 MGQAPSQSLLPPAQDQPRS@PVPSAFSD SRRM2 Q9UQ35 S2449 Yes 3.77 0.16 1.52 ± 0.37 QSR SRRM2 Q9UQ35 AQT@PPGPSLSGSK T1003 Yes 3.06 0.37 1.76 ± 0.03 SRRT Q9BXP5 HELS@PPQKR S74 Yes 2.57 0.41 1.07 ± 0.11 SRSF11 Q05519 LNHVAAGLVS@PSLK S207 Yes 5.04 0.12 0.87 ± 0.15 SRSF11 Q05519 KPIETGS@PK S449 Yes 2.87 0.1 0.91 ± 0.10 SRSF6 Q13247 SNS@PLPVPPSK S303 Yes 2.94 0.19 1.41 ± 0.16 SRSF9 Q13242 GS@PHYFSPFRPY S211 Yes 2.72 0.24 1.08 ± 0.56 SSB P05455 FAS@DDEHDEHDENGATGPVK S366 Yes 3.24 0.63 1.02 ± 0.18 SSH3 Q8TE77 RQS@FAVLR S37 Yes 3.01 0.32 0.49 ± 0.07 SSH3 Q8TE77 S@PPGSGASTPVGPWDQAVQR S9 Yes 4.98 0.26 0.79 ± 0.20 STAT3 P40763 FICVTPTTCSNTIDLPMS@PR S727 Yes 5.03 0.41 0.62 ± 0.05 STAT5A P42229 AVDGY@VKPQIK Y694 Yes 2.84 0.39 0.32 ± 0.03 STIM1 Q13586 LPDS@PALAK S575 Yes 2.77 0.4 0.70 ± 0.02 STMN1 P16949 KS@HEAEVLK S63 Yes 2.51 0.4 0.89 ± 0.05 STMN1 P16949 ASGQAFELILS@PR S25 Yes 3.26 0.37 1.13 ± 0.30 STMN1 P16949 AS@GQAFELILSPR S16 Yes 3.65 0.54 1.14 ± 0.41 STMN1 P16949 SKESVPEFPLS@PPK S38 Yes 2.69 0.44 1.31 ± 0.25 STRN3 Q13033 NLEQILNGGES@PKQK S229 Yes 3.91 0.5 0.64 ± 0.02 STRN4 Q9NRL3 S@LELNGAVEPSEGAPR S206 Yes 5.51 0.56 1.14 ± 0.15 STUB1 Q9UNE7 LGAGGGS@PEKSPSAQELK S19 Yes 3.8 0.34 1.60 ± 0.33 SYAP1 Q96A49 EQDLPLAEAVRPKT@PPVVIK T248 Yes 3.19 0.37 0.89 ± 0.09 TACC2 O95359 KS@TDSVPISK S2072 Yes 2.82 0.15 1.27 ± 0.01 TACC2 O95359 VQNS@PPVGR S2226 Yes 2.88 0.45 1.38 ± 0.14 LFAGSLAPLLQPGAAGGEIPAVQASSGS@ TACC2 O95359 S1313 Yes 4.18 0.17 1.38 ± 0.25 PK TACC2 O95359 SRQELASGLPS@PAATQELPVER S1562 Yes 4.04 0.21 1.44 ± 0.21 TACC2 O95359 MQES@PKLPQQSYNFDPDTCDESVDPFK S2359 Yes 5.75 0.22 1.50 ± 0.12 TACC2 O95359 TLPLTTAPEAGEVTPSDSGGQEDS@PAK S2256 Yes 5.03 0.28 1.57 ± 0.00 TACC2 O95359 HPGASEAADGCS@PLWGLSK S1025 Yes 3.7 0.18 1.98 ± 0.38 TBC1D10B Q4KMP7 HGAPAAPS@PPPR S7 No 2.88 0.61 1.26 ± 0.27 TBC1D10B Q4KMP7 AAGGAPS@PPPPVR S403 No 2.59 0.54 1.39 ± 0.28 TBC1D15 Q8TC07 KDS@SSVVEWTQAPK S70 Yes 4.89 0.12 1.45 ± 0.32 TBC1D4 O60343 HAS@APSHVQPSDSEK S341 Yes 3.66 0.15 2.58 ± 0.76 TCEA1 P23193 KKEPAITSQNS@PEAR S100 Yes 4.73 0.33 1.23 ± 0.27 KSAEPSANTTLVS@ETEEEGSVPAFGAAA TCOF1 Q13428 S171 Yes 4.84 0.12 0.90 ± 0.14 K TCOF1 Q13428 KLS@GDQPAAR S1350 Yes 2.76 0.52 0.97 ± 0.06 TCOF1 Q13428 TSQVGAASAPAKES@PR S381 Yes 3.85 0.37 1.06 ± 0.19 TCOF1 Q13428 TVANLLSGKS@PR S156 Yes 3.24 0.3 1.06 ± 0.23 TCOF1 Q13428 AALAPAKES@PR S906 Yes 2.75 0.59 1.09 ± 0.11 TCOF1 Q13428 KGAAPT@PPGK T914 Yes 3.14 0.56 1.11 ± 0.15 TCOF1 Q13428 PTSS@PAKGPPQK S583 Yes 3.12 0.1 1.39 ± 0.16 TCOF1 Q13428 KLGAGEGGEASVS@PEK S1378 Yes 4.95 0.19 1.44 ± 0.00 TERF2 Q15554 DLVLPTQALPAS@PALK S323 Yes 4.64 0.4 1.09 ± 0.09 THRAP3 Q9Y2W1 IDISPST@FR T685 Yes 2.95 0.13 1.04 ± 0.13 THRAP3 Q9Y2W1 IDIS@PSTFR S682 Yes 2.96 0.21 1.07 ± 0.14 THRAP3 Q9Y2W1 ASAVSELS@PR S243 Yes 3.17 0.25 1.08 ± 0.06 THRAP3 Q9Y2W1 SPALKS@PLQSVVVR S253 Yes 3.44 0.31 1.62 ± 0.00 TJAP1 Q5JTD0 KDS@LTQAQEQGNLLN S545 Yes 3.95 0.14 0.59 ± 0.05

145

Appendix 1. Quantified phosphopeptides (continued).

TJP1 Q07157 IDS@PGFKPASQQK S912 Yes 3.28 0.26 0.44 ± 0.09 TJP2 Q9UDY2 RAAS@SDQLR S978 Yes 2.73 0.1 0.70 ± 0.12 TJP2 Q9UDY2 GRS@IDQDYER S244 Yes 3.69 0.57 0.75 ± 0.07 TJP3 O95049 S@PGGGSEANGLALVSGFK S164 Yes 5.66 0.23 0.56 ± 0.09 TK1 P04183 KLFAPQQILQCS@PAN S231 Yes 4.33 0.6 0.64 ± 0.16 TLE3 Q04726 ESSANNSVS@PSESLR S203 Yes 4.76 0.1 0.78 ± 0.07 TMPO P42166 SS@TPLPTISSSAENTR S159 Yes 3.7 0.37 0.68 ± 0.04 TMPO P42166 SST@PLPTISSSAENTR T160 Yes 3.17 0.28 0.78 ± 0.08 TMPO P42166 S@STPLPTISSSAENTR S158 Yes 3.62 0.31 0.82 ± 0.10 TMPO P42166 GPPDFSSDEEREPT@PVLGSGAAAAGR T74 Yes 4.29 0.17 1.09 ± 0.39 TMPO P42167 AKT@PVTLK T208 Yes 2.64 0.29 1.08 ± 0.13 TMSB10 P63313 TET@QEKNTLPTK T23 Yes 3.96 0.1 0.38 ± 0.03 TMX1 Q9H3N1 KVEEEQEADEEDVS@EEEAESK S247 Yes 6.08 0.27 0.93 ± 0.15 TNKS1BP1 Q9C0C2 S@QEADVQDWEFR S836 Yes 4.31 0.49 0.58 ± 0.09 TNKS1BP1 Q9C0C2 NRS@AEEGELAESK S1666 Yes 4.73 0.65 0.63 ± 0.06 TNKS1BP1 Q9C0C2 RDS@LGTYSSR S872 Yes 2.68 0.32 0.68 ± 0.09 TNKS1BP1 Q9C0C2 HNGSLS@PGLEAR S1385 Yes 3.49 0.14 0.70 ± 0.10 TNKS1BP1 Q9C0C2 VSGAGFS@PSSK S1138 Yes 3.56 0.14 0.74 ± 0.07 TNKS1BP1 Q9C0C2 VSGAGFSPS@SK S1140 Yes 3.14 0.13 0.74 ± 0.07 TNKS1BP1 Q9C0C2 VSGAGFSPSS@K S1141 Yes 3.02 0.52 0.76 ± 0.08 TNKS1BP1 Q9C0C2 RFS@EGVLQSPSQDQEK S429 Yes 3.96 0.36 0.79 ± 0.12 TNKS1BP1 Q9C0C2 KEVLAS@PDRLWGSR S178 Yes 3.07 0.57 0.81 ± 0.17 TNKS1BP1 Q9C0C2 TEAQDLCRAS@PEPPGPESSSR S672 Yes 4.03 0.43 0.89 ± 0.16 PAVEASTGGEATQETGKEEAGKEEPPPLT TNKS1BP1 Q9C0C2 T131 No 4.45 0.44 0.98 ± 0.27 @PPAR TNS3 Q68CZ2 KLS@LGQYDNDAGGQLPFSK S776 Yes 4.41 0.26 0.35 ± 0.09 TNS3 Q68CZ2 GVGSGPHPPDTQQPS@PSK S660 Yes 3.18 0.11 0.59 ± 0.15 TOP2A P11388 S@VVSDLEADDVK S1374 Yes 4.61 0.19 0.83 ± 0.13 TOP2A P11388 IKNENTEGS@PQEDGVELEGLK S1247 Yes 4.09 0.21 1.06 ± 0.42 TOR1AIP1 Q5JTV8 LQQQHSEQPPLQPS@PVMTR S143 Yes 3.86 0.11 1.41 ± 0.57 TP53I11 O14683 KHS@QTDLVSR S2 No 3.03 0.3 0.81 ± 0.07 TPD52 P55327 ASAAFSSVGS@VITK S119 No 4.7 0.18 0.30 ± 0.06 TPD52 P55327 S@FEEKVENLK S136 Yes 3.31 0.51 0.73 ± 0.11 TPD52 P55327 LEDVKNS@PTFK S131 Yes 3.36 0.15 1.09 ± 0.13 TPD52L1 Q16890 HSIS@MPAMR S137 Yes 2.65 0.13 0.48 ± 0.10 VGGTNPNGGS@FEEVLSSTAHASAQSLA TPD52L1 Q16890 S174 Yes 4 0.2 0.57 ± 0.10 GGSR TPD52L1 Q16890 NSPTFKS@FEER S149 Yes 3.19 0.33 0.74 ± 0.09 TPD52L2 O43399 NSATFKS@FEDR S166 Yes 2.51 0.29 0.81 ± 0.08 TPI1 P60174 KQS@LGELIGTLNAAK S21 Yes 3.76 0.42 0.78 ± 0.14 TPR P12270 TDGFAEAIHS@PQVAGVPR S2141 Yes 4.7 0.28 0.94 ± 0.15 TPR P12270 TVPST@PTLVVPHR T2123 Yes 2.66 0.1 1.08 ± 0.32 GDLGPASPS@QELGSQPVPGGDGAPALG TPRN Q4KMQ1 S176 No 3.51 0.15 1.06 ± 0.11 K TRA2A Q13595 AHT@PTPGIYMGR T202 Yes 2.88 0.2 1.01 ± 0.19 TRA2B P62995 RPHT@PTPGIYMGR T201 Yes 3.39 0.2 1.45 ± 0.07 TRAFD1 O14545 ALPSLNT@GSSSPR T323 Yes 2.63 0.58 0.63 ± 0.05 TRAFD1 O14545 ALPSLNTGSSS@PR S327 Yes 3.11 0.11 0.69 ± 0.04 STAPSAAASASASAAASS@PAGGGAEALE TRIM28 Q13263 S50 Yes 3.93 0.15 0.68 ± 0.28 LLEHCGVCR TRIM28 Q13263 SRS@GEGEVSGLMR S473 Yes 3.63 0.22 1.07 ± 0.12 TRIM3 O75382 RPSS@MYSTGGK S455 Yes 3.01 0.13 1.44 ± 0.36 TRIM3 O75382 REDS@PGPEVQPMDK S7 Yes 4.33 0.68 1.73 ± 0.32 TRIM3 O75382 VKS@PGGPGSHVR S437 No 2.75 0.34 2.31 ± 0.00 TRIM47 Q96LD4 GGIPAS@PIDPFQSR S588 Yes 3.18 0.56 1.17 ± 0.23 TRPS1 Q9UHF7 GSSERGS@PIEK S1066 No 2.98 0.15 1.28 ± 0.17 TRPS1 Q9UHF7 SKTDLLVNDNPDPAPLS@PELQDFK S216 Yes 6.05 0.61 1.32 ± 0.51 RVS@GPDPKPGSNCSPAQSVLSEVPSVPT UBA1 P22314 S13 Yes 3.11 0.22 1.26 ± 0.20 NGMAK UBAP2L Q14157 NPSDSAVHS@PFTK S416 Yes 2.96 0.12 0.84 ± 0.12 UBE2J1 Q9Y385 RLS@TSPDVIQGHQPR S266 Yes 3.36 0.12 0.63 ± 0.11

146

Appendix 1. Quantified phosphopeptides (continued).

HAPSPEPAVQGT@GVAGVPEESGDAAAI UNG P13051 T31 Yes 4.62 0.15 1.31 ± 0.09 PAK HAPS@PEPAVQGTGVAGVPEESGDAAAI UNG P13051 S23 Yes 5.41 0.14 1.44 ± 0.27 PAK UPF1 Q92900 AYQHGGVTGLS@QY S1127 Yes 3.38 0.18 1.00 ± 0.00 USP14 P54578 AS@GEMASAQYITAALR S143 Yes 3.88 0.26 0.62 ± 0.40 USP24 Q9UPU5 VSDQNS@PVLPK S1297 No 3.23 0.37 0.82 ± 0.07 USP47 Q96K76 NSLIYELFS@VMAHSGSAAGGHYYACIK S491 No 3.18 0.18 0.68 ± 0.11 VIPAR Q9H9C1 TRPGS@FQSLSDALSDTPAK S121 Yes 4.68 0.17 0.86 ± 0.12 WAC Q9BTA9 QQGHEPVS@PR S511 Yes 2.68 0.26 1.16 ± 0.18 WDR26 Q9H7D7 RLS@QSDEDVIR S121 Yes 3.79 0.16 0.65 ± 0.10 WDR4 P57081 RS@PPPGPDGHAK S391 Yes 3.16 0.49 1.35 ± 0.61 WDR77 Q9BQA1 KET@PPPLVPPAAR T5 Yes 2.68 0.41 1.74 ± 0.30 WHSC2 Q9H3P2 EASRPPEEPSAPS@PTLPAQFK S384 No 3.05 0.17 0.63 ± 0.25 WIPF2 Q8TF74 EGPPAPPPVKPPPS@PVNIR S235 Yes 2.73 0.43 1.55 ± 0.60 WRNIP1 Q96S55 RPAAAAAAGSAS@PR S153 Yes 2.57 0.17 1.11 ± 0.14 ZC3H14 Q6PJT7 DLVQPDKPAS@PK S515 Yes 2.66 0.46 0.99 ± 0.13 ZC3H14 Q6PJT7 RPS@LPPSK S343 No 2.68 0.42 1.29 ± 0.31 ZC3H18 Q86VM9 LGVSVS@PSR S534 Yes 3.44 0.1 0.97 ± 0.05 ZC3H18 Q86VM9 LGVSVSPS@R S536 Yes 3.05 0.23 0.97 ± 0.05 ZC3HC1 Q86WB0 SQDATFSPGSEQAEKS@PGPIVSR S344 Yes 3.86 0.27 0.95 ± 0.16 ZDHHC5 Q9C0B5 GDS@LKEPTSIAESSR S380 Yes 2.72 0.62 0.88 ± 0.14 ZFYVE19 Q96K21 EHQTSAYS@PPR S463 Yes 2.99 0.11 1.79 ± 0.38 ZMYM4 Q5VZL5 KKS@IVAVEPR S1181 Yes 3.04 0.4 0.64 ± 0.28 ZMYM4 Q5VZL5 CGGVEQAS@SSPR S1239 Yes 3.18 0.64 0.87 ± 0.24 ZMYND8 Q9ULU4 ELSESVQQQSTPVPLIS@PKR S547 Yes 3.41 0.5 0.85 ± 0.35 ZMYND8 Q9ULU4 TPPSTTVGSHS@PPETPVLTR S756 Yes 3.26 0.23 1.11 ± 0.14 ZNF185 O15231 RES@CGSSVLTDFEGKDVATK S233 No 4.14 0.12 0.94 ± 0.25 ZNF326 Q5BKZ1 ISLSKS@PTK S270 Yes 2.62 0.12 1.08 ± 0.18 ZNF532 Q9HCE3 ET@EASSINLSVYEPFK T205 Yes 2.81 0.16 0.65 ± 0.11 ZNF687 Q8N1G0 HGLQLGAQS@PGR S1057 Yes 4.14 0.52 0.80 ± 0.18 ZNF828 Q96JM3 KPSGS@PDLWK S445 Yes 3.19 0.13 0.80 ± 0.23 ZNF828 Q96JM3 TAPTLS@PEHWK S405 Yes 3.18 0.24 0.90 ± 0.22 ZNF828 Q96JM3 LAPVPSPEPQKPAPVS@PESVK S214 Yes 4.17 0.15 1.23 ± 0.28 ZNRF2 Q8NHG8 DRPVGGS@PGGPR S135 Yes 2.52 0.32 1.12 ± 0.16 ZRANB2 O95218 YNLDAS@EEEDSNKK S188 Yes 3.68 0.33 0.98 ± 0.16 ZYX Q15942 GPPASS@PAPAPK S259 Yes 3 0.11 0.85 ± 0.07 *: Phosphorylation sites are indicated by "@" after the amino acid in the peptide sequence.

147

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells.

Degradation Rate Relative Synthesis Rate Constant Abundance

UniProt Gene Name Peptide Sequence Accession SKBR3_ SKBR3_ SKBR3_ SKBR3 SKBR3 SKBR3 shVPS4B shVPS4B shVPS4B

RPLP1 P05386 AAGVNVEPFWPGLFAK 0.078 0.074 0.019 0.023 4.08 3.67 FUS P35637 AAIDWFDGK 0.047 0.053 0.040 0.039 2.25 2.77 KRT19 P08727 AALEDTLAETEAR 0.049 0.086 0.029 0.025 2.88 4.09 RPS25 P62851 AALQELLSK 0.054 0.051 0.024 0.035 2.95 2.64 PRKDC P78527 AALSALESFLK 0.035 0.032 0.026 0.025 2.74 2.77 EIF4G1 Q04637 AALSEEELEK 0.090 0.079 0.058 0.058 3.03 2.58 ALDOA P04075 AAQEEYVK 0.055 0.068 0.019 0.041 3.07 2.91 HSPD1 P10809 AAVEEGIVLGGGCALLR 0.045 0.056 0.025 0.018 3.50 4.29 EWSR1 Q01844 AAVEWFDGK 0.038 0.035 0.027 0.027 2.34 2.82 ENO1 P06733 AAVPSGASTGIYEALELR 0.062 0.043 0.016 0.033 3.93 2.62 LMNA P02545 AAYEAELGDAR 0.026 0.038 0.024 0.025 1.92 2.90 SLC25A5 P05141 AAYFGIYDTAK 0.030 0.040 0.019 0.025 2.69 3.50 PGK1 P00558 ACANPAAGSVILLENLR 0.054 0.040 0.021 0.033 3.74 2.64 GNB2 P62879 ACGDSTLTQITAGLDPVGR 0.068 0.089 0.040 0.044 2.88 3.64 FASN P49327 ACLDTAVENMPSLK 0.066 0.045 0.023 0.038 3.74 2.48 RPS3A P61247 ACQSIYPLHDVFVR 0.078 0.072 0.024 0.027 4.10 3.66 SND1 Q7KZF4 ADDADEFGYSR 0.096 0.082 0.031 0.041 3.82 3.21 FLNB B2ZZ83 ADDTDSQSWR 0.055 0.072 0.033 0.044 2.28 3.00 PRDX1 Q06830 ADEGISFR 0.044 0.030 0.021 0.033 3.24 2.48 MYH10 P35580 ADEWLMK 0.054 0.055 0.023 0.038 2.59 2.33 MYH10 P35580 ADFCIIHYAGK 0.047 0.083 0.019 0.023 2.59 2.68 RPS8 P62241 ADGYVLEGK 0.047 0.054 0.034 0.038 2.44 2.51 FLNB O75369 ADIEMPFDPSK 0.055 0.071 0.038 0.047 2.41 2.89 HSP90AB2P Q58FF8 ADLINNLGTIAK 0.055 0.052 0.024 0.024 3.31 2.93 FLII Q13045 ADLTALFLPR 0.070 0.086 0.057 0.048 2.58 3.10 TRIM28 Q13263 ADVQSIIGLQR 0.038 0.042 0.021 0.028 3.08 3.09 KRT3 P12035 AEAEALYQTK 0.038 0.026 0.022 0.031 2.80 2.34 KRT79 Q5XKE5 AEAEAWYQTK 0.041 0.088 0.025 0.025 2.08 3.48 KRT8 P05787 AEAESM~YQIK 0.077 0.044 0.027 0.035 3.90 3.28 KRT8 P05787 AEAESMYQIK 0.055 0.082 0.028 0.036 3.23 3.77 TUFM P49411 AEAGDNLGALVR 0.027 0.036 0.057 0.022 1.76 3.46 AEASSGDHPTDTEMKEEQ FKBP4 Q02790 0.103 0.060 0.019 0.021 5.22 3.27 K ANXA2 P07355 AEDGSVIDYELIDQDAR 0.056 0.108 0.023 0.017 2.36 4.35 CANX P27824 AEEDEILNR 0.056 0.071 0.024 0.019 3.84 4.67 RPL11 P62913 AEEILEK 0.048 0.054 0.021 0.021 2.98 3.00 ACAA1 P09110 AEELGLPILGVLR 0.026 0.025 0.025 0.018 2.62 3.30 ARHGDIA P52565 AEEYEFLTPVEEAPK 0.097 0.050 0.021 0.029 4.46 2.80 RPS3 P23396 AELNEFLTR 0.060 0.062 0.031 0.028 3.18 3.43 DIAPH1 O60610 AEMDEVER 0.073 0.087 0.042 0.039 3.05 3.39 UBA1 P22314 AENYDIPSADR 0.071 0.049 0.027 0.028 4.01 2.96 EIF5B O60841 AETPTAAEDDNEGDK 0.117 0.079 0.023 0.030 4.65 3.35 RDX P35241 AFAAQEDLEK 0.073 0.052 0.026 0.050 3.35 2.28 ACLY P53396 AFDSGIIPMEFVNK 0.071 0.104 0.065 0.056 2.53 3.45 RNPEP Q9H4A4 AFFPCFDTPAVK 0.042 0.030 0.014 0.026 3.09 2.67 SRSF3 P84103 AFGYYGPLR 0.043 0.049 0.025 0.027 2.99 3.37 HNRNPM P52272 AFITNIPFDVK 0.044 0.050 0.026 0.027 2.74 3.31 TRAP1 Q12931 AFLDALQNQAEASSK 0.046 0.060 0.030 0.024 3.33 3.81 RAB10 P61026 AFLTLAEDILR 0.044 0.066 0.036 0.031 2.33 3.16 HSPA1L P34931 AFYPEEISSMVLTK 0.109 0.053 0.026 0.035 4.02 2.62

148

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

ZC3H18 Q86VM9 AGAEDDEEKGEGTPR 0.090 0.070 0.028 0.043 3.52 3.13 MDH2 P40926 AGAGSATLSMAYAGAR 0.032 0.033 0.026 0.019 2.73 3.60 HADH Q16836 AGDEFVEK 0.013 0.021 0.071 0.025 0.77 2.86 RPS18 P62269 AGELTEDEVER 0.095 0.072 0.030 0.028 4.23 3.55 ACTA2 P62736 AGFAGDDAPR 0.074 0.097 0.025 0.034 3.24 3.81 LRRFIP1 Q32MZ4 AGGEELDEGVAK 0.072 0.076 0.027 0.041 3.20 2.77 HSPA4 P34932 AGGIETIANEYSDR 0.084 0.051 0.023 0.030 3.92 2.78 PKM2 P14618 AGKPVICATQMLESMIK 0.073 0.050 0.014 0.038 3.61 2.32 ACADVL P49748 AGLGSGLSLSGLVHPELSR 0.028 0.040 0.027 0.032 2.15 2.72 HIST1H2AA Q96QV6 AGLQFPVGR 0.024 0.019 0.024 0.028 2.73 3.16 GSTK1 Q9Y2Q3 AGMSAEQAQGLLEK 0.017 0.028 0.025 0.015 1.71 2.86 SYNCRIP O60506 AGPIWDLR 0.062 0.062 0.033 0.031 3.04 3.34 PGD P52209 AGQAVDDFIEK 0.046 0.034 0.014 0.032 3.12 2.33 ACTN4 O43707 AGTQIENIDEDFRDGLK 0.048 0.048 0.024 0.020 2.45 2.97 PDCD6 O75340 AGVNFSEFTGVWK 0.070 0.047 0.018 0.036 4.33 2.60 ATP5B P06576 AIAELGIYPAVDPLDSTSR 0.039 0.039 0.023 0.022 3.45 4.09 MRPS9 P82933 AIAYLFPSGLFEK 0.040 0.050 0.025 0.026 3.01 3.41 ST13 P50502 AIDLFTDAIK 0.066 0.054 0.039 0.050 2.83 2.37 CTSD P07339 AIGAVPLIQGEYM~IPCEK 0.044 0.047 0.031 0.050 2.16 2.33 CTSD P07339 AIGAVPLIQGEYMIPCEK 0.037 0.045 0.041 0.046 1.92 2.31 SF3B1 O75533 AIGPHDVLATLLNNLK 0.078 0.084 0.041 0.047 3.05 3.07 TUBB2A Q13885 AILVDLEPGTMDSVR 0.050 0.039 0.016 0.034 3.73 2.57 FASN P49327 AINCATSGVVGLVNCLR 0.105 0.046 0.018 0.037 5.30 2.64 RPL24 P83731 AITGASLADIMAK 0.059 0.084 0.024 0.029 3.02 2.82 PCBP2 Q15366 AITIAGIPQSIIECVK 0.079 0.060 0.030 0.042 3.38 2.76 PCBP1 Q15365 AITIAGVPQSVTECVK 0.074 0.065 0.025 0.042 3.43 2.79 RPSA P08865 AIVAIENPADVSVISSR 0.068 0.069 0.026 0.028 3.63 3.63 KRT7 P08729 AKLEAAIAEAEER 0.043 0.084 0.029 0.028 1.97 3.51 HIST1H1A Q02539 ALAAAGYDVEK 0.028 0.018 0.026 0.032 2.18 2.34 HIST1H1B P16401 ALAAGGYDVEK 0.018 0.010 0.021 0.027 2.33 2.60 TALDO1 P37837 ALAGCDFLTISPK 0.055 0.030 0.033 0.035 2.30 2.05 ALDOA P04075 ALANSLACQGK 0.038 0.070 0.030 0.029 2.46 2.76 CCT3 P49368 ALDDMISTLK 0.049 0.052 0.034 0.039 2.67 2.60 PSMC2 P35998 ALDEGDIALLK 0.052 0.059 0.042 0.038 2.30 2.59 ACTN1 P12814 ALDFIASK 0.045 0.061 0.019 0.033 2.25 2.73 PABPC1 P11940 ALDTMNFDVIK 0.065 0.072 0.021 0.037 3.41 3.10 KRT19 P08727 ALEAANGELEVK 0.046 0.083 0.027 0.028 2.61 3.68 MYH9 P35579 ALEEAMEQK 0.068 0.061 0.017 0.028 3.18 2.70 MARS P56192 ALEEGLTPQEICDK 0.058 0.059 0.024 0.031 3.13 2.91 CLTC Q00610 ALEHFTDLYDIK 0.044 0.037 0.024 0.029 2.74 2.45 MYH9 P35579 ALELDSNLYR 0.041 0.046 0.037 0.053 2.03 2.03 ACADVL P49748 ALEQFATVVEAK 0.020 0.031 0.024 0.030 1.85 2.32 IQGAP1 P46940 ALESGDVNTVWK 0.045 0.067 0.026 0.029 2.02 2.33 PGK1 P00558 ALESPERPFLAILGGAK 0.054 0.064 0.013 0.034 3.49 2.52 RUVBL1 Q9Y265 ALESSIAPIVIFASNR 0.044 0.046 0.019 0.022 3.72 3.22 MYL6B P14649 ALGQNPTNAEVLK 0.125 0.105 0.037 0.043 4.19 3.31 AIFM1 O95831 ALGTEVIQLFPEK 0.028 0.028 0.024 0.019 2.82 3.49 CCT4 P50991 ALIAGGGAPEIELALR 0.076 0.060 0.026 0.027 4.27 3.22 CCT6A P40227 ALQFLEEVK 0.051 0.050 0.034 0.036 2.56 2.64 MVP Q14764 ALQPLEEGEDEEK 0.041 0.072 0.030 0.041 2.18 2.77 TUBB P07437 ALTVPELTQQVFDAK 0.055 0.034 0.014 0.034 3.23 2.19 MYBBP1A Q9BQG0 ALVDILSEVSK 0.049 0.037 0.035 0.027 2.60 2.73 GNAS Q5JWF2 ALWEDEGVR 0.064 0.066 0.033 0.035 2.93 3.33 EZR P15311 APDFVFYAPR 0.084 0.075 0.043 0.047 3.33 2.84 HGS O14964 APDWVDAEECHR 0.088 0.094 0.041 0.059 3.17 3.04 HSP90AA1 P07900 APFDLFENR 0.057 0.050 0.032 0.031 3.23 3.21 DDX17 Q92841 APILIATDVASR 0.089 0.132 0.047 0.050 3.25 4.48

149

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

RPL4 P36578 APIRPDIVNFVHTNLR 0.038 0.065 0.025 0.032 3.02 3.27 CAPN1 P07384 APSDLYQIILK 0.044 0.029 0.020 0.037 2.56 2.00 FLNB O75369 APSVATVGSICDLNLK 0.056 0.104 0.015 0.032 2.77 3.98 CANX P27824 APVPTGEVYFADSFDR 0.042 0.050 0.022 0.022 2.91 3.77 ASPH Q12797 AQCEDDLAEK 0.094 0.077 0.035 0.021 3.54 3.36 HADHB P55084 AQDEGLLSDVVPFK 0.021 0.031 0.025 0.019 1.96 3.22 AQFAQPEILIGTIPGAGGTQ ECHS1 P30084 0.023 0.034 0.024 0.020 2.70 3.91 R HSPA9 P38646 AQFEGIVTDLIR 0.078 0.060 0.028 0.030 4.59 3.97 LMNA P02545 AQHEDQVEQYK 0.035 0.061 0.022 0.029 2.13 3.47 KRT18 P05783 AQIFANTVDNAR 0.092 0.123 0.028 0.026 4.56 5.22 PRKCSH P14314 AQQEQELAADAFK 0.043 0.040 0.019 0.022 2.65 2.85 KRT18 P05783 AQYDELAR 0.109 0.124 0.025 0.024 5.09 5.29 KRT75 O95678 AQYEDIANR 0.076 0.105 0.032 0.029 3.97 4.97 HSPA1A P08107 ARFEELCSDLFR 0.100 0.057 0.036 0.032 3.53 2.82 HSPA2 P54652 ARFEELNADLFR 0.117 0.093 0.032 0.033 4.29 3.98 GOT2 P00505 ASAELALGENSEVLK 0.031 0.034 0.017 0.014 3.13 3.44 EIF6 P56537 ASFENNCEIGCFAK 0.070 0.079 0.030 0.039 3.12 2.93 HK2 P52789 ASGCEGEDVVTLLK 0.093 0.079 0.039 0.060 3.60 3.16 HIST1H1C P16403 ASGPPVSELITK 0.026 0.019 0.027 0.031 2.51 2.72 LGALS3BP Q08380 ASHEEVEGLVEK 0.020 0.024 0.022 0.036 1.23 1.96 ACTN1 P12814 ASIHEAWTDGK 0.037 0.058 0.027 0.026 2.15 3.00 KRT8 P05787 ASLEAAIADAEQR 0.089 0.107 0.026 0.027 4.76 5.04 COPA P53621 ASNLENSTYDLYTIPK 0.046 0.049 0.027 0.027 2.72 2.68 ACADVL P49748 ASNTAEVFFDGVR 0.031 0.033 0.039 0.028 1.97 2.70 COX4I1 P13073 ASWSSLSMDEK 0.034 0.053 0.014 0.051 2.98 2.66 HSPA1L P34931 ATAGDTHLGGEDFDNR 0.090 0.056 0.031 0.034 3.45 2.84 PRDX1 Q06830 ATAVMPDGQFK 0.045 0.030 0.021 0.033 3.15 2.52 HIST1H1B P16401 ATGPPVSELITK 0.018 0.013 0.019 0.019 2.64 3.04 MYBBP1A Q9BQG0 ATLQEILPEVLK 0.047 0.040 0.039 0.033 2.56 2.57 ACOT7 O00154 ATLWYVPLSLK 0.062 0.036 0.016 0.026 3.20 2.79 HSD17B4 P51659 AVANYDSVEEGEK 0.036 0.039 0.032 0.028 2.23 3.06 TUBA1A Q71U36 AVCMLSNTTAIAEAWAR 0.048 0.039 0.015 0.032 3.43 2.31 ATP5A1 P25705 AVDSLVPIGR 0.024 0.026 0.026 0.021 2.76 3.60 LGALS3BP Q08380 AVDTWSWGER 0.021 0.023 0.031 0.046 1.41 2.12 ATP1A1 P05023 AVFQANQENLPILK 0.058 0.051 0.027 0.041 3.06 2.74 DSTN P60981 AVIFCLSADK 0.059 0.036 0.016 0.035 2.79 2.16 S100A16 Q96FQ6 AVIVLVENFYK 0.089 0.122 0.035 0.038 3.63 4.32 CFL1 P23528 AVLFCLSEDK 0.059 0.050 0.019 0.038 3.02 2.33 TWF2 Q6IBS0 AVLPLLDAQQPCYLLYR 0.039 0.038 0.028 0.041 2.80 2.26 YWHAQ P27348 AVTEQGAELSNEER 0.057 0.048 0.021 0.033 3.38 2.84 YWHAB P31946 AVTEQGHELSNEER 0.044 0.046 0.037 0.029 2.38 2.58 FKBP4 Q02790 AWDIAIATMK 0.048 0.056 0.024 0.022 3.23 3.05 ILF3 Q12906 AYAALAALEK 0.029 0.033 0.036 0.032 1.76 2.15 ATP1B1 P05026 AYGENIGYSEK 0.037 0.060 0.029 0.040 2.10 2.51 ANXA2 P07355 AYTNFDAER 0.053 0.116 0.027 0.021 2.19 4.45 EPRS P07814 AYVDDTPAEQMK 0.061 0.031 0.019 0.039 2.87 2.05 C6orf115 Q9P1F3 CANLFEALVGTLK 0.039 0.035 0.028 0.034 2.16 2.12 PKM2 P14618 CCSGAIIVLTK 0.049 0.054 0.017 0.022 3.03 2.56 RAB3D O95716 CDLEDER 0.053 0.061 0.038 0.042 2.45 2.96 H2AFY O75367 CEELLEK 0.027 0.036 0.023 0.027 2.07 2.88 HSPD1 P10809 CEFQDAYVLLSEK 0.053 0.048 0.027 0.020 3.54 3.76 S100A11 P31949 CIESLIAVFQK 0.045 0.042 0.022 0.031 2.48 2.09 HSPD1 P10809 CIPALDSLTPANEDQK 0.057 0.055 0.024 0.023 4.07 4.22 RPL14 P50914 CMQLTDFILK 0.054 0.057 0.018 0.026 3.21 3.16 HSPA8 P11142 CNEIINWLDK 0.119 0.081 0.020 0.034 4.84 3.69 FUBP1 Q96AE4 CQHAAEIITDLLR 0.061 0.088 0.057 0.030 2.28 3.94 USP14 P54578 CTESEEEEVTK 0.063 0.048 0.015 0.032 3.52 2.67

150

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

FASN P49327 CTVFHGAQVEDAFR 0.084 0.042 0.024 0.039 4.15 2.48 VAPA Q9P0L0 CVFEMPNENDK 0.054 0.061 0.041 0.023 2.50 3.06 MFSD10 Q14728 DAADLLSPLALLR 0.029 0.044 0.029 0.021 2.07 3.12 CORO1B Q9BR76 DADPILISLR 0.057 0.051 0.037 0.031 2.84 3.30 DCI P42126 DADVQNFVSFISK 0.024 0.023 0.025 0.017 2.30 3.32 KRT19 P08727 DAEAWFTSR 0.041 0.074 0.030 0.028 2.61 3.57 ANXA6 P08133 DAFVAIVQSVK 0.052 0.041 0.025 0.040 3.92 3.36 HSPA9 P38646 DAGQISGLNVLR 0.073 0.060 0.027 0.028 4.34 4.05 HSPA8 P11142 DAGTIAGLNVLR 0.082 0.092 0.031 0.032 3.74 3.90 HSPA5 P11021 DAGTIAGLNVMR 0.055 0.071 0.028 0.020 3.14 3.94 HSPA1L P34931 DAGVIAGLNVLR 0.090 0.049 0.033 0.034 3.46 2.63 ANXA2 P07355 DALNIETAIK 0.047 0.101 0.029 0.017 1.97 4.03 CCT4 P50991 DALSDLALHFLNK 0.066 0.049 0.022 0.033 3.66 2.95 GANAB Q14697 DAQHYGGWEHR 0.035 0.041 0.018 0.029 2.68 3.14 ENO1 P06733 DATNVGDEGGFAPNILENK 0.058 0.045 0.014 0.032 3.79 2.64 HYOU1 Q9Y4L1 DAVITVPVFFNQAER 0.055 0.066 0.028 0.024 3.05 3.64 ARF4 P18085 DAVLLLFANK 0.068 0.062 0.034 0.042 2.82 2.48 ARF1 P84077 DAVLLVFANK 0.046 0.050 0.027 0.030 2.43 2.54 HIST1H4A P62805 DAVTYTEHAK 0.017 0.012 0.028 0.023 1.87 2.42 HYOU1 Q9Y4L1 DAVVYPILVEFTR 0.048 0.071 0.026 0.022 3.02 3.40 API5 Q9BZZ5 DAYQVILDGVK 0.026 0.048 0.017 0.031 2.58 2.55 TPI1 P60174 DCGATWVVLGHSER 0.046 0.030 0.016 0.034 3.34 2.30 ACAA1 P09110 DCLIPMGITSENVAER 0.030 0.022 0.023 0.017 2.92 3.40 ARPC2 O15144 DDETMYVESK 0.040 0.054 0.017 0.025 2.76 3.08 SUB1 P53999 DDNMFQIGK 0.056 0.040 0.029 0.033 2.68 2.27 TNKS1BP1 Q9C0C2 DEDGSTLFR 0.053 0.084 0.045 0.037 2.39 2.88 RPS3 P23396 DEILPTTPISEQK 0.073 0.072 0.016 0.030 3.75 3.30 UPF1 Q92900 DETGELSSADEK 0.100 0.091 0.034 0.054 3.44 3.18 PPA1 Q15181 DFAIDIIK 0.076 0.043 0.016 0.046 3.97 2.25 XPO1 O14980 DFEEYPEHR 0.058 0.042 0.023 0.041 3.69 2.99 SLC25A6 P12236 DFLAGGIAAAISK 0.030 0.046 0.032 0.029 2.47 3.03 SLC25A5 P05141 DFLAGGVAAAISK 0.029 0.038 0.024 0.021 2.56 3.49 PPIB P23284 DFMIQGGDFTR 0.052 0.067 0.032 0.027 2.75 3.51 HADHB P55084 DFMYVSQDPK 0.018 0.028 0.020 0.023 1.95 3.09 PDLIM5 Q96HC4 DFNMPLTISSLK 0.086 0.093 0.028 0.045 3.25 2.88 ATP5J2 P56134 DFSPSGIFGAFQR 0.030 0.022 0.029 0.020 2.74 3.44 HIBADH P31937 DFSSVFQFLR 0.021 0.022 0.025 0.021 2.42 2.90 PRDX6 P30041 DFTPVCTTELGR 0.052 0.029 0.015 0.038 3.71 2.34 EIF4A1 P60842 DFTVSAMHGDMDQK 0.096 0.078 0.029 0.042 3.68 3.12 DHX9 Q08211 DFVNYLVR 0.050 0.051 0.033 0.035 2.89 3.16 DHRS2 Q13268 DGAHVVISSR 0.014 0.017 0.026 0.027 2.12 3.00 FASN P49327 DGAWGAFR 0.062 0.044 0.034 0.040 3.23 2.59 ERP29 P30040 DGDFENPVPYTGAVK 0.035 0.034 0.037 0.019 2.68 3.50 PDIA3 P30101 DGEEAGAYDGPR 0.050 0.058 0.027 0.025 3.04 3.62 AIFM1 O95831 DGEQHEDLNEVAK 0.039 0.031 0.024 0.015 2.97 3.43 C8orf55 Q8WUY1 DGFVCALLR 0.013 0.030 0.020 0.023 2.99 3.30 CALU O43852 DGFVTVDELK 0.039 0.055 0.024 0.026 2.09 2.89 CNN3 Q15417 DGIILCELINK 0.120 0.128 0.054 0.062 3.55 3.73 G6PD P11413 DGLLPENTFIVGYAR 0.054 0.029 0.013 0.029 3.90 2.57 ACAT1 P24752 DGLTDVYNK 0.030 0.031 0.029 0.024 2.45 3.13 CALM1 P62158 DGNGYISAAELR 0.057 0.045 0.023 0.031 3.32 2.81 ATP1A1 P05023 DGPNALTPPPTTPEWIK 0.060 0.071 0.031 0.040 2.93 3.52 FLNB O75369 DGTYAVTYIPDK 0.064 0.123 0.024 0.035 3.10 3.69 SRSF1 Q07955 DGYDYDGYR 0.039 0.046 0.036 0.028 2.46 3.19 P4HB P07237 DHENIVIAK 0.026 0.049 0.022 0.026 1.98 2.81 VCP P55072 DHFEEAMR 0.058 0.054 0.026 0.033 3.23 3.26 ACTN4 O43707 DHGGALGPEEFK 0.042 0.043 0.025 0.023 2.53 3.05

151

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

TRIM28 Q13263 DHQYQFLEDAVR 0.024 0.042 0.043 0.031 2.57 3.11 YWHAZ P63104 DICNDVLSLLEK 0.057 0.050 0.021 0.034 3.08 2.57 CCT8 P50990 DIDEVSSLLR 0.071 0.057 0.028 0.032 3.76 3.16 AHNAK Q09666 DIDISSPEFK 0.067 0.088 0.050 0.051 2.40 3.38 SRSF1 Q07955 DIEDVFYK 0.045 0.058 0.020 0.026 2.90 3.40 NDUFV2 P19404 DIEEIIDELK 0.042 0.058 0.028 0.039 2.37 2.88 CCT4 P50991 DIEREDIEFICK 0.057 0.057 0.036 0.034 2.92 2.97 IDH2 P48735 DIFQEIFDK 0.019 0.019 0.021 0.019 1.96 2.69 RPL5 P46777 DIICQIAYAR 0.043 0.052 0.025 0.026 2.90 3.11 ANXA2 P07355 DIISDTSGDFR 0.055 0.096 0.028 0.029 2.27 3.63 CS O75390 DILADLIPK 0.045 0.052 0.021 0.023 3.16 3.67 RPS16 P62249 DILIQYDR 0.053 0.064 0.038 0.033 2.69 3.19 ALDH3A2 P51648 DILTAIAADLCK 0.042 0.050 0.035 0.033 2.53 2.94 PRDX6 P30041 DINAYNCEEPTEK 0.047 0.029 0.015 0.038 3.44 2.23 RPS6 P62753 DIPGLTDTTVPR 0.075 0.073 0.027 0.026 3.92 3.79 HSP90B1 P14625 DISTNYYASQK 0.042 0.053 0.026 0.028 2.77 3.03 TRIM28 Q13263 DIVENYFMR 0.038 0.050 0.033 0.040 2.90 2.86 CPNE2 Q96FN4 DIVQFVPFR 0.078 0.052 0.019 0.042 4.60 2.78 SPTAN1 Q13813 DLASVQALLR 0.063 0.066 0.038 0.045 3.07 2.71 LMNA P02545 DLEALLNSK 0.038 0.055 0.027 0.026 2.14 3.26 CTSA P10619 DLECVTNLQEVAR 0.036 0.049 0.044 0.036 2.05 3.04 EEF2 P13639 DLEEDHACIPIK 0.106 0.073 0.031 0.036 4.44 2.92 CLTB P09497 DLEEWNQR 0.062 0.117 0.054 0.059 2.23 3.55 PSMD3 O43242 DLESAEER 0.076 0.354 0.027 0.035 3.58 3.07 SLC25A13 Q9UJS0 DLGFFGIYK 0.028 0.037 0.019 0.024 2.25 3.01 GDI2 P50395 DLGTESQIFISR 0.037 0.033 0.023 0.032 2.58 2.38 SPTAN1 Q13813 DLIGVQNLLK 0.047 0.064 0.036 0.041 2.28 2.73 RPS27 P42677 DLLHPSPEEEK 0.088 0.063 0.027 0.030 4.03 3.36 PRKDC P78527 DLLLNTMSQEEK 0.028 0.030 0.024 0.024 2.75 2.84 PHGDH O43175 DLPLLLFR 0.037 0.018 0.019 0.029 2.27 1.34 HNRNPF P52597 DLSYCLSGMYDHR 0.052 0.057 0.029 0.030 2.93 3.34 ACTA2 P62736 DLTDYLM~K 0.055 0.071 0.020 0.026 2.67 3.08 ACTA2 P62736 DLTDYLMK 0.058 0.086 0.026 0.032 2.57 3.34 HNRPDL O14979 DLTEYLSR 0.051 0.066 0.035 0.037 2.54 3.39 DLYANTVLSGGTTM~YPGI ACTB P60709 0.069 0.102 0.027 0.026 3.14 4.12 ADR DLYANTVLSGGTTMYPGIA ACTB P60709 0.072 0.094 0.023 0.025 3.33 4.08 DR IDH3B O43837 DMGGYSTTTDFIK 0.040 0.041 0.050 0.029 1.99 3.32 SRI P30626 DMSGTMGFNEFK 0.050 0.042 0.030 0.041 2.45 1.95 EZR P15311 DNAMLEYLK 0.071 0.071 0.032 0.041 2.90 2.87 PDCD6IP Q8WUM4 DNDFIYHDR 0.091 0.068 0.018 0.044 4.31 2.83 HIST1H4A P62805 DNIQGITK 0.022 0.013 0.036 0.017 2.05 2.57 HIST1H4A P62805 DNIQGITKPAIR 0.020 0.010 0.032 0.022 2.00 2.97 FASN P49327 DNLEFFLAGIGR 0.095 0.043 0.028 0.041 4.38 2.36 HSP90AA1 P07900 DNSTMGYMAAK 0.052 0.041 0.029 0.031 2.99 2.61 HSP90AB3P Q58FF7 DNSTMGYMMAK 0.056 0.056 0.029 0.030 2.97 2.84 DPDAQPGGELM~LGGTDS CTSD P07339 0.051 0.030 0.038 0.037 1.99 2.42 K CTSD P07339 DPDAQPGGELMLGGTDSK 0.039 0.046 0.040 0.047 2.01 2.37 CALR P27797 DPDASKPEDWDER 0.047 0.056 0.029 0.026 2.56 3.24 RAB7A P51149 DPENFPFVVLGNK 0.100 0.147 0.032 0.051 4.00 4.69 FASN P49327 DPETLVGYSMVGCQR 0.071 0.052 0.038 0.037 3.48 2.82 XPO5 Q9HAV4 DPLLLAIIPK 0.080 0.065 0.031 0.028 3.83 3.08 PSMD12 O00232 DPNNLLNDWSQK 0.054 0.050 0.033 0.024 2.69 2.85 LMNB1 P20700 DQIAQLEASLAAAK 0.020 0.051 0.019 0.022 2.72 3.29 EIF4A1 P60842 DQIYDIFQK 0.098 0.068 0.020 0.038 4.17 2.72 HADHB P55084 DQLLLGPTYATPK 0.023 0.029 0.023 0.021 2.13 3.23

152

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

HSP90AA1 P07900 DQVANSAFVER 0.083 0.049 0.027 0.030 4.24 3.15 DQVTAQEIFQDNHEDGPT XRCC5 P13010 0.059 0.040 0.021 0.027 3.80 3.11 AK HSPA9 P38646 DSETGENIR 0.069 0.060 0.031 0.029 4.07 4.03 LRRFIP1 Q32MZ4 DSLAEVEEK 0.084 0.092 0.029 0.040 3.64 3.19 XRCC6 P12956 DSLIFLVDASK 0.039 0.034 0.026 0.025 2.85 2.76 PFN1 P07737 DSPSVWAAVPGK 0.086 0.052 0.015 0.026 4.39 2.69 PLEC Q15149 DSQDAGGFGPEDR 0.074 0.099 0.036 0.044 3.61 3.76 GSN P06396 DSQEEEKTEALTSAK 0.028 0.033 0.017 0.023 1.90 1.88 YWHAB P31946 DSTLIM~QLLR 0.062 0.052 0.021 0.030 3.42 2.72 YWHAB P31946 DSTLIMQLLR 0.065 0.054 0.017 0.031 3.59 2.81 RBMX P38159 DSYESYGNSR 0.039 0.045 0.027 0.032 2.75 3.42 ACTA2 P62736 DSYVGDEAQSK 0.061 0.086 0.026 0.026 2.58 3.31 RAP1B P61224 DTDDVPMILVGNK 0.054 0.060 0.041 0.032 2.27 3.23 CALM1 P62158 DTDSEEEIR 0.064 0.052 0.024 0.028 3.46 2.82 PPIB P23284 DTNGSQFFITTVK 0.062 0.072 0.036 0.018 2.79 3.32 FASN P49327 DTSFEQHVLWHTGGK 0.120 0.042 0.027 0.038 4.36 2.35 KRT7 P08729 DVDAAYMSK 0.044 0.086 0.026 0.026 2.06 3.44 KRT8 P05787 DVDEAYM~NK 0.068 0.090 0.028 0.036 3.55 3.87 KRT8 P05787 DVDEAYMNK 0.056 0.089 0.035 0.033 3.16 4.06 KRT8 P05787 DVDEAYMNKVELESR 0.086 0.101 0.028 0.024 3.81 4.58 MRPL49 Q13405 DVEDFLSPLLGK 0.023 0.030 0.023 0.025 2.64 3.36 EPHX1 P07099 DVELLYPVK 0.021 0.029 0.031 0.021 1.56 2.43 PDIA6 Q15084 DVIELTDDSFDK 0.041 0.053 0.025 0.022 2.41 3.26 GNB2L1 P63244 DVLSVAFSSDNR 0.064 0.069 0.043 0.048 2.68 2.70 TUBA1A Q71U36 DVNAAIATIK 0.035 0.039 0.017 0.041 2.78 2.20 RPS19 P39019 DVNQQEFVR 0.075 0.067 0.028 0.028 3.65 3.27 PPP1CA P62136 DVQGWGENDR 0.075 0.062 0.042 0.058 2.98 2.50 CDC37 Q16543 DVQMLQDAISK 0.050 0.063 0.050 0.042 2.22 2.39 PSAP P07602 DVVTAAGDMLK 0.024 0.047 0.030 0.042 1.30 2.37 GDI1 P31150 DWNVDLIPK 0.050 0.037 0.023 0.033 2.80 2.27 LAD1 O00515 DWSALSSLAR 0.073 0.080 0.050 0.055 2.54 2.66 HNRNPK P61978 DYDDMSPR 0.055 0.069 0.042 0.039 2.71 3.46 ACTR3 P61158 DYEEIGPSICR 0.053 0.053 0.020 0.028 3.27 3.18 PLEC Q15149 DYELQLVTYK 0.069 0.113 0.033 0.056 2.73 3.24 ACTN4 O43707 DYETATLSDIK 0.049 0.042 0.027 0.023 2.61 2.68 ACTN1 P12814 DYETATLSEIK 0.050 0.083 0.035 0.030 2.03 3.27 HNRNPA2B1 P22626 DYFEEYGK 0.043 0.047 0.039 0.027 2.27 3.09 PRDX3 P30048 DYGVLLEGSGLALR 0.032 0.028 0.024 0.018 2.77 3.53 CS O75390 DYIWNTLNSGR 0.038 0.041 0.018 0.017 3.43 3.68 RPS10 P46783 DYLHLPPEIVPATLR 0.086 0.076 0.023 0.025 4.57 3.85 CAPZB P47756 DYLLCDYNR 0.050 0.054 0.029 0.037 2.65 2.81 FAM82B Q96DB5 DYPAHTEEDK 0.023 0.034 0.022 0.022 2.30 3.15 EPB41L1 Q9H4G0 DYSEADGLSER 0.028 0.043 0.034 0.038 1.82 2.85 KRT19 P08727 DYSHYYTTIQDLR 0.048 0.079 0.026 0.025 2.90 3.83 DNAJB1 P25685 DYYQTLGLAR 0.058 0.066 0.046 0.044 2.50 2.81 YWHAE P62258 EAAENSLVAYK 0.041 0.040 0.011 0.029 3.21 2.50 RBBP4 Q09028 EAAFDDAVEER 0.037 0.040 0.029 0.029 2.55 3.15 LMNA P02545 EAALSTALSEK 0.036 0.061 0.037 0.025 1.83 3.22 P4HB P07237 EADDIVNWLK 0.033 0.041 0.023 0.027 2.11 2.84 SND1 Q7KZF4 EADGSETPEPFAAEAK 0.102 0.098 0.035 0.035 4.04 3.51 HDGF P51858 EAENPEGEEK 0.035 0.032 0.025 0.026 2.44 2.25 HSP90B1 P14625 EAESSPFVER 0.055 0.061 0.031 0.019 3.19 3.69 MYL6 P60660 EAFQLFDR 0.067 0.077 0.032 0.042 2.84 2.92 SRSF1 Q07955 EAGDVCYADVYR 0.042 0.047 0.031 0.030 2.70 3.32 G3BP1 Q13283 EAGEQGDIEPR 0.074 0.084 0.042 0.031 3.09 3.81 RPS11 P62280 EAIEGTYIDK 0.062 0.068 0.020 0.025 3.44 3.22 ANXA6 P08133 EAILDIITSR 0.062 0.036 0.028 0.036 6.32 4.07

153

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

RAB7A P51149 EAINVEQAFQTIAR 0.106 0.152 0.039 0.043 4.04 5.01 CCT2 P78371 EALLSSAVDHGSDEVK 0.077 0.065 0.020 0.030 4.06 3.21 EIF4H Q15056 EALTYDGALLGDR 0.099 0.072 0.032 0.044 3.91 2.90 NCL P19338 EAM~EDGEIDGNK 0.068 0.052 0.026 0.027 3.70 3.24 NCL P19338 EAMEDGEIDGNK 0.063 0.053 0.028 0.028 3.62 3.23 RPL7 P18124 EANNFLWPFK 0.045 0.052 0.022 0.035 3.12 3.24 CAPZA1 P52907 EASDPQPEEADGGLK 0.066 0.056 0.023 0.031 3.46 2.92 DYNC1I2 Q13409 EAVAPVQEESDLEK 0.069 0.056 0.014 0.028 3.55 3.00 EIF4G1 Q04637 EAVGDLLDAFK 0.059 0.069 0.051 0.070 2.27 2.18 RPS4X P62701 ECLPLIIFLR 0.081 0.078 0.025 0.032 4.03 3.67 ANXA6 A6NN80 EDAQEIADTPSGDK 0.100 0.028 0.049 0.023 5.36 4.31 SLC3A2 P08195 EDFDSLLQSAK 0.054 0.051 0.023 0.034 2.75 2.53 LMNA P02545 EDLQELNDR 0.041 0.060 0.029 0.033 2.34 3.54 MYH10 P35580 EDQSILCTGESGAGK 0.065 0.067 0.020 0.029 3.00 2.69 HSP90AB3P Q58FF7 EDQTEYLEER 0.071 0.051 0.029 0.035 3.84 2.80 HNRNPA3 P51991 EDTEEYNLR 0.050 0.075 0.037 0.033 2.68 3.47 HSP90B1 P14625 EEASDYLELDTIK 0.053 0.050 0.013 0.025 3.22 3.04 ALDH2 P05091 EEIFGPVMQILK 0.021 0.017 0.024 0.021 2.31 2.69 RPL36 Q9Y3U8 EELSNVLAAMR 0.045 0.055 0.031 0.033 2.72 3.17 DDX21 Q9NR30 EEYQLVQVEQK 0.106 0.137 0.031 0.033 4.03 4.83 RAB1A P62820 EFADSLGIPFLETSAK 0.048 0.054 0.025 0.028 2.83 3.01 SET Q01105 EFHLNESGDPSSK 0.039 0.039 0.024 0.023 2.82 2.76 PABPC1 P11940 EFSPFGTITSAK 0.081 0.082 0.038 0.032 3.19 3.44 DDX21 Q9NR30 EGAFSNFPISEETIK 0.129 0.167 0.040 0.042 4.55 5.45 TES Q9UGI8 EGDPAIYAER 0.052 0.047 0.033 0.037 2.61 3.04 DHX9 Q08211 EGETVEPYK 0.054 0.060 0.024 0.027 3.15 3.39 PRDX2 P32119 EGGLGPLNIPLLADVTR 0.058 0.046 0.018 0.033 4.33 2.94 HSP90AB3P Q58FF7 EGLELPEDEEEK 0.045 0.053 0.027 0.035 3.28 2.69 ACTN1 P12814 EGLLLWCQR 0.042 0.056 0.030 0.024 2.47 3.24 PPIA P62937 EGM~NIVEAMER 0.056 0.029 0.022 0.036 3.61 2.28 PPIA P62937 EGMNIVEAMER 0.045 0.032 0.023 0.033 3.20 2.34 MDH2 P40926 EGVVECSFVK 0.033 0.029 0.028 0.019 2.33 3.19 EEF1A1 P68104 EHALLAYTLGVK 0.099 0.063 0.018 0.033 4.64 2.87 SAR1A Q9NR31 EIFGLYGQTTGK 0.060 0.055 0.025 0.034 2.89 2.63 HSPD1 P10809 EIGNIISDAMK 0.053 0.050 0.022 0.021 3.73 3.89 TUBA1A Q71U36 EIIDLVLDR 0.058 0.045 0.019 0.035 3.61 2.52 GDI2 P50395 EIRPALELLEPIEQK 0.057 0.039 0.020 0.035 3.04 2.36 ACTA2 P62736 EITALAPSTM~K 0.041 0.091 0.017 0.027 2.52 3.54 ACTA2 P62736 EITALAPSTMK 0.052 0.094 0.027 0.029 2.53 3.54 TXN P10599 EKLEATINELV 0.059 0.041 0.015 0.039 3.73 2.29 RPS3 P23396 ELAEDGYSGVEVR 0.069 0.072 0.025 0.028 3.79 3.60 EIF3G O75821 ELAEQLGLSTGEK 0.052 0.076 0.041 0.031 2.48 3.04 COPG2 Q9UBF2 ELAPAVSVLQLFCSSPK 0.046 0.043 0.027 0.030 2.58 2.48 DDX5 P17844 ELAQQVQQVAAEYCR 0.110 0.123 0.048 0.076 3.48 3.85 DDX17 Q92841 ELAQQVQQVADDYGK 0.058 0.046 0.042 0.041 2.48 3.05 MYH9 P35579 ELEDATETADAMNR 0.078 0.066 0.022 0.030 3.48 2.90 HSPA5 P11021 ELEEIVQPIISK 0.056 0.067 0.030 0.022 2.82 3.55 RPL32 P62910 ELEVLLMCNK 0.039 0.047 0.021 0.027 2.57 2.41 ALDH2 P05091 ELGEYGLQAYTEVK 0.016 0.015 0.024 0.023 2.15 2.62 CCT3 P49368 ELGIWEPLAVK 0.075 0.063 0.029 0.035 3.43 3.06 HSP90B1 P14625 ELISNASDALDK 0.049 0.054 0.026 0.025 3.01 2.98 TRAP1 Q12931 ELISNASDALEK 0.058 0.052 0.019 0.029 3.97 3.76 HSP90AA2 Q14568 ELISNSSDALDK 0.068 0.044 0.018 0.030 3.88 2.69 PFDN5 Q99471 ELLVPLTSSMYVPGK 0.075 0.042 0.017 0.030 3.59 2.86 B2RCX0 ELNSNHDGADETSEK 0.046 0.035 0.021 0.035 2.76 2.40 VCP P55072 ELQELVQYPVEHPDK 0.069 0.067 0.024 0.023 3.48 3.66 PDIA3 P30101 ELSDFISYLQR 0.040 0.049 0.027 0.029 2.62 3.19

154

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

LGALS3BP Q08380 ELSEALGQIFDSQR 0.021 0.029 0.033 0.034 1.43 2.31 RNH1 P13489 ELSLAGNELGDEGAR 0.051 0.032 0.022 0.040 3.45 2.33 EHD1 Q9H4M9 ELVNNLGEIYQK 0.071 0.082 0.046 0.044 2.56 3.02 EIF4G1 Q04637 EMDEAATAEER 0.064 0.082 0.069 0.059 2.39 2.70 CAP1 Q01518 EMNDAAMFYTNR 0.071 0.062 0.020 0.031 3.57 3.01 ECHS1 P30084 EMQNLSFQDCYSSK 0.022 0.037 0.019 0.016 2.49 3.37 STT3A P46977 ENDYYTPTGEFR 0.058 0.064 0.027 0.030 3.49 3.82 P4HB P07237 ENLLDFIK 0.022 0.042 0.016 0.025 2.13 2.77 ACADM P11310 ENVLIGDGAGFK 0.035 0.016 0.032 0.024 2.28 2.81 CALR P27797 EQFLDGDGWTSR 0.044 0.049 0.024 0.021 2.93 3.38 SLC25A5 P05141 EQGVLSFWR 0.034 0.045 0.026 0.028 2.77 3.64 FASN P49327 EQGVTFPSGDIQEQLIR 0.127 0.045 0.018 0.040 6.20 2.67 DDX21 Q9NR30 EQLGEEIDSK 0.090 0.197 0.035 0.043 3.80 5.71 ATL3 Q6DD88 EQLQALIPYVLNPSK 0.033 0.057 0.025 0.027 2.42 3.28 LACTB2 Q53H82 EQQILTLFR 0.031 0.030 0.033 0.032 1.76 2.16 PSMD11 O00231 EQSILELGSLLAK 0.066 0.051 0.018 0.025 3.52 2.86 KTN1 Q86UP2 EREHLEMELEK 0.056 0.060 0.037 0.036 2.40 3.01 HSPA9 P38646 ERVEAVNMAEGIIHDTETK 0.055 0.060 0.034 0.030 3.03 3.48 KPNB1 Q14974 ESCLEAYTGIVQGLK 0.060 0.050 0.020 0.020 3.45 2.86 CPSF1 Q10570 ESLAEEHEGLVGEGQR 0.048 0.052 0.031 0.020 2.95 3.63 SRRT Q9BXP5 ESLSEEEAQK 0.052 0.065 0.030 0.029 2.72 3.39 CLTC Q00610 ESNCYDPER 0.049 0.050 0.033 0.025 2.85 3.06 SUPT16H Q9Y5B9 ESRYEEEEEQSR 0.045 0.032 0.033 0.025 2.79 3.28 UBA52 P62987 ESTLHLVLR 0.086 0.082 0.067 0.069 2.75 2.96 PPT1 P50897 ETIPLQETSLYTQDR 0.049 0.027 0.044 0.037 3.12 3.08 KRT18 P05783 ETMQSLNDR 0.094 0.111 0.029 0.031 4.48 4.93 ACTN4 O43707 ETTDTDTADQVIASFK 0.042 0.060 0.022 0.024 2.55 2.90 EEF2 P13639 ETVSEESNVLCLSK 0.124 0.081 0.019 0.035 5.13 3.16 SRRT Q9BXP5 EVAFFNNFLTDAK 0.063 0.062 0.025 0.025 3.28 3.55 VCP P55072 EVDIGIPDATGR 0.063 0.060 0.029 0.027 3.27 3.61 HYOU1 Q9Y4L1 EVEEEPGIHSLK 0.045 0.056 0.017 0.024 2.81 3.25 NCL P19338 EVFEDAAEIR 0.063 0.052 0.033 0.026 3.68 3.38 POR P16435 EVGETLLYYGCR 0.061 0.056 0.026 0.031 3.18 3.23 C1QBP Q07021 EVSFQSTGESEWK 0.053 0.055 0.026 0.017 3.61 3.83 KRT7 P08729 EVTINQSLLAPLR 0.046 0.086 0.030 0.023 2.18 3.56 KDM1A O60341 EYDELAETQGK 0.040 0.041 0.029 0.054 2.19 2.39 GOT2 P00505 EYLPIGGLAEFCK 0.035 0.041 0.021 0.021 3.05 3.33 KRT6A P02538 EYQELM~NVK 0.080 0.096 0.018 0.030 4.25 4.34 KRT6A P02538 EYQELMNVK 0.077 0.101 0.024 0.030 4.05 4.41 KRT4 P19013 EYQELMSVK 0.043 0.081 0.027 0.029 2.16 3.26 CCT8 P50990 FAEAFEAIPR 0.054 0.065 0.022 0.032 3.72 3.22 MYO6 Q9UM54 FAEFDQIMK 0.063 0.059 0.034 0.042 2.47 2.27 RBM8A Q9Y5S9 FAEYGEIK 0.066 0.054 0.033 0.052 2.97 2.46 ACTN1 P12814 FAIQDISVEETSAK 0.040 0.060 0.024 0.023 2.44 3.18 SFPQ P23246 FAQHGTFEYEYSQR 0.058 0.051 0.026 0.031 3.20 3.49 KRT2 P35908 FASFIDK 0.057 0.095 0.022 0.028 3.17 3.89 RPL4 P36578 FCIWTESAFR 0.056 0.052 0.024 0.029 3.39 3.03 TM9SF2 Q99805 FCNPGFPIGCYITDK 0.079 0.092 0.040 0.045 2.95 3.42 SULT1A1 P50225 FDADYAEK 0.067 0.033 0.019 0.037 3.20 2.08 PHB P35232 FDAGELITQR 0.037 0.035 0.028 0.017 3.05 3.71 FASN P49327 FDASFFGVHPK 0.091 0.044 0.010 0.040 4.90 2.42 HSPA8 P11142 FDDAVVQSDMK 0.106 0.085 0.010 0.034 4.67 3.62 PSMC2 P35998 FDDGAGGDNEVQR 0.078 0.065 0.040 0.037 3.44 3.42 EPRS P07814 FDDTNPEK 0.040 0.041 0.017 0.025 2.89 2.74 QARS P47897 FDDTNPEKEEAK 0.050 0.047 0.025 0.024 2.74 2.64 NDUFA8 P51970 FDECVLDK 0.025 0.038 0.020 0.025 2.35 3.21 CTSD P07339 FDGILGM~AYPR 0.035 0.042 0.032 0.041 2.02 2.41

155

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

CTSD P07339 FDGILGMAYPR 0.036 0.044 0.044 0.045 1.89 2.35 TUBA1A Q71U36 FDLMYAK 0.039 0.038 0.016 0.031 2.74 2.41 ACBD3 Q9H3P7 FDNSYSLWR 0.033 0.066 0.068 0.032 1.63 2.88 MYH10 P35580 FDQLLAEEK 0.039 0.033 0.027 0.026 2.15 2.29 RPS4X P62701 FDTGNLCMVTGGANLGR 0.042 0.063 0.039 0.033 2.40 3.10 SRPR P08240 FDTIDDK 0.084 0.063 0.033 0.048 3.40 2.66 TRIM25 Q14258 FDTIYQILLK 0.037 0.044 0.031 0.031 2.06 2.26 PPIA P62937 FEDENFILK 0.047 0.031 0.024 0.032 3.00 2.08 SEPT7 Q16181 FEDYLNAESR 0.068 0.058 0.033 0.037 2.93 3.02 HSPA2 P54652 FEELNADLFR 0.126 0.097 0.026 0.039 5.15 4.05 RAB5A P20339 FEIWDTAGQER 0.061 0.054 0.033 0.039 3.17 3.05 ASS1 P00966 FELSCYSLAPQIK 0.084 0.026 0.018 0.032 4.57 2.43 KRT17 Q04695 FETEQALR 0.046 0.077 0.025 0.026 3.04 3.81 KRT7 P08729 FETLQAQAGK 0.042 0.084 0.022 0.033 2.06 3.31 MAPRE1 Q15691 FFDANYDGK 0.063 0.064 0.022 0.047 2.97 2.51 ACADVL P49748 FFEEVNDPAK 0.029 0.044 0.026 0.027 2.02 2.78 DDT P30046 FFPLESWQIGK 0.043 0.026 0.017 0.033 3.61 2.37 PSMD12 O00232 FFQEENTEK 0.060 0.046 0.025 0.031 3.10 2.77 GART P22102 FGDPECQVILPLLK 0.073 0.034 0.016 0.034 4.11 2.59 HSPA1A P08107 FGDPVVQSDMK 0.071 0.056 0.037 0.039 2.70 2.56 GTF2I P78347 FGEAIGMGFPVK 0.051 0.054 0.033 0.090 2.34 1.81 SLC25A3 Q00325 FGFYEVFK 0.034 0.046 0.020 0.023 2.63 3.33 HADHA P40939 FGGGNPELLTQMVSK 0.035 0.036 0.023 0.024 2.18 3.21 SLC25A13 Q9UJS0 FGLYLPLFK 0.031 0.040 0.023 0.020 2.26 3.17 KRT19 P08727 FGPGVAFR 0.050 0.084 0.027 0.028 3.00 4.08 SCFD1 Q8WVM8 FGQDIISPLLSVK 0.057 0.042 0.029 0.034 2.75 2.72 CPNE3 O75131 FGVYDIDNK 0.043 0.038 0.032 0.034 2.72 2.13 PGK1 P00558 FHVEEEGK 0.055 0.038 0.009 0.037 3.43 2.23 SEPT9 Q9UHD8 FINDQYEK 0.038 0.051 0.042 0.037 1.87 2.52 TPR P12270 FLADQQSEIDGLK 0.045 0.055 0.033 0.028 2.43 3.42 STT3A P46977 FLAEEGFYK 0.034 0.054 0.020 0.034 2.69 2.99 RPL9 P32969 FLDGIYVSEK 0.052 0.058 0.022 0.024 3.11 3.08 DAD1 P61803 FLEEYLSSTPQR 0.034 0.057 0.040 0.028 2.11 3.36 IARS2 Q9NSE4 FLINLEGGDIR 0.028 0.031 0.028 0.024 2.45 3.01 YWHAZ P63104 FLIPNASQAESK 0.043 0.056 0.025 0.018 2.61 3.01 RAB31 Q13636 FLIWDTAGQER 0.065 0.084 0.037 0.039 2.97 3.42 PDIA3 P30101 FLQDYFDGNLK 0.043 0.050 0.021 0.025 2.52 3.02 HKDC1 Q2TB90 FLSQIESDR 0.036 0.044 0.030 0.024 2.54 3.60 ACTR3 P61158 FMEQVIFK 0.033 0.045 0.035 0.030 1.91 2.54 RPL10 P27635 FNADEFEDMVAEK 0.085 0.088 0.034 0.054 3.34 3.13 CLTC Q00610 FNALFAQGNYSEAAK 0.043 0.041 0.023 0.028 2.80 2.60 HNRNPM P52272 FNECGHVLYADIK 0.021 0.051 0.020 0.029 2.11 3.14 PRKDC P78527 FNNYVDCMK 0.029 0.032 0.029 0.019 2.50 2.90 DECR1 Q16698 FNVIQPGPIK 0.026 0.026 0.025 0.015 2.26 3.09 EIF3A Q14152 FNVLQYVVPEVK 0.107 0.086 0.023 0.032 4.18 3.34 RAN P62826 FNVWDTAGQEK 0.055 0.045 0.028 0.030 3.19 2.76 ARF6 P62330 FNVWDVGGQDK 0.066 0.070 0.027 0.038 3.16 3.20 TUBB1 Q9H4B7 FPGQLNADLR 0.055 0.043 0.021 0.033 3.55 2.64 FASN P49327 FPQLDSTSFANSR 0.119 0.045 0.019 0.039 5.90 2.65 SRP14 P37108 FQMAYSNLLR 0.043 0.066 0.040 0.068 2.11 2.64 HSP90B1 P14625 FQSSHHPTDITSLDQYVER 0.053 0.059 0.030 0.024 3.38 3.92 PSMB7 Q99436 FRPDMEEEEAK 0.082 0.059 0.016 0.025 4.15 3.39 ALDOA P04075 FSHEEIAMATVTALR 0.087 0.071 0.024 0.036 4.22 3.29 GNB2L1 P63244 FSPNSSNPIIVSCGWDK 0.108 0.099 0.026 0.040 4.38 3.88 RPL10A P62906 FSVCVLGDQQHCDEAK 0.051 0.058 0.015 0.024 3.37 3.24 PTPN1 P18031 FSYLAVIEGAK 0.048 0.066 0.026 0.050 2.49 2.48 CAPZA2 P47755 FTITPSTTQVVGILK 0.062 0.058 0.018 0.026 3.05 3.02

156

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

PRPF40A O75400 FTNMLGQPGSTALDLFK 0.058 0.055 0.037 0.038 2.64 2.78 RPSA P08865 FTPGTFTNQIQAAFR 0.072 0.072 0.021 0.034 3.92 3.37 ATP5B P06576 FTQAGSEVSALLGR 0.027 0.031 0.025 0.025 2.67 3.34 MDH1 P40925 FVEGLPINDFSR 0.034 0.025 0.019 0.033 3.66 2.58 MDH2 P40926 FVFSLVDAMNGK 0.026 0.033 0.018 0.021 2.49 3.20 PDIA3 P30101 FVMQEEFSR 0.048 0.049 0.028 0.021 2.72 3.36 GDI2 P50395 FVSISDLLVPK 0.051 0.035 0.015 0.035 3.14 2.15 GOT2 P00505 FVTVQTISGTGALR 0.035 0.034 0.023 0.020 3.12 3.52 RPL18A Q02543 FWYFVSQLK 0.053 0.055 0.020 0.028 2.97 2.49 CALR P27797 FYALSASFEPFSNK 0.039 0.049 0.019 0.021 2.89 3.34 NAP1L1 P55209 FYEEVHDLER 0.089 0.062 0.013 0.038 4.39 2.83 HSP90AA1 P07900 FYEQFSK 0.065 0.046 0.019 0.033 3.76 2.63 PGRMC1 O00264 FYGPEGPYGVFAGR 0.035 0.038 0.024 0.022 2.79 3.19 STT3A P46977 FYSLLDPSYAK 0.033 0.048 0.035 0.030 2.06 2.87 PKM2 P14618 GADFLVTEVENGGSLGSK 0.071 0.056 0.014 0.032 4.04 2.79 SLC25A4 P12235 GADIMYTGTVDCWR 0.041 0.048 0.024 0.020 3.01 3.84 ERP29 P30040 GALPLDTVTFYK 0.049 0.046 0.027 0.024 3.38 3.42 GAPDH P04406 GALQNIIPASTGAAK 0.057 0.037 0.014 0.030 3.84 2.50 TCP1 P17987 GANDFMCDEMER 0.076 0.052 0.035 0.033 3.65 3.04 CCT2 P78371 GATQQILDEAER 0.083 0.058 0.018 0.028 4.57 3.40 RAB11A P62491 GAVGALLVYDIAK 0.081 0.076 0.035 0.040 3.04 3.31 PSMB1 P20618 GAVYSFDPVGSYQR 0.062 0.049 0.017 0.022 3.90 3.22 MDH2 P40926 GCDVVVIPAGVPR 0.032 0.037 0.025 0.026 2.89 3.70 ACAA1 P09110 GCFQAEIVPVTTTVHDDK 0.030 0.035 0.019 0.020 2.56 3.12 PSAP P07602 GCSFLPDPYQK 0.029 0.052 0.037 0.047 1.50 2.42 PA2G4 Q9UQ80 GDAMIMEETGK 0.076 0.052 0.009 0.036 4.28 2.38 EWSR1 Q01844 GDATVSYEDPPTAK 0.055 0.064 0.028 0.021 2.87 3.26 H2AFV Q71UI9 GDEELDSLIK 0.032 0.032 0.033 0.030 2.36 2.99 HK1 P19367 GDFIALDLGGSSFR 0.043 0.056 0.022 0.020 3.03 3.97 DDX17 Q92841 GDGPICLVLAPTR 0.099 0.107 0.048 0.056 3.76 3.96 SOD1 P00441 GDGPVQGIINFEQK 0.050 0.042 0.014 0.037 3.45 2.57 PKM2 P14618 GDLGIEIPAEK 0.078 0.062 0.014 0.034 4.24 2.91 SEC11A P67812 GDLLFLTNR 0.034 0.058 0.034 0.024 2.19 3.12 PKM2 P14618 GDYPLEAVR 0.083 0.049 0.016 0.040 4.62 2.54 FUS P35637 GEATVSFDDPPSAK 0.059 0.052 0.036 0.029 2.82 3.12 RPN1 P04843 GEDEEENNLEVR 0.041 0.046 0.028 0.022 2.72 3.29 SFN P31947 GEELSCEER 0.146 0.136 0.019 0.041 4.97 4.33 CCT8 P50990 GEENLMDAQVK 0.062 0.075 0.017 0.030 3.98 2.97 AHNAK Q09666 GEGPDVDVNLPK 0.057 0.158 0.058 0.028 2.22 5.58 AHNAK Q09666 GEGPEFDVNLSK 0.086 0.064 0.032 0.037 2.65 3.09 AHNAK Q09666 GEGPEVDVNLPK 0.078 0.155 0.057 0.038 2.65 5.24 C10orf58 Q9BRX8 GEIFLDEK 0.054 0.050 0.026 0.031 2.55 2.78 PDIA6 Q15084 GESPVDYDGGR 0.047 0.058 0.026 0.025 2.96 3.77 HNRNPC P07910 GFAFVQYVNER 0.045 0.051 0.033 0.028 3.01 3.53 RBMXL2 O75526 GFAFVTFESPADAK 0.040 0.040 0.030 0.022 2.52 3.21 SRSF10 O75494 GFAYVQFEDVR 0.044 0.034 0.040 0.038 2.37 2.48 SYNCRIP O60506 GFCFLEYEDHK 0.053 0.041 0.028 0.031 2.78 2.57 EPPK1 P58107 GFFDPNTHENLTYVQLLR 0.075 0.083 0.029 0.021 3.50 3.80 HNRNPAB Q99729 GFGFILFK 0.045 0.052 0.033 0.032 2.53 3.04 PABPC1 P11940 GFGFVCFSSPEEATK 0.100 0.088 0.026 0.030 4.13 3.64 NCL P19338 GFGFVDFNSEEDAK 0.056 0.045 0.030 0.031 3.21 3.00 HNRPDL O14979 GFGFVLFK 0.035 0.049 0.022 0.032 2.37 2.94 PABPC1 P11940 GFGFVSFER 0.073 0.054 0.082 0.050 2.20 2.48 VKORC1L1 Q8N0U8 GFGLLGSIFGK 0.038 0.050 0.029 0.024 2.07 3.06 PDIA3 P30101 GFPTIYFSPANK 0.037 0.047 0.024 0.028 2.33 2.83 PARP1 P09874 GFSLLATEDK 0.057 0.040 0.026 0.029 2.80 2.81 HADHA P40939 GFYIYQEGVK 0.025 0.036 0.023 0.024 1.98 3.01

157

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

CCT7 Q99832 GGAEQFMEETER 0.085 0.057 0.022 0.033 4.63 3.23 HNRNPK P61978 GGDLMAYDR 0.052 0.056 0.040 0.044 2.46 2.87 PLEC Q15149 GGELVYTDSEAR 0.086 0.108 0.028 0.033 4.17 4.60 HNRNPA2B1 P22626 GGGGNFGPGPGSNFR 0.056 0.073 0.041 0.026 2.90 4.35 GGM~GSGGLATGIAGGLA KRT18 P05783 0.107 0.102 0.034 0.022 4.20 4.68 GMGGIQNEK HNRNPA2B1 P22626 GGNFGFGDSR 0.039 0.047 0.039 0.039 2.31 3.14 CSNK2A1 P68400 GGPNIITLADIVK 0.052 0.060 0.044 0.043 2.30 2.78 G6PD P11413 GGYFDEFGIIR 0.060 0.031 0.016 0.032 3.78 2.47 Q8NHW5 GHLENNPALEK 0.033 0.048 0.025 0.026 2.98 2.95 CCT6A P40227 GIDPFSLDALSK 0.073 0.056 0.029 0.036 3.52 2.75 FASN P49327 GILADEDSSRPVWLK 0.049 0.045 0.040 0.036 2.47 2.31 JUP P14923 GIMEEDEACGR 0.115 0.151 0.073 0.081 3.28 4.24 COX5A P20674 GINTLVTYDMVPEPK 0.066 0.095 0.025 0.031 3.25 4.12 PPA1 Q15181 GISCMNTTLSESPFK 0.039 0.043 0.024 0.037 3.02 2.44 DHX9 Q08211 GISHVIVDEIHER 0.045 0.052 0.035 0.025 3.14 3.53 SUB1 P53999 GISLNPEQWSQLK 0.080 0.046 0.022 0.038 4.19 2.46 ACTN4 O43707 GISQEQMQEFR 0.035 0.046 0.032 0.025 2.31 2.96 COPA P53621 GITGVDLFGTTDAVVK 0.040 0.044 0.029 0.033 2.17 2.35 FIS1 Q9Y3D6 GIVLLEELLPK 0.039 0.048 0.029 0.037 2.37 2.79 OAT P04181 GIYLWDVEGR 0.070 0.073 0.063 0.063 2.30 2.66 DDX39 O00148 GLAITFVSDENDAK 0.039 0.048 0.022 0.028 2.68 3.08 PGRMC1 O00264 GLATFCLDK 0.024 0.034 0.015 0.025 2.13 2.52 RPS3 P23396 GLCAIAQAESLR 0.054 0.066 0.033 0.026 2.88 3.57 HNRNPUL2 Q1KMD3 GLEEPEMDPK 0.038 0.042 0.034 0.027 2.26 2.98 HSP90B2P Q58FF3 GLFDEYGSK 0.037 0.051 0.029 0.032 2.48 2.88 PRDX1 Q06830 GLFIIDDK 0.047 0.033 0.015 0.029 3.32 2.44 PRDX3 P30048 GLFIIDPNGVIK 0.022 0.026 0.020 0.023 2.58 3.26 ANXA2 P07355 GLGTDEDSLIEIICSR 0.052 0.109 0.024 0.018 2.14 4.22 ETFA P13804 GLLPEELTPLILATQK 0.036 0.040 0.029 0.033 2.50 3.30 EIF4G1 Q04637 GLPLVDDGGWNTVPISK 0.113 0.094 0.057 0.050 3.52 3.20 HNRNPH1 P31943 GLPWSCSADEVQR 0.070 0.081 0.035 0.030 3.10 3.77 IQGAP1 P46940 GLQQQNSDWYLK 0.053 0.056 0.023 0.034 2.57 2.57 NCL P19338 GLSEDTTEETLK 0.060 0.052 0.024 0.027 3.70 3.31 EIF4G2 P78344 GLSFLFPLLK 0.133 0.125 0.045 0.048 4.03 3.83 PRKDC P78527 GLSSLLCNFTK 0.036 0.029 0.020 0.022 2.96 2.81 SLC25A1 P53007 GLSSLLYGSIPK 0.028 0.034 0.017 0.018 3.07 3.52 AKR1A1 P14550 GLVQALGLSNFNSR 0.030 0.023 0.025 0.029 2.83 2.30 CS O75390 GLVYETSVLDPDEGIR 0.044 0.052 0.024 0.019 3.42 4.23 ERBB2 P04626 GMSYLEDVR 0.055 0.053 0.057 0.057 2.12 2.11 MYL12A P19105 GNFNYIEFTR 0.056 0.095 0.036 0.035 2.46 3.04 ENO1 P06733 GNPTVEVDLFTSK 0.057 0.040 0.014 0.030 3.49 2.44 A8MWD9 GNSIIMLEALER 0.039 0.047 0.033 0.021 2.71 3.29 RPL4 P36578 GPCIIYNEDNGIIK 0.034 0.082 0.024 0.028 2.33 3.41 AHNAK Q09666 GPEVDIEGPEGK 0.084 0.109 0.030 0.037 3.45 4.33 VAPA Q9P0L0 GPFTDVVTTNLK 0.042 0.051 0.015 0.020 2.79 3.26 RPS16 P62249 GPLQSVQVFGR 0.069 0.068 0.024 0.029 3.71 3.45 NPM1 P06748 GPSSVEDIK 0.047 0.073 0.030 0.035 2.73 3.54 G6PD P11413 GPTEADELMK 0.053 0.025 0.013 0.029 3.41 2.35 CLTC Q00610 GQFSTDELVAEVEK 0.043 0.044 0.024 0.025 2.54 2.69 KRT7 P08729 GQLEALQVDGGR 0.048 0.082 0.028 0.025 2.28 3.57 PPA1 Q15181 GQYISPFHDIPIYADK 0.100 0.048 0.022 0.033 4.62 2.71 HNRNPK P61978 GSDFDCELR 0.049 0.054 0.043 0.042 2.43 2.84 PDIA6 Q15084 GSFSEQGINEFLR 0.034 0.051 0.023 0.022 2.26 3.32 IVD P26440 GSNTCELIFEDCK 0.027 0.033 0.016 0.017 2.92 3.52 HNRNPK P61978 GSYGDLGGPIITTQVTIPK 0.069 0.076 0.037 0.032 3.06 3.59 SLC25A5 P05141 GTDIMYTGTLDCWR 0.034 0.039 0.023 0.025 2.95 3.62 CANX P27824 GTLSGWILSK 0.034 0.046 0.023 0.023 2.21 2.92

158

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

EIF4A1 P60842 GVAINMVTEEDK 0.087 0.085 0.030 0.032 3.40 2.94 ANXA2 P07355 GVDEVTIVNILTNR 0.054 0.112 0.022 0.021 2.22 4.27 FASN P49327 GVDLVLNSLAEEK 0.123 0.041 0.015 0.040 5.12 2.33 SNRPF P62306 GVEEEEEDGEMRE 0.054 0.044 0.026 0.025 3.61 3.34 DLAT P10515 GVETIANDVVSLATK 0.048 0.041 0.024 0.020 3.24 3.83 ATP1A1 P05023 GVGIISEGNETVEDIAAR 0.034 0.047 0.062 0.035 1.73 2.73 PRDX5 P30044 GVLFGVPGAFTPGCSK 0.037 0.035 0.014 0.027 3.18 2.92 COPB2 P35606 GVNCIDYYSGGDK 0.051 0.053 0.028 0.030 2.63 2.48 PKM2 P14618 GVNLPGAAVDLPAVSEK 0.075 0.065 0.015 0.033 4.22 3.08 DDX21 Q9NR30 GVTFLFPIQAK 0.120 0.192 0.026 0.041 4.63 5.81 HSP90B1 P14625 GVVDSDDLPLNVSR 0.040 0.055 0.035 0.025 2.72 3.51 GVVPLAGTDGETTTQGLD ALDOC P09972 0.119 0.076 0.020 0.032 5.45 3.64 GLSER EEF1D P29692 GVVQELQQAISK 0.049 0.072 0.045 0.028 2.32 3.25 SNRNP70 P08621 GYAFIEYEHER 0.070 0.070 0.033 0.027 3.36 3.70 EIF4A1 P60842 GYDVIAQAQSGTGK 0.091 0.086 0.011 0.024 4.00 3.06 EPPK1 P58107 GYFDEEM~NR 0.067 0.073 0.030 0.032 3.08 3.26 EPPK1 P58107 GYFDEEMNR 0.053 0.071 0.028 0.034 2.67 3.17 HNRNPU Q00839 GYFEYIEENK 0.050 0.057 0.028 0.033 2.74 3.31 PUF60 Q9UHX1 GYGFIEYEK 0.062 0.058 0.047 0.065 2.51 2.18 RBM39 Q14498 GYGFITFSDSECAK 0.064 0.053 0.038 0.056 2.72 2.40 PABPC1 P11940 GYGFVHFETQEAAER 0.096 0.082 0.025 0.032 4.14 3.53 CSRP1 P21291 GYGYGQGAGTLSTDK 0.040 0.060 0.014 0.035 2.32 2.34 HSPD1 P10809 GYISPYFINTSK 0.056 0.051 0.018 0.021 4.01 3.89 MDH2 P40926 GYLGPEQLPDCLK 0.030 0.035 0.020 0.017 2.85 3.58 TXNDC5 Q8NBS9 GYPTLLLFR 0.049 0.048 0.028 0.026 2.82 3.04 TXNDC5 Q8NBS9 GYPTLLWFR 0.022 0.043 0.053 0.037 1.47 2.74 ACTB P60709 GYSFTTTAER 0.059 0.082 0.024 0.032 2.84 3.45 RBM8A Q9Y5S9 GYTLVEYETYK 0.059 0.064 0.022 0.033 3.28 3.07 ATP5A1 P25705 HALIIYDDLSK 0.025 0.028 0.022 0.025 2.42 3.09 C14orf166 Q9Y224 HDDYLVMLK 0.057 0.052 0.031 0.033 2.68 2.51 NDUFB7 P17568 HDWDYCEHR 0.030 0.032 0.026 0.028 2.69 3.43 ACTN1 P12814 HEAFESDLAAHQDR 0.048 0.053 0.026 0.029 2.79 3.05 MYH9 P35579 HEAMITDLEER 0.081 0.064 0.019 0.032 3.57 2.76 C17orf25 D3DTG9 HEEFEEGCK 0.044 0.022 0.022 0.035 3.09 1.93 PA2G4 Q9UQ80 HELLQPFNVLYEK 0.077 0.058 0.038 0.030 3.41 2.84 CFL1 P23528 HELQANCYEEVK 0.064 0.053 0.017 0.041 3.32 2.41 XAB2 Q9HCS7 HENYDEALR 0.053 0.065 0.051 0.037 2.39 3.44 TRIM28 Q13263 HEPLVLFCESCDTLTCR 0.053 0.051 0.033 0.023 3.24 3.39 TMED2 Q15363 HEQEYM~EVR 0.031 0.035 0.033 0.026 2.47 2.99 TMED2 Q15363 HEQEYMEVR 0.036 0.041 0.023 0.026 2.77 3.09 CALR P27797 HEQNIDCGGGYVK 0.039 0.049 0.023 0.010 2.65 3.48 REEP5 Q00765 HESQMDSVVK 0.029 0.039 0.019 0.025 2.46 2.92 RPL36A P83881 HFELGGDK 0.055 0.055 0.023 0.034 3.09 2.79 BAT1 Q13838 HFILDECDK 0.032 0.041 0.022 0.025 2.47 2.88 HFLLEEDKPEEPTAHAFVST FASN P49327 0.112 0.046 0.017 0.041 5.12 2.52 LTR CCT8 P50990 HFSGLEEAVYR 0.066 0.053 0.023 0.035 3.81 3.26 GPI P06744 HFVALSTNTTK 0.013 0.029 0.016 0.031 1.02 2.03 DDX39 O00148 HFVLDECDK 0.053 0.066 0.036 0.038 2.92 3.38 PRDX1 Q06830 HGEVCPAGWK 0.078 0.029 0.033 0.024 3.21 2.55 RPS15A P62244 HGYIGEFEIIDDHR 0.069 0.063 0.017 0.031 3.85 3.37 SOD2 P04179 HHAAYVNNLNVTEEK 0.063 0.056 0.024 0.015 3.32 3.16 CHD3 Q12873 HHYEQQQEDLAR 0.058 0.040 0.050 0.052 2.30 2.82 HK1 P19367 HIDLVEGDEGR 0.039 0.051 0.026 0.027 2.81 3.77 HK2 P52789 HIDMVEGDEGR 0.123 0.138 0.040 0.050 5.03 5.38 STX3 Q13277 HIEEDEVR 0.077 0.110 0.036 0.034 3.06 4.35 MAT2A P31153 HIGYDDSSK 0.107 0.083 0.043 0.049 3.68 2.90

159

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

HSP90AA5P Q58FG0 HIYYITGETK 0.064 0.044 0.016 0.032 3.85 2.64 CCT5 P48643 HKLDVTSVEDYK 0.091 0.070 0.024 0.031 3.76 2.91 IVL P07476 HLEEEEGQLK 0.043 0.049 0.013 0.030 1.68 1.95 RRBP1 Q9P2E9 HLEEIVEK 0.071 0.083 0.056 0.064 2.38 2.70 HSP90AB1 P08238 HLEINPDHPIVETLR 0.114 0.071 0.018 0.031 5.85 3.65 HSP90AA1 P07900 HLEINPDHSIIETLR 0.076 0.051 0.023 0.032 4.40 3.12 CTSA P10619 HLHYWFVESQK 0.038 0.045 0.036 0.042 1.99 2.56 PTGES3 Q15185 HLNEIDLFHCIDPNDSK 0.070 0.042 0.018 0.036 4.01 2.76 HIST1H2AA Q96QV6 HLQLAIR 0.020 0.017 0.025 0.024 2.46 3.09 RPL6 Q02878 HLTDAYFK 0.061 0.057 0.021 0.030 3.23 2.74 ACTR2 P61160 HLWDYTFGPEK 0.059 0.050 0.018 0.029 2.92 2.98 AARS P49588 HNDLDDVGK 0.050 0.179 0.030 0.045 2.80 1.88 NMNAT1 Q9HAN9 HNLYSSESEDR 0.025 0.031 0.029 0.018 2.34 3.14 GSTM3 P21266 HNMCGETEEEK 0.030 0.019 0.009 0.033 2.91 2.07 P4HB P07237 HNQLPLVIEFTEQTAPK 0.041 0.048 0.023 0.020 2.41 3.35 HMGB1 P09429 HPDASVNFSEFSK 0.032 0.028 0.017 0.026 2.81 2.17 PPP4R2 Q9NY27 HPDEDAVEAEGHEVK 0.105 0.095 0.041 0.044 3.39 3.50 G3BP1 Q13283 HPDSHQLFIGNLPHEVDK 0.080 0.085 0.027 0.033 3.56 3.54 RTN4 Q9NQC3 HQAQIDHYLGLANK 0.049 0.093 0.022 0.020 2.31 3.65 RPL6 Q02878 HQEGEIFDTEK 0.063 0.058 0.023 0.019 3.43 3.15 ACTA2 P62736 HQGVMVGMGQK 0.059 0.078 0.024 0.037 2.60 3.00 SPTAN1 Q13813 HQLLEADISAHEDR 0.044 0.074 0.048 0.034 2.32 3.60 ACTN4 O43707 HRDYETATLSDIK 0.036 0.065 0.028 0.025 2.09 2.85 ACTN4 O43707 HRPELIEYDK 0.044 0.055 0.028 0.023 2.34 3.11 AP3D1 O14617 HRPSEADEEELAR 0.090 0.057 0.032 0.031 3.78 2.85 RPL12 P30050 HSGNITFDEIVNIAR 0.056 0.058 0.026 0.024 3.18 3.28 MYH9 P35579 HSQAVEELAEQLEQTK 0.076 0.068 0.017 0.031 3.26 2.86 HSP90AA5P Q58FG0 HSQFIGYPITLFVEK 0.082 0.049 0.019 0.030 4.42 2.90 PPA1 Q15181 HTGCCGDNDPIDVCEIGSK 0.065 0.048 0.028 0.039 3.45 2.59 ACTN1 P12814 HTNYTMEHIR 0.040 0.059 0.034 0.023 2.33 3.36 TPI1 P60174 HVFGESDELIGQK 0.042 0.031 0.013 0.035 2.76 2.00 SOD1 P00441 HVGDLGNVTADK 0.044 0.025 0.014 0.026 3.30 2.40 TUFM P49411 HYAHTDCPGHADYVK 0.040 0.037 0.024 0.022 3.37 3.45 TECR Q9NZ01 HYEVEILDAK 0.036 0.039 0.033 0.031 2.13 2.58 PPIB P23284 HYGPGWVSMANAGK 0.049 0.060 0.049 0.026 2.04 3.25 VPS26A O75436 HYLFYDGESVSGK 0.068 0.055 0.024 0.039 3.19 2.68 DNAJA1 P31689 HYNGEAYEDDEHHPR 0.244 0.149 0.052 0.073 6.84 4.50 DDX1 Q92499 HYYEVSCHDQGLCR 0.049 0.049 0.026 0.025 2.96 2.97 GOT2 P00505 IAAAILNTPDLR 0.040 0.043 0.028 0.024 3.24 3.90 SPTAN1 Q13813 IAALQAFADQLIAAGHYAK 0.052 0.066 0.034 0.027 2.67 3.20 GSTM3 P21266 IAAYLQSDQFCK 0.035 0.018 0.012 0.031 3.19 2.07 IDH3A P50213 IAEFAFEYAR 0.019 0.036 0.034 0.021 2.31 3.66 TXNDC5 Q8NBS9 IAEVDCTAER 0.037 0.052 0.023 0.020 2.78 2.99 RPL7 P18124 IALTDNALIAR 0.066 0.054 0.025 0.033 3.73 3.56 S100A14 Q9HCY8 IANLGSCNDSK 0.091 0.070 0.031 0.063 3.66 2.64 ABCD3 P28288 IANPDQLLTQDVEK 0.028 0.024 0.015 0.017 2.53 3.10 SLC25A13 Q9UJS0 IAPLEEGTLPFNLAEAQR 0.035 0.046 0.026 0.018 2.67 3.96 ACAA1 P09110 IAQFLSDIPETVPLSTVNR 0.033 0.025 0.026 0.020 3.16 3.52 MYH9 P35579 IAQLEEQLDNETK 0.071 0.063 0.018 0.033 3.23 2.63 ACTN4 O43707 ICDQWDALGSLTHSR 0.046 0.054 0.027 0.027 2.79 3.22 ACTN1 P12814 ICDQWDNLGALTQK 0.051 0.091 0.030 0.019 2.20 3.45 PPP1CA P62136 ICGDIHGQYYDLLR 0.105 0.106 0.042 0.046 3.75 3.90 HNRNPD Q14103 IDASKNEEDEGHSNSSPR 0.051 0.065 0.031 0.027 2.57 3.37 HNRNPK P61978 IDEPLEGSEDR 0.054 0.068 0.040 0.036 2.71 3.35 RAD23B P54727 IDIDPEETVK 0.068 0.049 0.023 0.033 3.17 2.69 HSP90AB3P Q58FF7 IDIIPNPQER 0.106 0.063 0.022 0.033 5.25 3.30 LMNA P02545 IDSLSAQLSQLQK 0.035 0.051 0.026 0.024 2.03 3.27

160

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

HNRNPA2B1 P22626 IDTIEIITDR 0.041 0.047 0.031 0.027 2.73 3.37 AHNAK Q09666 IDVTAPDVSIEEPEGK 0.050 0.063 0.053 0.076 2.08 2.38 SSBP1 Q04837 IDYGEYMDK 0.036 0.039 0.028 0.024 2.49 3.29 FLNA P21333 IECDDKGDGSCDVR 0.046 0.086 0.024 0.023 1.66 2.98 CDC73 Q6P1J9 IEDEECVR 0.073 0.077 0.069 0.087 2.51 2.67 RPS9 P46781 IEDFLER 0.064 0.064 0.024 0.028 3.68 3.47 NACA Q13765 IEDLSQQAQLAAAEK 0.089 0.073 0.022 0.040 3.93 3.15 RPS14 P62263 IEDVTPIPSDSTR 0.085 0.082 0.032 0.034 3.73 3.81 NAPA P54920 IEEACEIYAR 0.055 0.050 0.048 0.056 2.49 2.36 NUP133 Q8WUM0 IEEMAEQER 0.042 0.053 0.033 0.033 2.75 3.31 RPL4 P36578 IEEVPELPLVVEDK 0.069 0.068 0.015 0.021 4.04 3.56 RPL5 P46777 IEGDMIVCAAYAHELPK 0.054 0.067 0.015 0.016 3.13 3.27 AHNAK Q09666 IEGEMQVPDVDIR 0.106 0.127 0.049 0.032 3.71 5.28 ARCN1 P48444 IEGLLAAFPK 0.053 0.048 0.026 0.028 2.68 2.38 HNRNPA3 P51991 IETIEVMEDR 0.043 0.053 0.031 0.033 2.58 3.30 HNRNPA1L2 Q32P51 IEVIEIM~TDR 0.055 0.065 0.033 0.032 2.98 3.65 HNRNPA1L2 Q32P51 IEVIEIMTDR 0.053 0.061 0.034 0.034 2.90 3.49 HSPA5 P11021 IEWLESHQDADIEDFK 0.050 0.074 0.023 0.019 3.02 3.75 FLNB O75369 IEYNDQNDGSCDVK 0.062 0.111 0.023 0.028 3.14 4.36 PPP1CA P62136 IFCCHGGLSPDLQSMEQIR 0.094 0.080 0.037 0.041 3.53 3.57 GRB7 Q14451 IFCSEDEQSR 0.107 0.087 0.037 0.053 3.78 3.14 SLC44A2 Q8IWA5 IFDDSPCPFTAK 0.031 0.054 0.037 0.039 1.83 2.40 ACADVL P49748 IFEGTNDILR 0.038 0.037 0.029 0.028 2.27 2.72 FLNB O75369 IFFAGDTIPK 0.048 0.087 0.036 0.036 2.35 3.57 ANP32B Q92688 IFGGLDMLAEK 0.035 0.026 0.016 0.030 2.88 2.17 ACADVL P49748 IFGSEAAWK 0.032 0.031 0.027 0.025 1.94 2.43 MDH2 P40926 IFGVTTLDIVR 0.028 0.034 0.022 0.020 2.68 3.45 PDIA3 P30101 IFRDGEEAGAYDGPR 0.118 0.082 0.032 0.021 4.22 4.22 HNRPAB D3DWP7 IFVGGLNPEATEEK 0.053 0.061 0.028 0.032 2.95 3.41 HNRNPD Q14103 IFVGGLSPDTPEEK 0.039 0.044 0.022 0.022 2.77 3.22 ENO1 P06733 IGAEVYHNLK 0.049 0.034 0.018 0.028 2.98 2.41 RHOA P61586 IGAFGYMECSAK 0.068 0.070 0.058 0.051 2.12 2.47 PPIB P23284 IGDEDVGR 0.054 0.067 0.030 0.025 2.87 3.66 TXNRD1 Q16881 IGEHMEEHGIK 0.039 0.023 0.035 0.024 2.41 1.90 IGEHTPSALAIMENANVLA ALDOA P04075 0.107 0.071 0.022 0.034 4.99 3.60 R ABCF1 Q8NE71 IGFFNQQYAEQLR 0.088 0.067 0.023 0.040 4.01 3.10 EZR P15311 IGFPWSEIR 0.084 0.077 0.030 0.038 3.58 3.11 EEF1A1 P68104 IGGIGTVPVGR 0.091 0.072 0.016 0.030 4.83 3.32 PHB2 Q99623 IGGVQQDTILAEGLHFR 0.038 0.037 0.026 0.020 3.19 3.74 ATP2A2 P16615 IGIFGQDEDVTSK 0.058 0.067 0.030 0.027 2.70 3.10 ATP5B P06576 IGLFGGAGVGK 0.024 0.026 0.019 0.019 2.46 3.07 ACLY P53396 IGNTGGMLDNILASK 0.130 0.092 0.039 0.047 4.49 3.39 MATR3 P43243 IGPYQPNVPVGIDYVIPK 0.101 0.085 0.033 0.034 4.13 4.01 TUBA1A Q71U36 IHFPLATYAPVISAEK 0.063 0.046 0.013 0.031 3.88 2.64 PSMD4 P55036 IIAFVGSPVEDNEK 0.081 0.065 0.038 0.064 2.52 2.27 MYO1C O00159 IICDLVEEK 0.037 0.047 0.031 0.029 2.17 2.59 UGDH O60701 IIDSLFNTVTDK 0.086 0.098 0.026 0.027 3.09 3.41 RPS8 P62241 IIDVVYNASNNELVR 0.065 0.064 0.025 0.027 3.57 3.28 HNRNPK P61978 IILDLISESPIK 0.048 0.064 0.038 0.036 2.37 2.99 HSPA5 P11021 IINEPTAAAIAYGLDK 0.111 0.094 0.022 0.028 4.71 3.99 HSPA1A P08107 IINEPTAAAIAYGLDR 0.097 0.058 0.034 0.033 3.61 3.03 PPIAL4A Q9Y536 IIPGFMCQGGDFTR 0.052 0.032 0.021 0.035 3.66 2.33 Q8NHW5 IIQLLDDYPK 0.050 0.057 0.026 0.031 2.86 3.12 PLEC Q15149 IISLETYNLLR 0.054 0.132 0.049 0.026 2.57 5.46 GAPDH P04406 IISNASCTTNCLAPLAK 0.052 0.037 0.007 0.031 3.87 2.60 IITITGTQDQIQNAQYLLQN HNRNPK P61978 0.058 0.062 0.030 0.031 2.89 3.23 SVK

161

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

PCBP2 Q15366 IITLAGPTNAIFK 0.069 0.060 0.040 0.045 2.70 2.52 PCBP1 Q15365 IITLTGPTNAIFK 0.079 0.060 0.033 0.040 3.14 2.66 GNB2L1 P63244 IIVDELKQEVISTSSK 0.093 0.071 0.049 0.033 3.04 3.03 TPI1 P60174 IIYGGSVTGATCK 0.030 0.026 0.017 0.023 2.53 2.10 SUCLA2 Q9P2R7 ILACDDLDEAAR 0.036 0.036 0.038 0.026 2.42 3.26 TKT P29401 ILATPPQEDAPSVDIANIR 0.055 0.027 0.017 0.026 4.24 3.32 MVP Q14764 ILDQSEAEK 0.062 0.065 0.015 0.032 2.25 2.45 P4HB P07237 ILEFFGLK 0.030 0.042 0.021 0.025 2.03 2.73 PRKDC P78527 ILELSGSSSEDSEK 0.035 0.029 0.023 0.025 2.84 2.67 ATP5A1 P25705 ILGADTSVDLEETGR 0.029 0.029 0.024 0.022 2.82 3.49 KRT19 P08727 ILGATIENSR 0.043 0.083 0.027 0.028 2.79 4.01 EEF1G P26641 ILGLLDAYLK 0.077 0.062 0.025 0.024 3.59 3.07 NSUN2 Q08J23 ILLTQENPFFR 0.095 0.074 0.033 0.035 4.28 3.46 SCYL1 Q96KG9 ILPVLCGLTVDPEK 0.061 0.051 0.028 0.030 3.03 2.52 RPL18 Q07020 ILTFDQLALDSPK 0.055 0.062 0.017 0.027 3.46 3.21 DDX39 O00148 ILVATNLFGR 0.030 0.043 0.025 0.025 2.61 3.26 AGR2 O95994 IMFVDPSLTVR 0.089 0.055 0.022 0.021 4.54 2.98 CCDC47 Q96A33 IMNEEDPEK 0.112 0.122 0.032 0.035 4.18 4.49 TUBB2C P68371 IMNTFSVVPSPK 0.056 0.042 0.011 0.034 3.65 2.49 ATP5B P06576 IMNVIGEPIDER 0.036 0.035 0.026 0.026 3.04 3.58 EPRS P07814 INEAVECLLSLK 0.037 0.039 0.023 0.025 2.56 2.62 HNRNPM P52272 INEILSNALK 0.035 0.050 0.028 0.030 2.23 2.68 SUCLG2 Q96I99 INFDDNAEFR 0.021 0.028 0.019 0.024 2.49 3.40 PCBP1 Q15365 INISEGNCPER 0.098 0.060 0.029 0.041 4.19 2.95 TUBB2C P68371 INVYYNEATGGK 0.067 0.049 0.020 0.029 3.41 2.58 PRKDC P78527 IPALDLLIK 0.029 0.033 0.025 0.023 2.67 2.99 IPDPEAVKPDDWDEDAPA CANX P27824 0.047 0.058 0.038 0.028 2.18 3.38 K TMED10 P49755 IPDQLVILDMK 0.030 0.046 0.017 0.020 2.61 3.01 RPS18 P62269 IPDWFLNR 0.085 0.069 0.025 0.035 4.09 3.31 FLNA P21333 IPEISIQDMTAQVTSPSGK 0.046 0.100 0.019 0.029 2.02 3.37 CCT3 P49368 IPGGIIEDSCVLR 0.068 0.082 0.030 0.034 3.30 3.31 CSE1L P55060 IPGLLGVFQK 0.047 0.030 0.036 0.027 2.75 2.34 PRDX4 Q13162 IPLLSDLTHQISK 0.074 0.062 0.023 0.015 3.28 3.61 IPNIYAIGDVVAGPM~LAH DLD P09622 0.036 0.026 0.024 0.028 2.71 3.36 K DLD P09622 IPNIYAIGDVVAGPMLAHK 0.054 0.045 0.023 0.024 3.33 3.74 VPS35 Q96QK1 IPVDTYNNILTVLK 0.046 0.040 0.020 0.028 3.13 2.56 FLNB O75369 IPYLPITNFNQNWQDGK 0.082 0.123 0.019 0.028 3.80 4.55 TPM4 P67936 IQALQQQADEAEDR 0.046 0.061 0.018 0.022 2.41 3.09 MDH2 P40926 IQEAGTEVVK 0.027 0.033 0.026 0.028 2.21 3.01 ARF4 P18085 IQEVADELQK 0.078 0.074 0.017 0.035 3.44 3.00 PGK1 P00558 IQLINNMLDK 0.052 0.036 0.019 0.039 2.91 2.24 DRG1 Q9Y295 IQLLDLPGIIEGAK 0.059 0.074 0.057 0.041 2.23 2.89 TPM1 P09493 IQLVEEELDR 0.049 0.071 0.025 0.025 2.34 3.05 SLC25A3 Q00325 IQTQPGYANTLR 0.045 0.058 0.018 0.026 3.42 4.15 A6NL28 IQVLQQQADDAEER 0.064 0.071 0.029 0.025 2.87 3.48 TUBB1 Q9H4B7 IREEYPDR 0.043 0.039 0.025 0.038 2.70 2.42 HSP90AB2P Q58FF8 IRYESLTDPSK 0.077 0.045 0.019 0.030 3.85 2.76 RHOC P08134 ISAFGYLECSAK 0.108 0.126 0.046 0.059 3.52 3.66 SHMT1 P34896 ISATSIFFESMPYK 0.063 0.042 0.029 0.023 3.54 2.95 MFAP1 P55081 ISEDVEER 0.082 0.082 0.061 0.059 2.70 3.29 TUBB2A Q13885 ISEQFTAM~FR 0.036 0.028 0.021 0.027 2.42 2.24 KIF5A Q12840 ISFLENNLEQLTK 0.055 0.059 0.026 0.037 2.93 2.46 HIST1H4A P62805 ISGLIYEETR 0.016 0.012 0.022 0.020 2.46 2.95 ISLGLPVGAVINCADNTGA RPL23 P62829 0.064 0.067 0.024 0.029 3.59 3.44 K CPNE3 O75131 ISLNSLCYGDMDK 0.055 0.040 0.025 0.035 3.34 2.29

162

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

AHNAK Q09666 ISMPDFDLHLK 0.107 0.141 0.026 0.033 4.26 5.08 AHNAK Q09666 ISMPDVDLHLK 0.094 0.139 0.029 0.036 3.71 4.88 AHNAK Q09666 ISMQDVDLSLGSPK 0.056 0.100 0.054 0.048 1.91 3.71 CACYBP Q9HB71 ISNYGWDQSDK 0.110 0.066 0.018 0.035 5.32 3.12 HSPD1 P10809 ISSIQSIVPALEIANAHR 0.061 0.054 0.025 0.019 4.32 4.39 RPS8 P62241 ISSLLEEQFQQGK 0.077 0.066 0.027 0.029 3.48 3.30 CTSD P07339 ISVNNVLPVFDNLM~QQK 0.036 0.044 0.036 0.047 1.92 2.25 TUBB P07437 ISVYYNEATGGK 0.049 0.032 0.012 0.031 3.11 2.15 TMED10 P49755 ITDSAGHILYSK 0.031 0.038 0.021 0.023 2.34 2.67 LMNA P02545 ITESEEVVSR 0.039 0.054 0.032 0.027 2.25 3.54 AIMP1 Q12904 ITFDAFPGEPDK 0.032 0.048 0.023 0.017 2.51 2.77 PKM2 P14618 ITLDNAYM~EK 0.070 0.044 0.010 0.027 3.71 2.36 PKM2 P14618 ITLDNAYMEK 0.073 0.058 0.012 0.031 4.11 2.76 EIF3B P55884 ITNDFYPEEDGK 0.142 0.090 0.026 0.042 4.94 3.49 HSPA5 P11021 ITPSYVAFTPEGER 0.069 0.058 0.027 0.018 3.63 4.31 MARS P56192 ITQDIFQQLLK 0.053 0.053 0.028 0.028 2.72 2.83 PA2G4 Q9UQ80 ITSGPFEPDLYK 0.083 0.056 0.029 0.030 3.79 2.93 RPL22 P35268 ITVTSEVPFSK 0.032 0.055 0.013 0.033 2.97 2.86 MYO6 Q9UM54 IVEANPLLEAFGNAK 0.063 0.077 0.026 0.030 2.81 3.05 RARS P54136 IVFVPGCSIPLTIVK 0.059 0.046 0.017 0.022 3.10 2.94 CCT3 P49368 IVLLDSSLEYK 0.052 0.051 0.042 0.034 2.45 2.79 KRT18 P05783 IVLQIDNAR 0.064 0.094 0.028 0.034 3.42 4.30 DSG2 Q14126 IVSLEPAYPPVFYLNK 0.045 0.128 0.043 0.061 2.20 4.21 ACO2 Q99798 IVYGHLDDPASQEIER 0.038 0.033 0.023 0.027 3.43 3.40 POTEKP Q9BYX7 IWHHTFYNELR 0.068 0.094 0.023 0.024 3.04 3.71 PPP1CA P62136 IYGFYDECK 0.078 0.079 0.038 0.047 3.00 3.01 XPO1 O14980 IYLDMLNVYK 0.040 0.031 0.015 0.033 3.10 2.44 ACADM P11310 IYQIYEGTSQIQR 0.029 0.015 0.020 0.028 2.88 3.11 KHDRBS1 Q07666 KDDEENYLDLFSHK 0.040 0.057 0.041 0.026 1.88 2.85 CANX P27824 KDDTDDEIAK 0.103 0.065 0.032 0.030 3.42 3.19 PSMC4 P43686 KDEQEHEFYK 0.079 0.054 0.027 0.030 3.39 2.70 KRT8 P05787 KDVDEAYMNK 0.091 0.098 0.022 0.029 4.07 4.19 HIST1H2BC P62807 KESYSVYVYK 0.013 0.013 0.017 0.018 1.99 2.66 MYH10 P35580 KFDQLLAEEK 0.047 0.052 0.029 0.036 2.09 2.33 TUFM P49411 KGDECELLGHSK 0.031 0.034 0.024 0.027 2.64 2.67 RPL4 P36578 KLDELYGTWR 0.050 0.060 0.034 0.019 2.40 3.02 EEF1G P26641 KLDPGSEETQTLVR 0.053 0.056 0.038 0.019 2.43 3.03 SCP2 P22307 KLEEEGEQFVK 0.022 0.032 0.015 0.030 1.87 2.46 MYH9 P35579 KLEEEQIILEDQNCK 0.064 0.062 0.027 0.031 2.60 2.42 CAPZB P47756 KLEVEANNAFDQYR 0.051 0.054 0.019 0.018 2.72 2.81 HMGB1L1 B2RPK0 KLGEMWNNTAADDK 0.028 0.021 0.019 0.037 2.25 1.69 DES P17661 KLLEGEESR 0.050 0.090 0.032 0.024 2.14 3.74 MRPS7 Q9Y2R9 KPVEELTEEEK 0.042 0.063 0.033 0.030 2.39 3.25 MYH9 P35579 KQELEEICHDLEAR 0.072 0.065 0.021 0.023 2.93 2.73 XRCC6 P12956 KQELLEALTK 0.047 0.038 0.022 0.022 2.77 2.68 HSPA5 P11021 KSDIDEIVLVGGSTR 0.050 0.058 0.039 0.027 2.19 3.29 HNRNPCL1 O60812 KSDVEAIFSK 0.037 0.040 0.034 0.032 2.14 2.85 RPL10A P62906 KYDAFLASESLIK 0.052 0.053 0.020 0.025 2.89 2.91 BCAP31 P51572 KYDDVTEK 0.044 0.047 0.044 0.025 1.79 2.78 EIF5A P63241 KYEDICPSTHNMDVPNIK 0.091 0.057 0.024 0.037 4.00 2.53 TUFM P49411 KYEEIDNAPEER 0.045 0.048 0.027 0.025 3.13 3.53 KRT10 P13645 LAADDFR 0.069 0.089 0.025 0.030 3.68 4.12 CLIC1 O00299 LAALNPESNTAGLDIFAK 0.115 0.077 0.018 0.042 4.77 3.13 KPNB1 Q14974 LAATNALLNSLEFTK 0.047 0.049 0.040 0.038 2.38 2.54 PDIA6 Q15084 LAAVDATVNQVLASR 0.047 0.055 0.024 0.020 2.73 3.51 LMNA P02545 LADALQELR 0.036 0.050 0.033 0.034 2.14 3.19 MRPL38 Q96DV4 LAEYYGLYR 0.041 0.046 0.028 0.023 3.04 3.35

163

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

UBA1 P22314 LAGTQPLEVLEAVQR 0.069 0.049 0.019 0.028 4.36 3.07 ENO1 P06733 LAM~QEFMILPVGAANFR 0.063 0.039 0.014 0.022 4.02 2.76 ATL3 Q6DD88 LAMDEIFQK 0.031 0.046 0.032 0.028 1.90 2.83 ENO1 P06733 LAMQEFM~ILPVGAANFR 0.064 0.040 0.015 0.031 4.02 2.72 RPL23A P62750 LAPDYDALDVANK 0.063 0.068 0.021 0.025 3.57 3.46 PES1 O00541 LAQEEESEAK 0.065 0.099 0.041 0.042 2.61 3.68 OXSR1 O95747 LASGVEGSDIPDDGK 0.065 0.048 0.018 0.034 3.65 2.62 AP1B1 Q10567 LASQANIAQVLAELK 0.045 0.048 0.024 0.019 2.77 2.86 CLTC Q00610 LASTLVHLGEYQAAVDGAR 0.048 0.045 0.031 0.025 2.83 2.88 LATDLLSLMPSLTSGEVAHC TBRG4 Q969Z0 0.062 0.095 0.060 0.045 2.45 3.57 AK HSPB1 P04792 LATQSNEITIPVTFESR 0.107 0.046 0.016 0.032 5.05 2.50 TUBB1 Q9H4B7 LAVNMVPFPR 0.056 0.047 0.024 0.040 3.55 2.74 STIP1 P31948 LAYINPDLALEEK 0.105 0.065 0.016 0.032 5.34 3.38 MATR3 P43243 LCSLFYTNEEVAK 0.052 0.059 0.028 0.035 2.72 3.38 ACTR2 P61160 LCYVGYNIEQEQK 0.056 0.062 0.011 0.032 3.37 3.16 KRT7 P08729 LDADPSLQR 0.050 0.100 0.029 0.026 2.41 4.09 NAP1L1 P55209 LDGLVETPTGYIESLPR 0.129 0.090 0.026 0.041 5.24 3.64 PKM2 P14618 LDIDSPPITAR 0.060 0.060 0.029 0.028 3.22 3.00 DHX15 O43143 LDLGEDYPSGK 0.080 0.071 0.026 0.039 3.89 3.41 TKT P29401 LDNLVAILDINR 0.040 0.021 0.019 0.024 3.32 2.74 MYH10 P35580 LDPHLVLDQLR 0.079 0.070 0.021 0.023 3.66 3.02 VCP P55072 LDQLIYIPLPDEK 0.055 0.056 0.026 0.031 3.02 3.09 RPS8 P62241 LDVGNFSWGSECCTR 0.075 0.065 0.025 0.028 3.92 3.42 RPS9 P46781 LDYILGLK 0.059 0.060 0.019 0.030 3.33 3.06 KRT7 P08729 LEAAIAEAEER 0.049 0.083 0.032 0.033 2.33 3.40 LMNA P02545 LEAALGEAK 0.037 0.056 0.026 0.022 2.07 3.26 KRT18 P05783 LEAEIATYR 0.098 0.097 0.027 0.028 4.58 4.28 KRT8 P05787 LEAELGNMQGLVEDFK 0.084 0.101 0.024 0.028 4.32 4.52 EIF1AX P47813 LEAMCFDGVK 0.066 0.063 0.038 0.035 3.04 2.69 KRT8 P05787 LEGLTDEINFLR 0.060 0.088 0.034 0.033 3.54 4.10 CAPN1 P07384 LEICNLTPDALK 0.039 0.029 0.012 0.035 2.78 2.03 HADHB P55084 LEQDEYALR 0.021 0.029 0.022 0.022 2.16 3.31 KRT13 P13646 LEQEIATYR 0.049 0.084 0.028 0.025 2.96 4.10 KRT8 P05787 LESGMQNMSIHTK 0.076 0.095 0.023 0.028 3.97 4.42 ETFA P13804 LEVAPISDIIAIK 0.032 0.048 0.029 0.026 2.32 3.50 C14orf166 Q9Y224 LEYGDNAEK 0.061 0.060 0.022 0.028 3.21 3.11 HSPB1 P04792 LFDQAFGLPR 0.083 0.044 0.019 0.035 4.11 2.32 RPS9 P46781 LFEGNALLR 0.071 0.062 0.020 0.026 3.99 3.36 RBMX P38159 LFIGGLNTETNEK 0.034 0.051 0.022 0.021 2.52 3.15 U2AF2 P26368 LFIGGLPNYLNDDQVK 0.061 0.059 0.029 0.033 3.14 3.23 ILF3 Q12906 LFPDTPLALDANK 0.043 0.042 0.023 0.023 2.77 3.15 ARCN1 P48444 LFTAESLIGLK 0.065 0.053 0.026 0.029 2.63 2.35 SFPQ P23246 LFVGNLPADITEDEFK 0.049 0.072 0.013 0.032 3.48 3.87 DHCR24 Q15392 LGCQDAFPEVYDK 0.056 0.064 0.031 0.033 2.79 3.31 PGK1 P00558 LGDVYVNDAFGTAHR 0.055 0.035 0.014 0.037 3.84 2.40 ATP5J2 P56134 LGELPSWILM~R 0.023 0.032 0.038 0.020 2.17 3.56 ATP5J2 P56134 LGELPSWILMR 0.029 0.040 0.030 0.030 2.66 3.33 HMGB1L1 B2RPK0 LGEMWNNTAADDK 0.034 0.023 0.016 0.028 2.77 2.12 TBCB Q99426 LGEYEDVSR 0.068 0.050 0.031 0.039 3.28 2.65 DHX9 Q08211 LGGIGQFLAK 0.043 0.048 0.025 0.033 2.58 3.06 TALDO1 P37837 LGGSQEDQIK 0.035 0.030 0.017 0.029 2.62 2.06 S100A9 P06702 LGHPDTLNQGEFK 0.103 0.050 0.018 0.062 4.11 1.56 TPD52 P55327 LGINSLQELK 0.063 0.040 0.018 0.032 3.29 2.32 IQGAP1 P46940 LGLAPQIQDLYGK 0.050 0.050 0.020 0.029 2.55 2.51 PHB2 Q99623 LGLDYEER 0.044 0.041 0.022 0.026 3.51 3.67 EPPK1 P58107 LGLLDTQTSQVLTAVDK 0.063 0.069 0.023 0.029 3.02 3.00 TMEM65 Q6PI78 LGLSIPDLTPK 0.040 0.039 0.020 0.023 2.50 3.10

164

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

PSAP P07602 LGPGMADICK 0.019 0.033 0.029 0.025 1.43 2.85 MOGS Q13724 LGPLLDILADSR 0.051 0.055 0.030 0.023 2.71 3.54 CKMT2 P17540 LGYILTCPSNLGTGLR 0.020 0.023 0.025 0.025 2.57 3.64 DIAPH1 O60610 LHDEKEETAGSYDSR 0.148 0.089 0.023 0.038 5.05 3.19 TUBB2A Q13885 LHFFM~PGFAPLTSR 0.056 0.039 0.018 0.028 3.81 2.69 TUBB2A Q13885 LHFFMPGFAPLTSR 0.054 0.042 0.009 0.028 4.02 2.74 LHLSGIDANPNALFPPVEFP FASN P49327 0.185 0.060 0.008 0.038 9.75 3.56 APR FAM3C Q92520 LIADLGSTSITNLGFR 0.040 0.070 0.041 0.037 1.87 2.70 FLNA P21333 LIALLEVLSQK 0.053 0.088 0.022 0.027 2.33 3.27 IDH2 P48735 LIDDMVAQVLK 0.019 0.023 0.022 0.020 1.99 2.71 TRAPPC3 O43617 LIEDFLAR 0.060 0.045 0.039 0.030 2.89 2.57 DNAJC3 Q13217 LIESAEELIR 0.073 0.099 0.055 0.037 2.56 3.80 RPS9 P46781 LIGEYGLR 0.067 0.054 0.021 0.037 3.80 2.88 ARPC3 O15145 LIGNMALLPIR 0.057 0.061 0.030 0.029 3.01 3.24 GRB7 Q14451 LIGQQGLVDGLFLVR 0.056 0.074 0.060 0.061 2.26 2.69 GNB1 P62873 LIIWDSYTTNK 0.045 0.081 0.047 0.054 1.96 2.91 P4HB P07237 LITLEEEM~TK 0.034 0.054 0.015 0.022 2.13 3.12 P4HB P07237 LITLEEEMTK 0.029 0.042 0.023 0.022 2.05 2.75 MT‐ATP6 P00846 LITTQQWLIK 0.018 0.026 0.019 0.014 2.26 3.20 PRDX5 P30044 LLADPTGAFGK 0.043 0.029 0.023 0.015 2.93 3.11 PRKDC P78527 LLALNSLYSPK 0.038 0.034 0.015 0.026 2.95 2.66 MIF P14174 LLCGLLAER 0.049 0.027 0.013 0.038 3.91 2.19 TUFM P49411 LLDAVDTYIPVPAR 0.047 0.040 0.022 0.019 4.06 3.92 PSMA4 P25789 LLDEVFFSEK 0.054 0.051 0.024 0.025 3.02 2.77 NHP2L1 P55769 LLDLVQQSCNYK 0.042 0.054 0.021 0.021 2.88 3.15 UBE2V1 Q13404 LLEELEEGQK 0.081 0.053 0.026 0.051 3.49 2.23 LMNA P02545 LLEGEEER 0.036 0.054 0.030 0.028 2.31 3.41 DES P17661 LLEGEESR 0.068 0.099 0.026 0.028 3.67 4.46 KRT4 P19013 LLEGEEYR 0.036 0.035 0.029 0.032 2.97 2.72 RPS16 P62249 LLEPVLLLGK 0.063 0.072 0.020 0.031 3.47 3.30 HNRNPU Q00839 LLEQYKEESK 0.048 0.056 0.033 0.016 2.27 4.13 S100A8 P05109 LLETECPQYIR 0.072 0.031 0.020 0.031 3.77 1.59 RG9MTD1 Q7L0Y3 LLETTEEDK 0.062 0.067 0.041 0.030 3.01 3.71 TALDO1 P37837 LLGELLQDNAK 0.039 0.028 0.022 0.026 2.52 2.24 CAPZA1 P52907 LLLNNDNLLR 0.036 0.054 0.025 0.030 2.63 2.87 APMAP Q9HDC9 LLLSSETPIEGK 0.029 0.045 0.025 0.022 2.24 3.15 B4E3B6 LLQDFFDGR 0.085 0.051 0.030 0.031 3.50 2.80 HSPA2 P54652 LLQDFFNGK 0.076 0.078 0.036 0.042 3.13 3.09 HSPA1A P08107 LLQDFFNGR 0.070 0.047 0.043 0.040 2.67 2.53 RARS P54136 LLQQEEEIK 0.043 0.044 0.022 0.019 2.70 2.74 SLC3A2 P08195 LLTSFLPAQLLR 0.055 0.059 0.045 0.039 2.39 2.65 SLC9A3R1 O14745 LLVVDPETDEQLQK 0.094 0.038 0.040 0.055 3.65 1.91 EIF3M Q7L2H7 LLYEALVDCK 0.081 0.062 0.022 0.045 3.64 2.68 CLTC Q00610 LLYNNVSNFGR 0.037 0.043 0.030 0.024 2.41 2.81 ENO1 P06733 LM~IEMDGTENK 0.048 0.039 0.018 0.028 3.03 2.42 RARS P54136 LMDLLGEGLK 0.046 0.036 0.028 0.038 2.47 2.12 STIP1 P31948 LMDVGLIAIR 0.063 0.050 0.038 0.033 3.11 2.83 S100A6 P06703 LMEDLDR 0.079 0.050 0.018 0.042 4.09 2.51 ENO1 P06733 LMIEMDGTENK 0.053 0.041 0.015 0.037 3.24 2.30 SET Q01105 LNEQASEEILK 0.034 0.033 0.025 0.027 2.40 2.49 PKM2 P14618 LNFSHGTHEYHAETIK 0.059 0.057 0.010 0.028 3.78 2.77 MYL12A P19105 LNGTDPEDVIR 0.078 0.074 0.022 0.039 3.57 2.98 RPS6 P62753 LNISFPATGCQK 0.065 0.049 0.028 0.031 3.17 2.62 PPP1CA P62136 LNLDSIIGR 0.067 0.069 0.053 0.048 2.43 2.84 RPS25 P62851 LNNLVLFDK 0.051 0.055 0.025 0.030 2.88 2.74 CCDC47 Q96A33 LNQENEHIYNLWCSGR 0.113 0.111 0.041 0.042 4.01 4.49 CNDP2 Q96KP4 LPDGSEIPLPPILLGR 0.038 0.028 0.018 0.025 3.43 2.45

165

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

KRT7 P08729 LPDIFEAQIAGLR 0.050 0.094 0.028 0.024 2.41 3.90 FASN P49327 LPEDPLLSGLLDSPALK 0.156 0.054 0.012 0.040 6.94 2.78 PRDX6 P30041 LPFPIIDDR 0.045 0.030 0.024 0.030 3.27 2.44 TMCO1 Q9UM00 LPFTPLSYIQGLSHR 0.086 0.102 0.032 0.026 3.78 4.46 CCT7 Q99832 LPIGDVATQYFADR 0.075 0.054 0.016 0.033 4.61 3.26 EEF1A1 P68104 LPLQDVYK 0.107 0.073 0.009 0.034 5.08 3.16 CAPN2 P17655 LPPGEYILVPSTFEPNK 0.079 0.067 0.009 0.035 4.77 3.35 TRIM25 Q14258 LPTFGAPEQLVDLK 0.053 0.043 0.036 0.031 2.41 2.40 IQGAP1 P46940 LPYDVTPEQALAHEEVK 0.070 0.064 0.025 0.032 2.81 2.98 KRT7 P08729 LQAEIDNIK 0.042 0.084 0.028 0.025 2.07 3.36 KRT8 P05787 LQAEIEGLK 0.044 0.089 0.020 0.029 3.34 3.98 S100A6 P06703 LQDAEIAR 0.077 0.052 0.018 0.042 4.06 2.68 P4HA1 P13674 LQDTYNLDTDTISK 0.091 0.148 0.039 0.039 3.43 5.13 EZR P15311 LQDYEEK 0.099 0.077 0.034 0.039 3.68 2.87 SWAP70 Q9UH65 LQEALEDER 0.068 0.059 0.029 0.036 3.20 2.96 SMC2 O95347 LQEELSENDK 0.056 0.050 0.036 0.035 3.17 2.74 LMNA P02545 LQEKEDLQELNDR 0.036 0.058 0.027 0.027 1.93 3.39 DHRS2 Q13268 LQGEGLSVAGIVCHVGK 0.011 0.015 0.024 0.019 1.83 2.84 EEF1B2 P24534 LQIQCVVEDDK 0.083 0.064 0.028 0.034 3.73 2.90 RAB2A P61019 LQIWDTAGQESFR 0.068 0.063 0.028 0.032 3.26 3.22 PSMD3 O43242 LQLDSPEDAEFIVAK 0.083 0.062 0.018 0.031 3.79 2.92 KRT18 P05783 LQLETEIEALK 0.048 0.079 0.046 0.055 2.37 2.87 EIF4A1 P60842 LQMEAPHIIVGTPGR 0.062 0.074 0.052 0.035 2.51 3.14 ALDOA P04075 LQSIGTENTEENR 0.116 0.073 0.017 0.031 5.38 3.52 LMNA P02545 LQTMKEELDFQK 0.043 0.049 0.030 0.018 2.07 3.34 FASN P49327 LQVVDQPLPVR 0.095 0.043 0.025 0.039 4.86 2.61 RPS4X P62701 LRECLPLIIFLR 0.067 0.072 0.032 0.032 3.08 3.41 CD59 P13987 LRENELTYYCCK 0.080 0.127 0.038 0.034 2.61 4.35 CAP1 Q01518 LSDLLAPISEQIK 0.068 0.065 0.010 0.032 3.64 2.89 PDCD6 O75340 LSDQFHDILIR 0.088 0.050 0.018 0.039 4.76 2.85 SND1 Q7KZF4 LSECEEQAK 0.074 0.067 0.031 0.048 3.04 2.49 KRT8 P05787 LSELEAALQR 0.077 0.094 0.029 0.032 4.19 4.47 GANAB Q14697 LSFQHDPETSVLVLR 0.037 0.040 0.025 0.023 2.63 3.16 ETHE1 O95571 LSGAQADLHIEDGDSIR 0.030 0.042 0.021 0.023 2.69 3.64 PRDX6 P30041 LSILYPATTGR 0.042 0.029 0.030 0.029 2.87 2.55 FASN P49327 LSIPTYGLQCTR 0.080 0.043 0.025 0.041 4.18 2.50 PTBP1 P26599 LSLDGQNIYNACCTLR 0.052 0.054 0.025 0.021 3.04 3.71 CAT P04040 LSQEDPDYGIR 0.034 0.044 0.033 0.032 2.19 3.26 LONP1 P36776 LSSDVLTLLIK 0.045 0.040 0.030 0.021 2.47 2.99 TALDO1 P37837 LSSTWEGIQAGK 0.046 0.031 0.017 0.032 2.62 2.11 IQGAP1 P46940 LTAEEMDER 0.059 0.055 0.024 0.037 2.81 2.71 CLTC Q00610 LTDQLPLIIVCDR 0.039 0.125 0.031 0.028 2.63 2.79 PDHX O00330 LTEDEEGNAK 0.041 0.036 0.025 0.024 3.14 3.48 VDAC1 P21796 LTFDSSFSPNTGK 0.023 0.028 0.027 0.021 2.06 2.84 VDAC2 P45880 LTFDTTFSPNTGK 0.039 0.049 0.021 0.020 2.71 3.55 PTGES3 Q15185 LTFSCLGGSDNFK 0.057 0.041 0.017 0.031 3.51 2.55 CAND1 Q86VP6 LTLIDPETLLPR 0.048 0.040 0.036 0.029 2.87 2.98 VDAC2 P45880 LTLSALVDGK 0.033 0.048 0.015 0.020 2.67 3.46 LTLYDIAHTPGVAADLSHIE MDH2 P40926 0.031 0.043 0.020 0.020 2.89 3.66 TK RPS8 P62241 LTPEEEEILNK 0.069 0.075 0.016 0.029 3.84 3.46 PPP2R1A P30153 LTQDQDVDVK 0.043 0.068 0.032 0.038 2.34 2.92 RPL14 P50914 LVAIVDVIDQNR 0.042 0.051 0.021 0.030 2.92 3.10 HK1 P19367 LVDEYSLNAGK 0.035 0.042 0.023 0.032 2.47 3.03 RPS12 P25398 LVEALCAEHQINLIK 0.048 0.051 0.012 0.030 3.64 2.84 CAPZB P47756 LVEDMENK 0.039 0.055 0.022 0.031 2.37 2.75 SAR1A Q9NR31 LVFLGLDNAGK 0.068 0.053 0.016 0.029 3.39 2.68 TPM3 P06753 LVIIEGDLER 0.053 0.063 0.024 0.026 2.79 3.24

166

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

TPM1 P09493 LVIIESDLER 0.042 0.068 0.037 0.028 1.66 2.65 RPL30 P62888 LVILANNCPALR 0.055 0.064 0.034 0.028 3.14 3.55 TPM2 P07951 LVILEGELER 0.049 0.070 0.025 0.024 2.33 2.96 GAPDH P04406 LVINGNPITIFQER 0.059 0.038 0.015 0.029 4.21 2.68 ATP5B P06576 LVLEVAQHLGESTVR 0.025 0.025 0.018 0.020 2.77 3.39 RAB5A P20339 LVLLGESAVGK 0.057 0.048 0.027 0.039 2.84 2.61 SELENBP1 Q13228 LVLPSLISSR 0.036 0.016 0.014 0.031 4.27 2.59 HSPA1A P08107 LVNHFVEEFK 0.085 0.048 0.033 0.029 3.11 2.56 PRDX1 Q06830 LVQAFQFTDK 0.055 0.030 0.016 0.030 3.19 2.31 PRDX4 Q13162 LVQAFQYTDK 0.030 0.061 0.013 0.022 2.73 3.26 RPS27L Q71UM5 LVQSPNSYFMDVK 0.083 0.079 0.026 0.035 3.72 3.41 KRT8 P05787 LVSESSDVLPK 0.086 0.109 0.020 0.023 4.38 4.74 LRRC59 Q96AG4 LVTLPVSFAQLK 0.065 0.062 0.038 0.030 2.70 3.24 PCBP2 Q15366 LVVPASQCGSLIGK 0.083 0.060 0.015 0.034 3.69 2.78 PCBP1 Q15365 LVVPATQCGSLIGK 0.062 0.057 0.021 0.043 3.22 2.60 GNB2L1 P63244 LWDLTTGTTTR 0.087 0.071 0.030 0.038 3.70 3.16 PRKCSH P14314 LWEEQLAAAK 0.034 0.041 0.018 0.025 2.50 2.93 RPL27A P46776 LWTLVSEQTR 0.058 0.057 0.019 0.028 3.53 3.12 ANXA5 P08758 LYDAYELK 0.093 0.144 0.013 0.035 4.26 5.04 PSMD11 O00231 LYDNLLEQNLIR 0.046 0.051 0.039 0.018 2.49 3.20 TMEM70 Q9BUB7 LYHEATTDTYK 0.070 0.065 0.020 0.035 4.00 3.46 TCEB2 Q15370 LYKDDQLLDDGK 0.079 0.068 0.044 0.058 2.92 2.44 FAM82B Q96DB5 LYQLLTQYK 0.016 0.028 0.026 0.027 1.89 2.57 RPL31 P62899 LYTLVTYVPVTTFK 0.056 0.061 0.015 0.033 3.40 3.23 ACTN4 O43707 MAPYQGPDAVPGALDYK 0.061 0.066 0.027 0.027 3.28 3.48 EIF3B P55884 MAQELYMEQK 0.083 0.077 0.033 0.034 3.22 2.96 CCDC6 Q16204 MAQYLEEER 0.060 0.046 0.029 0.037 3.09 2.80 PDIA3 P30101 MDATANDVPSPYEVR 0.054 0.058 0.025 0.019 3.30 3.76 FLNB O75369 MDCQETPEGYK 0.066 0.097 0.020 0.026 3.30 3.90 VCP P55072 MDELQLFR 0.053 0.056 0.033 0.030 2.81 3.28 FLNB O75369 MDGTYACSYTPVK 0.067 0.096 0.041 0.031 2.62 3.72 CNP P09543 MDLVTYFGK 0.011 0.026 0.015 0.025 1.05 2.02 P4HB P07237 MDSTANEVEAVK 0.034 0.043 0.025 0.021 1.96 2.94 HSPA9 P38646 MEEFKDQLPADECNK 0.087 0.060 0.037 0.033 3.80 3.62 GSTK1 Q9Y2Q3 MELLAHLLGEK 0.018 0.031 0.028 0.017 1.92 2.71 GLB1 P16278 MFEIDYSR 0.011 0.017 0.021 0.016 1.73 2.65 RPS21 P63220 MGESDDSILR 0.079 0.071 0.022 0.029 3.91 3.40 MDH2 P40926 MISDAIPELK 0.029 0.034 0.019 0.015 2.68 3.51 CALM1 P62158 MKDTDSEEEIR 0.058 0.047 0.025 0.032 2.86 2.59 ARF1 P84077 MLAEDELR 0.072 0.054 0.019 0.036 3.87 3.08 ARF4 P18085 MLLVDELR 0.058 0.079 0.043 0.035 2.52 3.22 EHD1 Q9H4M9 MQELLQTQDFSK 0.091 0.090 0.009 0.039 4.17 3.37 EPPK1 P58107 MSIYQAMWK 0.052 0.053 0.039 0.025 2.24 2.42 KRT14 P02533 MSVEADINGLR 0.043 0.074 0.033 0.019 2.53 4.27 HSPA8 P11142 MVNHFIAEFK 0.109 0.090 0.022 0.034 4.57 3.62 LDHB P07195 MVVESAYEVIK 0.091 0.044 0.017 0.033 4.81 2.65 FASN P49327 MVVPGLDGAQIPR 0.133 0.052 0.008 0.039 6.44 2.84 SNRNP70 P08621 MWDPHNDPNAQGDAFK 0.087 0.079 0.032 0.023 3.80 4.35 PHGDH O43175 NAGNCLSPAVIVGLLK 0.041 0.019 0.020 0.026 2.20 1.33 UQCRC2 P22695 NALANPLYCPDYR 0.036 0.035 0.020 0.020 3.54 4.02 PRCP P42785 NALDPMSVLLAR 0.030 0.021 0.030 0.031 2.26 2.80 HSPA1L P34931 NALESYAFNMK 0.088 0.051 0.025 0.035 3.44 2.60 HSPA9 P38646 NAVITVPAYFNDSQR 0.052 0.065 0.023 0.023 4.06 4.53 RPS8 P62241 NCIVLIDSTPYR 0.070 0.071 0.023 0.032 3.89 3.23 HIST1H2AA Q96QV6 NDEELNK 0.021 0.018 0.030 0.028 2.03 2.60 USO1 O60763 NDGVLLLQALTR 0.032 0.041 0.023 0.023 2.87 2.89 NCL P19338 NDLAVVDVR 0.050 0.063 0.024 0.019 3.41 3.63

167

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

CORO1B Q9BR76 NDQCYEDIR 0.051 0.046 0.026 0.033 2.99 2.96 UBA1 P22314 NEEDAAELVALAQAVNAR 0.073 0.034 0.020 0.046 4.47 2.29 ZMPSTE24 O75844 NEEEGNSEEIK 0.049 0.070 0.017 0.024 3.11 3.72 SUPT16H Q9Y5B9 NEGNIFPNPEATFVK 0.051 0.033 0.026 0.028 3.19 3.16 HSPA5 P11021 NELESYAYSLK 0.053 0.063 0.028 0.024 2.77 3.41 ACAT1 P24752 NEQDAYAINSYTR 0.030 0.034 0.023 0.021 3.05 3.86 EIF3A Q14152 NETDEDGWTTVR 0.122 0.090 0.030 0.033 4.84 3.71 U2AF2 P26368 NFAFLEFR 0.060 0.055 0.039 0.035 2.85 3.18 ANXA5 P08758 NFATSLYSMIK 0.064 0.054 0.032 0.048 2.85 2.37 P4HB P07237 NFEDVAFDEK 0.029 0.046 0.026 0.027 1.97 2.70 PABPC1 P11940 NFGEDMDDER 0.081 0.066 0.030 0.046 3.57 2.79 S100A14 Q9HCY8 NFHQYSVEGGK 0.054 0.118 0.038 0.034 2.51 4.38 HNRNPU Q00839 NFILDQTNVSAAAQR 0.054 0.057 0.034 0.030 2.99 3.43 MYH9 P35579 NFINNPLAQADWAAK 0.072 0.065 0.021 0.029 3.23 2.91 DERL1 Q9BUN8 NFLSTPQFLYR 0.068 0.089 0.031 0.028 3.30 4.08 DHX9 Q08211 NFLYAWCGK 0.038 0.043 0.029 0.033 2.43 2.94 TAGLN2 P37802 NFSDNQLQEGK 0.052 0.057 0.016 0.038 2.82 2.53 HSPA4 P34932 NFTTEQVTAMLLSK 0.052 0.051 0.025 0.039 2.47 2.47 EIF4G1 Q04637 NHDEESLECLCR 0.137 0.099 0.031 0.053 4.79 3.29 KRT19 P08727 NHEEEISTLR 0.051 0.089 0.026 0.028 2.99 4.24 ANXA4 P09525 NHLLHVFDEYK 0.032 0.026 0.014 0.033 2.39 2.05 RAB10 P61026 NIDEHANEDVER 0.059 0.072 0.029 0.030 3.01 3.69 RPLP2 P05387 NIEDVIAQGIGK 0.063 0.066 0.017 0.026 3.53 3.31 RPN1 P04843 NIEIDSPYEISR 0.049 0.057 0.023 0.022 3.17 3.70 S100A9 P06702 NIETIINTFHQYSVK 0.099 0.045 0.028 0.055 3.66 1.50 SLC1A5 Q15758 NIFPSNLVSAAFR 0.123 0.110 0.056 0.052 4.02 4.02 RPL4 P36578 NIPGITLLNVSK 0.044 0.054 0.023 0.029 2.88 2.86 ARF1 P84077 NISFTVWDVGGQDK 0.061 0.055 0.024 0.041 3.06 2.66 XRCC6 P12956 NIYVLQELDNPGAK 0.053 0.039 0.030 0.023 3.27 3.12 RPS6 P62753 NKEEAAEYAK 0.069 0.064 0.027 0.040 3.13 2.89 XRN2 Q9H0D6 NKFDVDAADEK 0.062 0.069 0.043 0.039 2.38 2.98 VAMP8 Q9BV40 NKTEDLEATSEHFK 0.075 0.098 0.038 0.065 2.57 3.12 KRT1 P04264 NKYEDEINK 0.085 0.100 0.033 0.030 3.68 4.23 SYNCRIP O60506 NLANTVTEEILEK 0.063 0.067 0.024 0.035 2.86 2.99 HNRNPR O43390 NLATTVTEEILEK 0.046 0.050 0.030 0.031 2.54 3.08 PABPC1 P11940 NLDDGIDDER 0.101 0.083 0.028 0.040 4.30 3.48 PABPC4L P0CB38 NLDDTIDDEK 0.078 0.069 0.025 0.044 3.73 2.87 TUBA1A Q71U36 NLDIERPTYTNLNR 0.066 0.045 0.023 0.033 3.69 2.61 PDIA6 Q15084 NLEPEWAAAASEVK 0.045 0.056 0.023 0.013 2.67 3.75 CAMK2B Q13554 NLINQMLTINPAK 0.040 0.042 0.021 0.028 2.20 2.63 CLTC Q00610 NLQNLLILTAIK 0.035 0.039 0.030 0.029 2.23 2.48 RAN P62826 NLQYYDISAK 0.061 0.049 0.016 0.027 3.70 2.83 GPI P06744 NLVTEDVMR 0.016 0.028 0.021 0.038 1.18 2.14 RPL3 P39023 NNASTDYDLSDK 0.057 0.058 0.023 0.029 3.42 3.25 CLTC Q00610 NNLAGAEELFAR 0.041 0.042 0.031 0.027 2.63 2.85 CD81 P60033 NNLCPSGSNIISNLFK 0.067 0.105 0.055 0.091 2.18 2.97 NNQFQALLQYADPVSAQH PTBP1 P26599 0.054 0.058 0.024 0.030 3.30 3.38 AK HSP90AA1 P07900 NPDDITNEEYGEFYK 0.086 0.052 0.022 0.032 4.62 3.11 HSP90AB1 P08238 NPDDITQEEYGEFYK 0.108 0.068 0.015 0.030 5.30 3.40 CMPK1 P30085 NPDSQYGELIEK 0.035 0.036 0.017 0.029 2.81 2.24 DNAJB1 P25685 NPFDTFFGQR 0.059 0.064 0.051 0.053 2.48 2.67 ACADVL P49748 NPFGNAGLLLGEAGK 0.029 0.039 0.025 0.029 1.93 2.75 MYO1C O00159 NPQSYLYLVK 0.043 0.056 0.021 0.032 2.91 2.96 IPO7 O95373 NPVWYQALTHGLNEEQR 0.135 0.089 0.021 0.024 5.51 3.48 SF3B4 Q15427 NQDATVYVGGLDEK 0.067 0.056 0.054 0.062 2.41 2.41 HNRNPA1 P09651 NQGGYGGSSSSSSYGSGR 0.059 0.064 0.031 0.026 3.20 3.87 HSPA5 P11021 NQLTSNPENTVFDAK 0.038 0.065 0.036 0.023 2.25 3.70

168

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

HSPA8 P11142 NQTAEKEEFEHQQK 0.108 0.093 0.036 0.036 3.85 3.57 HSPA1A P08107 NQVALNPQNTVFDAK 0.122 0.054 0.036 0.028 4.04 2.92 HSPA8 P11142 NQVAMNPTNTVFDAK 0.121 0.104 0.025 0.038 4.88 4.01 SNRNP200 O75643 NSAFESLYQDK 0.055 0.068 0.053 0.042 1.98 2.83 HSPA8 P11142 NSLESYAFNM~K 0.051 0.061 0.046 0.049 2.19 2.38 HSPA8 P11142 NSLESYAFNMK 0.087 0.074 0.033 0.039 3.59 3.14 NAPA P54920 NSQSFFSGLFGGSSK 0.040 0.034 0.047 0.047 2.05 2.22 TUBB2A Q13885 NSSYFVEWIPNNVK 0.070 0.043 0.013 0.036 3.63 2.40 PDIA6 Q15084 NSYLEVLLK 0.040 0.052 0.019 0.027 2.40 2.74 HNRNPK P61978 NTDEMVELR 0.048 0.062 0.048 0.040 2.27 3.04 DARS2 Q6PI48 NTEIGFLQDALSK 0.022 0.025 0.022 0.018 2.45 3.27 PKM2 P14618 NTGIICTIGPASR 0.083 0.066 0.014 0.034 4.42 3.14 ARCN1 P48444 NTLEWCLPVIDAK 0.041 0.039 0.032 0.030 2.23 2.20 FLNB O75369 NTVELLVEDK 0.044 0.073 0.044 0.040 2.09 2.68 SLC25A1 P53007 NTWDCGLQILK 0.030 0.036 0.016 0.018 2.74 3.56 DEK P35659 NVGQFSGFPFEK 0.033 0.031 0.030 0.030 2.32 2.75 TAGLN2 P37802 NVIGLQMGTNR 0.059 0.064 0.032 0.033 2.83 2.91 YWHAG P61981 NVTELNEPLSNEER 0.080 0.055 0.019 0.032 3.99 3.08 YBX1 P67809 NYQQNYQNSESGEK 0.119 0.094 0.071 0.038 3.41 3.70 HNRNPA2B1 P22626 NYYEQWGK 0.044 0.046 0.030 0.027 2.60 3.19 HNRNPUL2 Q1KMD3 NYYGYQGYR 0.034 0.039 0.040 0.037 2.29 2.75 SUMF2 Q8NBJ7 PFAIDIFPVTNK 0.036 0.051 0.038 0.026 2.03 3.01 FLNB O75369 PFDLVIPFAVR 0.093 0.126 0.032 0.029 3.83 5.03 TUFM P49411 PFLLPVEAVYSVPGR 0.046 0.042 0.023 0.021 4.12 3.94 RAN P62826 PFLWLAR 0.069 0.047 0.026 0.030 3.98 2.97 KRT7 P08729 PGGLGSSSLYGLGASR 0.048 0.095 0.024 0.023 2.31 3.94 HEXB P07686 PGPALWPLPLSVK 0.037 0.042 0.028 0.012 2.69 3.37 FLNB O75369 PGTYVIYVR 0.065 0.099 0.046 0.032 2.61 3.78 FAM114A2 Q9NRY5 PLAENEEGEK 0.040 0.039 0.022 0.030 2.62 2.43 ANXA2 P07355 PLYFADR 0.053 0.078 0.026 0.040 2.21 2.91 VDAC2 P45880 PMCIPPSYADLGK 0.047 0.533 0.026 0.023 2.93 3.62 EEF1A1 P68104 PMCVESFSDYPPLGR 0.122 0.092 0.022 0.028 5.57 4.04 PGAM1 P18669 PMQFLGDEETVR 0.061 0.051 0.018 0.024 4.36 3.59 ARL8A Q96BM9 PQLQGIPVLVLGNK 0.074 0.103 0.041 0.034 2.70 3.91 BANF1 O75531 PVGSLAGIGEVLGK 0.033 0.031 0.021 0.028 2.36 2.40 KRT18 P05783 PVSSAASVYAGAGGSGSR 0.097 0.101 0.027 0.024 4.42 4.55 COPB2 P35606 PYLISGADDR 0.050 0.043 0.035 0.029 2.52 2.55 HSPA9 P38646 QAASSLQQASLK 0.052 0.063 0.023 0.024 3.37 4.01 RPL12 P30050 QAQIEVVPSASALIIK 0.056 0.063 0.018 0.024 3.38 3.32 ANXA2 P07355 QDIAFAYQR 0.045 0.070 0.035 0.034 1.90 2.68 ARF1 P84077 QDLPNAMNAAEITDK 0.072 0.063 0.017 0.031 3.78 3.15 KRT7 P08729 QEELEAALQR 0.047 0.086 0.031 0.029 2.32 3.65 POTEKP Q9BYX7 QEYDESGPSIVHR 0.067 0.097 0.029 0.025 2.95 3.90 DKC1 O60832 QEYVDYSESAK 0.039 0.050 0.027 0.024 2.55 2.99 PRDX1 Q06830 QGGLGPMNIPLVSDPK 0.056 0.038 0.010 0.033 4.38 2.58 ACO2 Q99798 QGLLPLTFADPADYNK 0.031 0.021 0.016 0.023 3.19 3.48 P4HA1 P13674 QLDEGEISTIDK 0.094 0.151 0.053 0.043 2.94 5.03 TPM1 P09493 QLEDELVSLQK 0.041 0.065 0.032 0.034 1.45 2.24 PRKDC P78527 QLFSSLFSGILK 0.032 0.032 0.027 0.023 2.69 2.71 ALDOA P04075 QLLLTADDR 0.107 0.068 0.014 0.032 5.11 3.25 MYH11 P35749 QLLQANPILEAFGNAK 0.068 0.071 0.020 0.030 3.12 2.84 EZR P15311 QLLTLSSELSQAR 0.055 0.052 0.043 0.040 2.48 2.50 PSME1 Q06323 QLVHELDEAEYR 0.026 0.050 0.019 0.031 2.08 2.04 KRT8 P05787 QLYEEEIR 0.079 0.089 0.031 0.031 4.19 4.28 TAGLN2 P37802 QMEQISQFLQAAER 0.059 0.056 0.022 0.035 3.24 2.83 COPA P53621 QQPLFVSGGDDYK 0.046 0.045 0.018 0.031 2.88 2.42 TPI1 P60174 QSLGELIGTLNAAK 0.049 0.033 0.014 0.032 3.19 2.17

169

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

KRT19 P08727 QSSATSSFGGLGGGSVR 0.049 0.089 0.024 0.025 2.93 4.15 LDHA P00338 QVVESAYEVIK 0.116 0.085 0.015 0.035 5.38 3.53 RPS9 P46781 QVVNIPSFIVR 0.072 0.063 0.026 0.036 3.92 3.37 PTMA P06454 RAAEDDEDDDVDTK 0.049 0.031 0.035 0.044 2.49 2.06 RPS18 P62269 RAGELTEDEVER 0.060 0.069 0.040 0.032 2.69 3.30 MYH9 P35579 RALEQQVEEMK 0.053 0.063 0.036 0.036 2.18 2.52 HSP90AB3P Q58FF7 RAPFDLFENK 0.056 0.067 0.040 0.034 2.55 2.80 ACTN4 O43707 RDHALLEEQSK 0.050 0.048 0.033 0.020 2.22 3.03 DHX15 O43143 REVDDLGPEVGDIK 0.066 0.060 0.030 0.039 3.35 2.87 HSPA8 P11142 RFDDAVVQSDMK 0.057 0.106 0.048 0.031 2.31 3.38 HNRNPA1L2 Q32P51 RGFAFVTFDDHDSVDK 0.051 0.059 0.032 0.020 2.55 3.36 HNRNPA2B1 P22626 RGFGFVTFDDHDPVDK 0.046 0.049 0.031 0.023 2.56 3.25 RGTGGVDTAATGGVFDIS CKMT1A P12532 0.019 0.026 0.025 0.020 2.17 3.52 NLDR HIST1H4A P62805 RISGLIYEETR 0.016 0.032 0.024 0.024 2.20 2.64 PSMD6 Q15008 RLDEELEDAEK 0.050 0.045 0.033 0.024 2.34 2.79 RNQDLAPNSAEQASILSLV ILF2 Q12905 0.049 0.038 0.022 0.020 2.51 3.02 TK HNRNPK P61978 RPAEDMEEEQAFK 0.055 0.060 0.037 0.037 2.45 3.04 ILF3 Q12906 RPMEEDGEEK 0.036 0.054 0.025 0.023 2.02 3.01 FASN P49327 RPTPQDSPIFLPVDDTSFR 0.145 0.056 0.020 0.039 6.07 3.00 MYH9 P35579 RQLEEAEEEAQR 0.056 0.065 0.035 0.036 2.45 2.68 POR P16435 RSDEDYLYR 0.062 0.058 0.034 0.021 2.78 3.35 KRT8 P05787 RTEMENEFVLIK 0.077 0.096 0.028 0.027 3.63 4.34 ATP5A1 P25705 RTGAIVDVPVGEELLGR 0.024 0.030 0.029 0.021 2.34 3.51 HSPA9 P38646 RYDDPEVQK 0.082 0.060 0.045 0.030 3.44 3.73 LDHB P07195 SADTLWDIQK 0.089 0.039 0.022 0.025 5.15 2.78 LDHA P00338 SADTLWGIQK 0.097 0.081 0.018 0.035 4.70 3.47 S100A14 Q9HCY8 SANAEDAQEFSDVER 0.127 0.162 0.034 0.033 5.11 5.92 HK1 P19367 SANLVAATLGAILNR 0.043 0.051 0.027 0.021 2.93 3.92 WDR36 Q8NI36 SAPFFIPTIPGLVPR 0.069 0.066 0.026 0.023 4.03 3.70 KRT7 P08729 SAYGGPVGAGIR 0.039 0.075 0.027 0.033 2.16 3.45 HNRNPL P14866 SDALETLGFLNHYQMK 0.054 0.057 0.033 0.032 2.85 3.15 RPSA P08865 SDGIYIINLK 0.060 0.055 0.029 0.031 3.06 2.80 HSPA9 P38646 SDIGEVILVGGMTR 0.060 0.032 0.034 0.069 3.79 1.85 LGALS3BP Q08380 SDLAVPSELALLK 0.024 0.028 0.027 0.032 1.35 2.19 PRKDC P78527 SDPGLLTNTMDVFVK 0.034 0.029 0.022 0.020 3.12 2.89 GNG12 Q9UBI6 SDPLLIGIPTSENPFK 0.064 0.090 0.033 0.030 2.99 4.19 PA2G4 Q9UQ80 SDQDYILK 0.073 0.048 0.018 0.025 3.82 2.64 HNRNPCL1 O60812 SDVEAIFSK 0.045 0.045 0.042 0.027 2.34 3.21 PSAP P07602 SDVYCEVCEFLVK 0.019 0.064 0.030 0.056 1.26 2.53 SND1 Q7KZF4 SEAVVEYVFSGSR 0.075 0.077 0.039 0.040 3.07 2.83 AHNAK Q09666 SEDGVEGDLGETQSR 0.123 0.112 0.026 0.033 4.71 4.28 RPN1 P04843 SEDLLDYGPFR 0.037 0.046 0.038 0.034 2.33 2.96 DLD P09622 SEEQLKEEGIEYK 0.035 0.036 0.037 0.040 2.04 2.65 FASN P49327 SEGVVAVLLTK 0.053 0.053 0.036 0.036 2.65 2.22 ANXA5 P08758 SEIDLFNIR 0.063 0.063 0.025 0.042 3.30 3.06 ANXA6 P08133 SEIDLLNIR 0.093 0.034 0.034 0.034 6.55 4.23 PA2G4 Q9UQ80 SEM~EVQDAELK 0.045 0.051 0.030 0.027 2.62 2.70 PA2G4 Q9UQ80 SEMEVQDAELK 0.076 0.054 0.024 0.039 3.85 2.74 ANXA11 P50995 SETDLLDIR 0.041 0.051 0.038 0.043 2.13 2.26 CACYBP Q9HB71 SFDLLVK 0.088 0.065 0.024 0.024 3.94 2.94 DDX3X O00571 SFLLDLLNATGK 0.081 0.044 0.051 0.056 2.74 2.64 FASN P49327 SFYGSTLFLCR 0.055 0.043 0.048 0.046 2.65 2.42 HSPA8 P11142 SFYPEEVSSM~VLTK 0.134 0.086 0.013 0.029 5.40 3.50 HSPA8 P11142 SFYPEEVSSMVLTK 0.123 0.100 0.024 0.033 4.92 3.97 LMNA P02545 SGAQASSTPLSPTR 0.036 0.061 0.023 0.023 2.54 3.88 DDX6 P26196 SGAYLIPLLER 0.059 0.071 0.058 0.069 2.33 2.46

170

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

EEF1A1 P68104 SGDAAIVDMVPGK 0.127 0.062 0.029 0.041 4.60 2.46 SSBP1 Q04837 SGDSEVYQLGDVSQK 0.035 0.041 0.021 0.025 2.83 3.21 SGPFGQIFRPDNFVFGQSG TUBB2A Q13885 0.066 0.042 0.013 0.025 3.60 2.38 AGNNWAK RCC1 P18754 SGQVYSFGCNDEGALGR 0.023 0.030 0.027 0.024 2.18 3.32 DHRS7 Q9Y394 SGVDADSSYFK 0.074 0.080 0.032 0.041 2.79 3.05 HIST1H1C P16403 SGVSLAALK 0.018 0.020 0.020 0.027 2.06 2.51 CKMT1A P12532 SGYFDER 0.023 0.026 0.023 0.021 2.56 3.66 UBE2O Q9C0C9 SGYPDIGFPLFPLSK 0.089 0.072 0.033 0.062 3.85 2.39 YWHAQ P27348 SICTTVLELLDK 0.050 0.042 0.022 0.029 2.55 2.40 TPM1 B7Z722 SIDDLEEK 0.043 0.080 0.029 0.039 1.61 2.65 NSF P46459 SIDSNPYDTDK 0.036 0.039 0.019 0.026 2.70 2.89 HSP90B1 P14625 SILFVPTSAPR 0.078 0.058 0.028 0.021 3.74 3.78 TUBA1B P68363 SIQFVDWCPTGFK 0.059 0.037 0.012 0.032 3.51 2.31 EEF1D P29692 SIQLDGLVWGASK 0.104 0.072 0.023 0.029 4.05 3.24 NCL P19338 SISLYYTGEK 0.044 0.047 0.036 0.028 2.62 2.97 HSP90AB2P Q58FF8 SIYYITGESK 0.067 0.055 0.017 0.027 3.84 2.91 SFPQ P23246 SLDEMEK 0.040 0.060 0.031 0.033 2.23 3.02 KRT7 P08729 SLDLDGIIAEVK 0.043 0.076 0.027 0.029 2.05 3.10 KRT8 P05787 SLDM~DSIIAEVK 0.077 0.088 0.027 0.029 3.66 4.07 KRT8 P05787 SLDMDSIIAEVK 0.064 0.082 0.031 0.027 3.47 3.97 S100A13 Q99584 SLDVNQDSELK 0.069 0.042 0.034 0.035 3.40 2.14 PRKCSH P14314 SLEDQVEMLR 0.041 0.038 0.030 0.025 2.55 3.00 SF3A3 Q12874 SLESLDTSLFAK 0.038 0.042 0.036 0.030 2.06 2.78 ELAVL1 Q15717 SLFSSIGEVESAK 0.053 0.057 0.033 0.035 2.51 2.88 KRT18 P05783 SLGSVQAPSYGAR 0.117 0.118 0.055 0.030 3.75 5.07 CCT2 P78371 SLHDALCVLAQTVK 0.081 0.059 0.021 0.034 3.97 3.01 SEC24C P53992 SLLDFLPR 0.072 0.059 0.043 0.050 2.85 2.46 KRT19 P08727 SLLEGQEDHYNNLSASK 0.050 0.091 0.025 0.027 2.95 4.06 FASN P49327 SLLVNPEGPTLMR 0.133 0.051 0.015 0.045 6.57 2.84 ERP29 P30040 SLNILTAFQK 0.043 0.042 0.028 0.019 2.89 3.40 CBX3 Q13185 SLSDSESDDSK 0.068 0.048 0.046 0.059 2.19 2.02 VAPB O95292 SLSSSLDDTEVK 0.040 0.051 0.023 0.023 2.33 3.05 HSP90AA1 P07900 SLTNDWEDHLAVK 0.082 0.041 0.017 0.026 4.36 2.67 SNRNP200 O75643 SLVQEMVGSFGK 0.057 0.043 0.051 0.052 2.04 2.44 SLYQSAGVAPESFEYIEAHG FASN P49327 0.128 0.047 0.015 0.043 6.00 2.70 TGTK ANXA2 P07355 SLYYYIQQDTK 0.051 0.107 0.023 0.019 2.03 3.98 KRT7 P08729 SM~QDVVEDFK 0.040 0.086 0.027 0.032 2.03 3.23 C10orf58 Q9BRX8 SMLDQLGVPLYAVVK 0.047 0.058 0.026 0.028 2.51 3.11 KRT7 P08729 SMQDVVEDFK 0.042 0.074 0.024 0.033 2.06 3.00 LMNA P02545 SNEDQSMGNWQIK 0.037 0.056 0.023 0.027 2.16 3.36 KPNB1 Q14974 SNEILTAIIQGMR 0.040 0.051 0.024 0.031 3.18 3.09 P4HB P07237 SNFAEALAAHK 0.023 0.042 0.015 0.020 2.00 2.89 GGCT O75223 SNLNSLDEQEGVK 0.023 0.020 0.012 0.022 3.45 1.91 DHRS2 Q13268 SPALLLSQLLPYM~ENR 0.014 0.018 0.023 0.021 2.41 3.43 NACA Q13765 SPASDTYIVFGEAK 0.088 0.075 0.018 0.038 4.14 3.13 ATP1A1 P05023 SPDFTNENPLETR 0.048 0.036 0.033 0.031 2.72 3.16 FLNA P21333 SPFEVYVDK 0.041 0.074 0.030 0.022 1.58 2.74 FLNB O75369 SPFTVGVAAPLDLSK 0.050 0.088 0.032 0.036 2.49 3.40 AHNAK Q09666 SPQISMSDIDLNLK 0.100 0.152 0.031 0.034 3.76 5.20 RPS2 P15880 SPYQEFTDHLVK 0.068 0.061 0.019 0.027 3.61 3.20 EZR P15311 SQEQLAAELAEYTAK 0.077 0.076 0.021 0.041 3.54 2.89 MATR3 P43243 SQESGYYDR 0.056 0.068 0.040 0.039 2.77 3.61 MDH2 P40926 SQETECTYFSTPLLLGK 0.030 0.037 0.022 0.024 2.75 3.53 HSPA8 P11142 SQIHDIVLVGGSTR 0.105 0.079 0.028 0.036 4.29 3.55 KRT19 P08727 SQYEVMAEQNR 0.035 0.086 0.029 0.026 2.56 4.13 KRT8 P05787 SRAEAESMYQIK 0.068 0.093 0.026 0.029 3.34 4.24

171

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

KRT19 P08727 SRLEQEIATYR 0.052 0.084 0.029 0.027 2.70 3.99 BCLAF1 Q9NYF8 SSFYPDGGDQETAK 0.040 0.059 0.031 0.037 2.31 3.00 RPL18A Q02543 SSGEIVYCGQVFEK 0.055 0.057 0.018 0.024 3.28 3.16 IDH2 P48735 SSGGFVWACK 0.027 0.027 0.029 0.011 1.84 2.69 HNRNPU Q00839 SSGPTSLFAVTVAPPGAR 0.066 0.075 0.028 0.029 3.67 4.38 HNRNPA1L2 Q32P51 SSGPYGGGGQYFAK 0.048 0.050 0.028 0.042 2.72 2.37 SND1 Q7KZF4 SSHYDELLAAEAR 0.088 0.074 0.033 0.038 3.65 3.13 DDOST P39656 SSLNPILFR 0.043 0.042 0.034 0.024 2.77 3.21 CPNE3 O75131 SSPVEFECINEK 0.094 0.049 0.021 0.040 4.21 2.56 HNRNPL P14866 SSSGLLEWESK 0.043 0.047 0.026 0.032 2.65 2.87 HSPA2 P54652 STAGDTHLGGEDFDNR 0.098 0.097 0.028 0.031 4.47 4.10 HNRNPR O43390 STAYEDYYYHPPPR 0.062 0.066 0.028 0.018 3.60 3.96 HIST3H3 Q16695 STELLIR 0.008 0.013 0.021 0.022 2.35 3.20 EEF1G P26641 STFVLDEFK 0.064 0.058 0.021 0.028 3.47 2.81 HNRNPH1 P31943 STGEAFVQFASQEIAEK 0.057 0.063 0.031 0.031 2.76 3.27 PFN1 P07737 STGGAPTFNVTVTK 0.058 0.048 0.017 0.039 3.45 2.50 EEF1A1 P68104 STTTGHLIYK 0.076 0.065 0.020 0.048 3.66 2.62 DDX21 Q9NR30 STYEQVDLIGK 0.091 0.155 0.035 0.040 3.55 4.72 RRBP1 Q9P2E9 SVEEEEQVWR 0.042 0.056 0.046 0.057 2.00 2.48 TKT P29401 SVPTSTVFYPSDGVATEK 0.040 0.023 0.013 0.024 3.55 2.81 YWHAZ P63104 SVTEQGAELSNEER 0.070 0.057 0.019 0.033 3.88 2.97 ATP1B1 P05026 SYEAYVLNIVR 0.034 0.057 0.030 0.032 2.16 2.80 ACTA2 P62736 SYELPDGQVITIGNER 0.070 0.098 0.025 0.026 3.22 3.96 TALDO1 P37837 SYEPLEDPGVK 0.038 0.029 0.026 0.034 2.46 2.32 CACYBP Q9HB71 SYSMIVNNLLK 0.089 0.054 0.040 0.032 3.44 3.08 EIF3I Q13347 SYSSGGEDGYVR 0.117 0.102 0.050 0.035 3.83 3.80 SLC1A5 Q15758 SYSTTYEER 0.126 0.061 0.056 0.074 4.31 2.49 DDOST P39656 TADDPSLSLIK 0.030 0.067 0.022 0.021 2.41 3.36 CCT8 P50990 TAEELMNFSK 0.055 0.046 0.037 0.034 2.65 2.64 SDF4 Q9BRK5 TAEHFQEAMEESK 0.085 0.079 0.054 0.090 2.51 2.62 TXN P10599 TAFQEALDAAGDK 0.073 0.037 0.016 0.039 4.19 2.29 IARS2 Q9NSE4 TALAEAELEYNPEHVSR 0.032 0.040 0.015 0.021 2.89 3.48 LMNA P02545 TALINSTGEEVAMR 0.040 0.045 0.027 0.025 2.39 3.23 RAB5C P51148 TAMNVNEIFM~AIAK 0.059 0.054 0.031 0.034 2.86 2.96 TATESFASDPILYRPVAVAL PKM2 P14618 0.073 0.062 0.017 0.031 3.85 2.95 DTK CCT3 P49368 TAVETAVLLLR 0.058 0.051 0.038 0.044 3.01 2.77 GOT2 P00505 TCGFDFTGAVEDISK 0.034 0.034 0.024 0.017 2.71 3.39 HNRNPU Q00839 TCNCETEDYGEK 0.052 0.062 0.026 0.034 2.67 3.26 C11orf59 Q6IAA8 TDEQALLSSILAK 0.055 0.053 0.036 0.046 2.43 2.37 CAPNS1 P04632 TDGFGIDTCR 0.044 0.041 0.025 0.036 2.86 2.38 KRT16 P08779 TDLEM~QIEGLK 0.048 0.084 0.031 0.024 2.49 3.39 KRT16 P08779 TDLEMQIEGLK 0.045 0.070 0.025 0.033 2.64 3.17 MYH9 P35579 TDLLLEPYNK 0.071 0.068 0.024 0.041 3.01 2.58 PPP2R1A P30153 TDLVPAFQNLMK 0.093 0.087 0.027 0.043 3.64 3.22 XRCC5 P13010 TDTLEDLFPTTK 0.042 0.046 0.022 0.019 3.21 2.92 HNRNPK P61978 TDYNASVSVPDSSGPER 0.064 0.068 0.041 0.035 3.05 3.60 VAMP8 Q9BV40 TEDLEATSEHFK 0.075 0.103 0.044 0.060 2.64 3.36 CDC37 Q16543 TEEDSEEVR 0.064 0.073 0.047 0.037 2.71 3.23 DDX21 Q9NR30 TEEIAEEEETVFPK 0.058 0.083 0.053 0.065 2.14 2.85 HMGN1 P05114 TEESPASDEAGEK 0.056 0.052 0.034 0.040 3.04 2.69 KRT8 P05787 TEISEMNR 0.077 0.101 0.026 0.031 4.32 4.73 MYH9 P35579 TEMEDLMSSK 0.063 0.063 0.024 0.035 2.87 2.50 KRT8 P05787 TEMENEFVLIK 0.068 0.087 0.026 0.029 3.74 4.08 PSME1 Q06323 TENLLGSYFPK 0.025 0.023 0.018 0.029 1.95 1.94 LCP1 P13796 TENLNDDEK 0.069 0.080 0.035 0.046 2.77 2.88 ALDH2 P05091 TEQGPQVDETQFK 0.018 0.012 0.021 0.017 2.33 2.81 CCT6B Q92526 TEVNSGFFYK 0.065 0.061 0.020 0.037 3.61 2.79

172

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

DYNC1H1 Q14204 TEYLSNADER 0.060 0.054 0.013 0.029 3.92 2.74 HSPA5 P11021 TFAPEEISAMVLTK 0.054 0.064 0.023 0.026 2.95 3.34 EEF2 P13639 TFCQLILDPIFK 0.131 0.081 0.019 0.039 5.56 3.04 SLC2A1 P11166 TFDEIASGFR 0.091 0.164 0.038 0.056 3.43 5.06 PDIA4 P13667 TFDSIVMDPK 0.033 0.040 0.037 0.021 1.95 2.71 HADH Q16836 TFESLVDFSK 0.015 0.019 0.019 0.021 2.74 3.23 GARS P41250 TFFSFPAVVAPFK 0.061 0.063 0.025 0.038 3.17 2.61 XRCC6 P12956 TFNTSTGGLLLPSDTK 0.060 0.041 0.022 0.025 3.60 3.03 DDX21 Q9NR30 TFSFAIPLIEK 0.137 0.194 0.042 0.041 4.70 5.89 ARCN1 P48444 TFTEMDSHEEK 0.052 0.042 0.020 0.026 2.87 2.29 GPI P06744 TFTTQETITNAETAK 0.011 0.027 0.014 0.029 1.10 2.21 PFN1 P07737 TFVNITPAEVGVLVGK 0.063 0.050 0.018 0.036 3.53 2.56 ATP5A1 P25705 TGAIVDVPVGEELLGR 0.028 0.038 0.024 0.020 2.89 3.72 TGDFQLHTNVNDGTEFGG VDAC2 P45880 0.098 0.062 0.020 0.015 3.13 4.17 SIYQK PDIA6 Q15084 TGEAIVDAALSALR 0.041 0.055 0.028 0.024 2.58 3.50 HADHA P40939 TGIEQGSDAGYLCESQK 0.027 0.023 0.029 0.022 2.07 3.19 RPN2 P04844 TGQEVVFVAEPDNK 0.041 0.068 0.027 0.025 2.39 3.37 ATP5A1 P25705 TGTAEMSSILEER 0.027 0.028 0.022 0.019 2.82 3.49 PRDX5 P30044 THLPGFVEQAEALK 0.029 0.035 0.018 0.023 2.83 2.63 CAPNS1 P04632 THYSNIEANESEEVR 0.068 0.037 0.024 0.033 3.54 2.61 RPS5 P46782 TIAECLADELINAAK 0.068 0.067 0.021 0.030 3.64 3.31 ACLY P53396 TIAIIAEGIPEALTR 0.122 0.112 0.033 0.043 4.85 4.45 ATP5B P06576 TIAMDGTEGLVR 0.027 0.029 0.020 0.023 2.67 3.37 PRDX1 Q06830 TIAQDYGVLK 0.047 0.028 0.018 0.027 3.22 2.29 TUBA1A Q71U36 TIGGGDDSFNTFFSETGAGK 0.054 0.038 0.022 0.035 3.20 2.30 TUFM P49411 TIGTGLVTNTLAMTEEEK 0.036 0.030 0.022 0.021 3.32 3.40 MDH2 P40926 TIIPLISQCTPK 0.026 0.035 0.020 0.020 2.64 3.44 TUBA1A Q71U36 TIQFVDWCPTGFK 0.057 0.085 0.025 0.035 2.60 2.31 NAPA P54920 TIQGDEEDLR 0.058 0.044 0.039 0.047 2.91 2.63 UBA52 P62987 TITLEVEPSDTIENVK 0.126 0.125 0.050 0.067 3.97 3.72 KRT17 Q04695 TKFETEQALR 0.045 0.084 0.028 0.025 2.38 3.81 TLN1 Q9Y490 TLAESALQLLYTAK 0.062 0.064 0.025 0.039 2.77 2.47 DHRS2 Q13268 TLALELAPK 0.014 0.018 0.023 0.023 1.98 2.92 GPI P06744 TLAQLNPESSLFIIASK 0.017 0.032 0.012 0.032 1.17 2.27 DHX15 O43143 TLATDILM~GVLK 0.059 0.063 0.031 0.030 3.00 3.30 COPA P53621 TLDLPIYVTR 0.051 0.040 0.024 0.028 2.93 2.55 PARP1 P09874 TLGDFAAEYAK 0.030 0.075 0.044 0.032 2.03 2.90 TAGLN2 P37802 TLMNLGGLAVAR 0.055 0.054 0.026 0.044 2.93 2.58 HSPD1 P10809 TLNDELEIIEGMK 0.057 0.048 0.014 0.024 3.88 3.61 EPPK1 P58107 TLQEVTEMDSVK 0.062 0.070 0.024 0.025 2.97 3.03 CLTC Q00610 TLQIFNIEMK 0.036 0.039 0.025 0.022 2.35 2.57 UBA52 P62987 TLSDYNIQK 0.111 0.116 0.045 0.070 3.60 3.40 EIF1B O60739 TLTTVQGIADDYDK 0.203 0.155 0.054 0.116 5.74 3.82 PFN1 P07737 TLVLLMGK 0.065 0.057 0.009 0.034 3.45 2.23 KRT4 P19013 TMQDSVEDFK 0.026 0.031 0.025 0.034 2.53 2.38 RPL18 Q07020 TNSTFNQVVLK 0.043 0.062 0.021 0.032 2.86 2.90 CSNK2A1 P68400 TPALVFEHVNNTDFK 0.063 0.087 0.034 0.038 3.02 3.43 ANXA2 P07355 TPAQYDASELK 0.047 0.107 0.030 0.020 1.83 3.91 CANX P27824 TPELNLDQFHDK 0.036 0.053 0.018 0.023 2.51 3.22 HADHB P55084 TPFLLSGTSYK 0.018 0.020 0.013 0.014 2.04 3.03 ACAT1 P24752 TPIGSFLGSLSLLPATK 0.032 0.040 0.024 0.020 2.98 3.80 RBBP4 Q09028 TPSSDVLVFDYTK 0.036 0.042 0.038 0.032 2.21 2.65 ANXA11 P50995 TPVLFDIYEIK 0.057 0.046 0.025 0.043 2.38 2.10 CANX P27824 TPYTIMFGPDK 0.043 0.056 0.024 0.021 2.62 3.19 NOP58 Q9Y2X3 TQLYEYLQNR 0.029 0.045 0.039 0.019 2.34 3.88 CUTA O60888 TQSSLVPALTDFVR 0.035 0.023 0.019 0.035 3.39 2.33 ATP1A1 P05023 TSATWLALSR 0.041 0.049 0.035 0.038 2.33 2.66

173

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

TARDBP Q13148 TSDLIVLGLPWK 0.048 0.054 0.038 0.041 2.36 2.59 Q8NHW5 TSFFQALGITTK 0.047 0.053 0.019 0.024 3.00 3.01 ATP5A1 P25705 TSIAIDTIINQK 0.024 0.028 0.020 0.017 2.40 3.23 TSRPENAIIYNNNEDFQVGQ TKT P29401 0.041 0.021 0.016 0.023 3.09 2.66 AK HSPA9 P38646 TTPSVVAFTADGER 0.046 0.068 0.042 0.018 2.96 4.46 HSPA1L P34931 TTPSYVAFTDTER 0.103 0.089 0.031 0.033 4.25 3.88 DDX5 P17844 TTYLVLDEADR 0.097 0.120 0.072 0.075 2.89 3.78 BLVRB P30043 TVAGQDAVIVLLGTR 0.026 0.028 0.016 0.033 3.19 2.29 PPIB P23284 TVDNFVALATGEK 0.048 0.064 0.025 0.023 2.59 3.31 RPL3 P39023 TVFAEHISDECK 0.051 0.059 0.018 0.028 3.19 3.17 PRKDC P78527 TVGALQVLGTEAQSSLLK 0.024 0.026 0.015 0.025 2.89 2.73 HSPD1 P10809 TVIIEQSWGSPK 0.049 0.049 0.018 0.020 3.75 3.89 TVLGTPEVLLGALPGAGGTQ HADHA P40939 0.031 0.042 0.025 0.022 2.50 3.79 R ATP5B P06576 TVLIM~ELINNVAK 0.025 0.032 0.023 0.026 2.41 2.97 ATP5B P06576 TVLIMELINNVAK 0.027 0.028 0.023 0.022 2.47 3.16 KRT18 P05783 TVQSLEIDLDSM~R 0.090 0.101 0.030 0.025 4.04 4.52 KRT18 P05783 TVQSLEIDLDSMR 0.096 0.106 0.025 0.023 4.60 4.82 PRKDC P78527 TVSLLDENNVSSYLSK 0.037 0.029 0.023 0.025 2.92 2.81 HIST1H4A P62805 TVTAM~DVVYALK 0.018 0.013 0.020 0.019 2.21 2.78 HIST1H4A P62805 TVTAMDVVYALK 0.017 0.012 0.018 0.019 2.27 2.83 SYPL1 Q16563 TVTATFGYPFR 0.066 0.041 0.032 0.025 3.23 3.14 HSPA8 P11142 TVTNAVVTVPAYFNDSQR 0.102 0.101 0.043 0.031 3.84 4.31 HSPA5 P11021 TWNDPSVQQDIK 0.055 0.058 0.016 0.019 3.32 3.47 GDI2 P50395 TYDATTHFETTCDDIK 0.047 0.033 0.015 0.034 3.07 2.22 RNPEP Q9H4A4 TYQLVYFLDK 0.053 0.028 0.026 0.034 2.62 2.30 RPS2 P15880 TYSYLTPDLWK 0.050 0.059 0.031 0.036 2.57 2.70 FH P07954 VAALTGLPFVTAPNK 0.030 0.027 0.019 0.020 2.84 3.46 RPN1 P04843 VACITEQVLTLVNK 0.029 0.078 0.026 0.020 2.22 3.15 AHCY P23526 VADIGLAAWGR 0.041 0.054 0.025 0.035 3.17 2.60 EIF3D O15371 VADWTGATYQDK 0.080 0.082 0.033 0.034 3.43 3.14 SLC3A2 P08195 VAEDEAEAAAAAK 0.064 0.056 0.033 0.038 2.66 2.69 NOP58 Q9Y2X3 VAEEEETSVK 0.045 0.050 0.019 0.021 2.72 3.17 ALDH2 P05091 VAEQTPLTALYVANLIK 0.023 0.023 0.030 0.021 2.21 2.67 ATP5B P06576 VALTGLTVAEYFR 0.031 0.031 0.026 0.021 2.72 3.30 ACTB P60709 VAPEEHPVLLTEAPLNPK 0.065 0.126 0.026 0.025 3.04 4.79 SNRPD3 P62318 VAQLEQVYIR 0.037 0.040 0.036 0.022 2.51 3.31 MDH2 P40926 VAVLGASGGIGQPLSLLLK 0.027 0.032 0.020 0.018 2.63 3.41 RAN P62826 VCENIPIVLCGNK 0.057 0.052 0.028 0.032 3.03 2.80 RPL30 P62888 VCTLAIIDPGDSDIIR 0.062 0.064 0.020 0.023 4.02 3.68 KRT7 P08729 VDALNDEINFLR 0.047 0.072 0.027 0.030 2.32 3.03 ACLY P53396 VDATADYICK 0.159 0.105 0.020 0.020 5.61 3.94 P4HB P07237 VDATEESDLAQQYGVR 0.035 0.045 0.025 0.019 2.34 3.20 MDH2 P40926 VDFPQDQLTALTGR 0.030 0.034 0.024 0.023 2.80 3.38 AHNAK Q09666 VDIDAPDVDVHGPDWHLK 0.107 0.151 0.035 0.035 4.15 5.41 PSMC3 P17980 VDILDPALLR 0.071 0.054 0.044 0.035 2.99 2.95 ATP2A1 O14983 VDQSILTGESVSVIK 0.052 0.071 0.021 0.022 2.62 3.16 HNRNPCL1 O60812 VDSLLENLEK 0.041 0.050 0.034 0.032 2.57 3.16 KRT4 P19013 VDSLNDEINFLK 0.036 0.032 0.034 0.032 2.29 2.60 AHNAK Q09666 VDTNAPDLSLEGPEGK 0.105 0.420 0.036 0.037 3.96 4.95 AHNAK Q09666 VDVDVPDVNIEGPDAK 0.108 0.167 0.032 0.032 4.22 5.74 AHNAK Q09666 VDVEVPDVSLEGPEGK 0.109 0.155 0.026 0.036 4.47 5.44 TPD52 P55327 VEEEIQTLSQVLAAK 0.063 0.045 0.018 0.029 3.52 2.35 RPS28 P62857 VEFMDDTSR 0.065 0.062 0.028 0.028 3.54 3.33 AHNAK Q09666 VEGDMQVPDLDIK 0.087 0.078 0.038 0.062 3.12 2.91 PDIA4 P13667 VEGFPTIYFAPSGDK 0.040 0.050 0.024 0.019 2.49 3.16 RPL24 P83731 VELCSFSGYK 0.056 0.062 0.017 0.035 3.25 3.05

174

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

VAPB O95292 VEQVLSLEPQHELK 0.038 0.058 0.018 0.022 2.55 3.18 NASP P49321 VESLDVDSEAK 0.056 0.024 0.035 0.045 3.01 2.23 SET Q01105 VEVTEFEDIK 0.042 0.031 0.024 0.025 2.75 2.57 TFRC P02786 VEYHFLSPYVSPK 0.125 0.158 0.074 0.073 3.40 4.32 MGST1 P10620 VFANPEDCVAFGK 0.051 0.044 0.020 0.021 3.39 2.99 EEF2 P13639 VFDAIMNFK 0.103 0.081 0.019 0.038 4.59 3.11 RALA P11233 VFFDLMR 0.050 0.085 0.037 0.030 2.57 3.88 DHRS2 Q13268 VFHGNESLWK 0.011 0.018 0.022 0.018 1.99 2.77 HNRNPCL1 O60812 VFIGNLNTLVVK 0.038 0.045 0.030 0.029 2.65 3.18 HIST1H4A P62805 VFLENVIR 0.017 0.013 0.021 0.016 2.57 3.21 HNRNPL P14866 VFNVFCLYGNVEK 0.047 0.051 0.023 0.026 2.78 3.43 RPL24 P83731 VFQFLNAK 0.052 0.058 0.013 0.030 3.24 2.82 EEF2 P13639 VFSGLVSTGLK 0.081 0.063 0.022 0.041 3.72 2.74 HNRPDL O14979 VFVGGLSPDTSEEQIK 0.052 0.077 0.025 0.030 2.80 3.67 MAGOHB Q96A72 VFYYLVQDLK 0.038 0.041 0.036 0.031 2.15 2.65 FASN P49327 VGDPQELNGITR 0.111 0.046 0.016 0.035 5.58 2.80 ATP2A2 P16615 VGEATETALTCLVEK 0.040 0.057 0.019 0.027 2.70 3.32 HSPD1 P10809 VGGTSDVEVNEK 0.052 0.050 0.023 0.018 3.70 3.97 TUBA1A Q71U36 VGINYQPPTVVPGGDLAK 0.082 0.051 0.028 0.033 3.72 2.81 HSPD1 P10809 VGLQVVAVK 0.057 0.049 0.025 0.016 3.28 3.77 DDX3X O00571 VGNLGLATSFFNER 0.087 0.118 0.046 0.045 3.21 3.98 NANS Q9NR45 VGSGDTNNFPYLEK 0.032 0.020 0.017 0.027 2.34 1.97 AHNAK Q09666 VGVEVPDVNIEGPEGK 0.046 0.092 0.051 0.054 2.16 3.27 ACTN1 P12814 VGWEQLLTTIAR 0.041 0.056 0.031 0.032 2.38 2.92 EIF5A P63241 VHLVGIDIFTGK 0.075 0.044 0.012 0.040 3.84 2.20 KRT18 P05783 VIDDTNITR 0.095 0.094 0.028 0.034 4.46 4.31 EPPK1 P58107 VIEETEER 0.071 0.079 0.026 0.027 3.32 3.39 S100A9 P06702 VIEHIMEDLDTNADK 0.111 0.047 0.036 0.055 3.75 1.62 GPD2 P43304 VIFFLPWQK 0.028 0.031 0.019 0.016 2.59 3.15 LDHAL6A Q6ZMR3 VIGSGCNLDSAR 0.115 0.075 0.012 0.036 6.07 3.56 SLC3A2 P08195 VILDLTPNYR 0.057 0.064 0.042 0.041 2.40 2.44 HSPA9 P38646 VINEPTAAALAYGLDK 0.061 0.065 0.019 0.025 4.31 4.08 RPL13 P26373 VITEEEK 0.044 0.049 0.026 0.015 2.68 2.77 SND1 Q7KZF4 VITEYLNAQESAK 0.078 0.077 0.029 0.042 3.35 2.95 RPS18 P62269 VITIMQNPR 0.060 0.086 0.047 0.037 2.46 3.10 ILF2 Q12905 VKPAPDETSFSEALLK 0.041 0.043 0.026 0.025 2.38 2.93 NOP58 Q9Y2X3 VKVEEEEEEK 0.045 0.061 0.032 0.032 2.45 2.92 KRT18 P05783 VKYETELAMR 0.080 0.098 0.029 0.028 3.44 4.27 EPPK1 P58107 VLADPSDDTK 0.068 0.089 0.018 0.028 3.45 3.58 CCT6A P40227 VLAQNSGFDLQETLVK 0.076 0.052 0.016 0.016 3.95 3.03 TCP1 P17987 VLCELADLQDK 0.065 0.053 0.020 0.036 3.74 2.89 KRT14 P02533 VLDELTLAR 0.048 0.086 0.026 0.027 2.93 4.08 MYBBP1A Q9BQG0 VLDLVEVLVTK 0.049 0.057 0.042 0.027 2.51 2.78 FASN P49327 VLEALLPLK 0.118 0.045 0.018 0.039 4.67 2.38 HSPA5 P11021 VLEDSDLK 0.059 0.061 0.029 0.016 2.99 3.53 CALD1 Q05682 VLEEEEQR 0.038 0.067 0.032 0.035 1.69 2.64 PPIB P23284 VLEGMEVVR 0.056 0.056 0.028 0.019 2.92 3.63 RPL11 P62913 VLEQLTGQTPVFSK 0.054 0.064 0.022 0.033 3.15 3.30 SPTAN1 Q13813 VLETAEDIQER 0.053 0.093 0.034 0.033 2.94 3.47 HSPH1 Q92598 VLGTAFDPFLGGK 0.116 0.120 0.030 0.032 4.72 4.31 EIF4A1 P60842 VLITTDLLAR 0.086 0.064 0.035 0.038 3.48 2.92 RPL15 P61313 VLNSYWVGEDSTYK 0.053 0.046 0.017 0.038 3.27 2.50 HSPE1 P61604 VLQATVVAVGSGSK 0.040 0.042 0.023 0.016 2.94 3.45 ILF3 Q12906 VLQDMGLPTGAEGR 0.041 0.051 0.030 0.024 2.77 3.38 TCOF1 Q13428 VLTELLEQER 0.047 0.073 0.032 0.026 2.93 3.53 COPA P53621 VLTIDPTEFK 0.046 0.043 0.024 0.024 2.59 2.46 VM~TIAPGLFGTPLLTSLPE HSD17B10 Q99714 0.038 0.050 0.018 0.019 3.36 4.24 K

175

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

SNRPEL1 Q5VYJ4 VM~VQPINLIFR 0.043 0.046 0.037 0.034 2.66 3.11 LARP7 Q4G0J3 VMEEESTEK 0.029 0.033 0.027 0.023 2.18 3.18 SNRPEL1 Q5VYJ4 VMVQPINLIFR 0.040 0.048 0.039 0.031 2.51 3.34 DHRS2 Q13268 VNCVVPGIIK 0.013 0.016 0.015 0.025 2.06 2.78 DYNC1H1 Q14204 VNFLPEIITLSK 0.069 0.054 0.012 0.030 3.67 2.68 PRDX5 P30044 VNLAELFK 0.042 0.028 0.016 0.024 2.88 2.78 EIF3B P55884 VNLFTDFDK 0.107 0.076 0.041 0.034 3.70 3.02 C10orf58 Q9BRX8 VNLLSVLEAAK 0.047 0.044 0.025 0.027 2.46 2.75 VNNSSLIGVGYTQTLRPGV VDAC2 P45880 0.043 0.048 0.027 0.017 2.56 3.57 K RPS5 P46782 VNQAIWLLCTGAR 0.060 0.064 0.025 0.026 3.27 3.38 AHNAK Q09666 VNVEAPDVNLEGLGGK 0.086 0.127 0.029 0.035 3.60 4.71 FUBP1 Q96AE4 VPDGMVGFIIGR 0.066 0.087 0.047 0.034 2.69 3.26 RPS19 P39019 VPEWVDTVK 0.048 0.066 0.029 0.030 2.86 3.21 OSTC Q9NRP0 VPFLVLECPNLK 0.045 0.053 0.030 0.028 2.68 3.29 VARS P26640 VPLEVQEADEAK 0.042 0.037 0.027 0.026 2.72 2.30 ACADVL P49748 VPSENVLGEVGSGFK 0.035 0.040 0.026 0.025 2.13 2.68 GAPDH P04406 VPTANVSVVDLTCR 0.056 0.037 0.020 0.030 3.93 2.65 GAR1 Q9NY12 VPYFNAPVYLENK 0.065 0.065 0.029 0.030 3.17 3.42 AHNAK Q09666 VQANLGAPDINIEGLDAK 0.083 0.143 0.060 0.040 2.67 5.15 HSPA9 P38646 VQQTVQDLFGR 0.072 0.060 0.026 0.029 4.46 4.01 NOLC1 Q14978 VREEEIEVDSR 0.094 0.113 0.050 0.062 3.18 4.21 SDHA P31040 VRIDEYDYSK 0.029 0.033 0.020 0.028 2.73 2.75 KRT7 P08729 VRQEESEQIK 0.044 0.079 0.024 0.031 2.00 3.19 TFRC P02786 VSASPLLYTLIEK 0.104 0.128 0.057 0.081 3.67 3.79 GGCT O75223 VSEEIEDIIK 0.034 0.014 0.008 0.034 3.57 1.98 HNRNPU Q00839 VSELKEELK 0.049 0.057 0.044 0.024 2.17 3.00 PDCD5 O14737 VSEQGLIEILK 0.062 0.072 0.025 0.051 2.55 2.49 PPIA P62937 VSFELFADK 0.044 0.031 0.016 0.034 3.19 2.08 AHNAK Q09666 VSGPDLDLNLK 0.106 0.135 0.023 0.047 4.06 4.54 HSPB1 P04792 VSLDVNHFAPDELTVK 0.102 0.042 0.015 0.031 4.63 2.19 DNAJA2 O60884 VSLEDLYNGK 0.058 0.066 0.041 0.054 2.48 2.31 AHNAK Q09666 VSVGAPDLSLEASEGSIK 0.079 0.158 0.032 0.034 3.08 5.27 SRP9 P49458 VTDDLVCLVYK 0.052 0.059 0.055 0.060 2.09 2.26 TIMM44 O43615 VTDLLGGLFSK 0.042 0.044 0.030 0.018 2.50 3.24 HIST1H2AB P04908 VTIAQGGVLPNIQAVLLPK 0.022 0.020 0.024 0.021 2.66 3.29 LDHA P00338 VTLTSEEEAR 0.103 0.088 0.015 0.030 5.26 3.85 VDAC1 P21796 VTQSNFAVGYK 0.020 0.019 0.035 0.018 1.66 2.56 HSPA4 P34932 VTYMEEER 0.072 0.053 0.021 0.034 3.69 2.86 PUF60 Q9UHX1 VVAEVYDQER 0.084 0.076 0.028 0.040 3.69 3.09 ATP5B P06576 VVDLLAPYAK 0.030 0.036 0.024 0.021 2.59 3.27 GAPDH P04406 VVDLMAHMASKE 0.044 0.031 0.014 0.031 3.41 2.36 PARP1 P09874 VVDRDSEEAEIIR 0.048 0.055 0.039 0.027 2.83 3.68 PRDX6 P30041 VVFVFGPDK 0.045 0.020 0.018 0.035 2.68 1.84 MTHFD1 P11586 VVGDVAYDEAK 0.046 0.024 0.022 0.027 3.19 2.47 TPI1 P60174 VVLAYEPVWAIGTGK 0.043 0.033 0.013 0.035 3.31 2.21 EIF2S1 P05198 VVTDTDETELAR 0.083 0.066 0.028 0.033 3.89 3.15 PDIA3 P30101 VVVAENFDEIVNNENK 0.041 0.041 0.041 0.028 2.21 2.77 CKMT1A P12532 VVVDALSGLK 0.018 0.023 0.019 0.020 2.23 3.30 FASN P49327 VVVQVLAEEPEAVLK 0.105 0.044 0.026 0.038 4.14 2.39 SRSF1 Q07955 VVVSGLPPSGSWQDLK 0.035 0.054 0.020 0.019 2.60 3.60 DDX3X O00571 VVWVEESDK 0.105 0.117 0.057 0.047 3.23 3.70 FASN P49327 VYATILNAGTNTDGFK 0.137 0.044 0.020 0.039 5.31 2.37 FASN P49327 VYQWDDPDPR 0.090 0.054 0.037 0.038 4.31 2.91 CACYBP Q9HB71 WDYLTQVEK 0.094 0.060 0.026 0.044 4.30 2.83 KTN1 Q86UP2 WEEVQSYIR 0.058 0.076 0.039 0.027 2.58 3.78 ILF2 Q12905 WFEENASQSTVK 0.041 0.039 0.028 0.025 2.52 3.04 EEF1G P26641 WFLTCINQPQFR 0.071 0.038 0.027 0.039 3.60 2.61

176

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

ATP2A2 P16615 WGSNELPAEEGK 0.060 0.072 0.030 0.027 2.76 3.35 HNRNPA3 P51991 WGTLTDCVVMR 0.036 0.083 0.038 0.031 2.21 3.08 ANXA2 P07355 WISIMTER 0.052 0.096 0.032 0.026 2.05 3.74 CBX3 Q13185 WKDSDEADLVLAK 0.052 0.059 0.036 0.030 2.50 2.98 PRMT1 Q99873 WLAPDGLIFPDR 0.083 0.076 0.035 0.025 3.73 3.35 BOP1 Q14137 WLEASEEER 0.103 0.039 0.034 0.036 4.47 3.70 TALDO1 P37837 WLHNEDQMAVEK 0.041 0.027 0.014 0.025 2.61 1.97 SPCS1 Q9Y6A9 WLPVQESSTDDK 0.066 0.095 0.031 0.038 2.89 3.58 FASN P49327 WLSTSIPEAQWHSSLAR 0.117 0.047 0.018 0.035 5.87 2.76 ERBB2 P04626 WMALESILR 0.050 0.049 0.054 0.060 2.11 2.20 CAPN1 P07384 WNTTLYEGTWR 0.036 0.027 0.016 0.037 3.02 2.08 KRT8 P05787 WSLLQQQK 0.080 0.102 0.025 0.035 3.87 4.15 ATAD3A Q9NVI7 WSNFDPTGLER 0.049 0.058 0.036 0.024 2.86 3.81 CCT3 P49368 WSSLACNIALDAVK 0.081 0.060 0.024 0.035 3.78 3.01 LMAN2 Q12907 WTELAGCTADFR 0.038 0.046 0.032 0.029 2.51 3.10 KRT7 P08729 WTLLQEQK 0.038 0.079 0.029 0.031 1.84 3.15 CNDP2 Q96KP4 WVAIQSVSAWPEK 0.030 0.023 0.013 0.029 2.67 1.98 UQCRB P14927 WYYNAAGFNK 0.033 0.027 0.030 0.014 2.66 3.39 NAP1L4 Q99733 YAALYQPLFDK 0.105 0.036 0.039 0.026 3.18 2.61 DHX29 Q7Z478 YAEQQNEEEK 0.090 0.072 0.044 0.037 3.01 3.29 LCP1 P13796 YAFVNWINK 0.084 0.088 0.023 0.031 3.45 3.38 RPS4X P62701 YALTGDEVK 0.061 0.066 0.022 0.033 3.21 3.16 DSTN P60981 YALYDASFETK 0.065 0.046 0.031 0.044 2.91 2.17 CFL1 P23528 YALYDATYETK 0.052 0.052 0.017 0.037 2.99 2.38 WDR1 O75083 YAPSGFYIASGDVSGK 0.067 0.067 0.021 0.034 3.00 3.00 SEC24C P53992 YASFQVENDQER 0.068 0.056 0.045 0.042 2.67 2.59 ATP5O P48047 YATALYSAASK 0.021 0.022 0.023 0.016 2.29 3.07 NAP1L1 P55209 YAVLYQPLFDK 0.111 0.081 0.019 0.031 4.56 3.41 ECH1 Q13011 YCAQDAFFQVK 0.021 0.028 0.032 0.024 2.35 3.02 TOR1AIP1 Q5JTV8 YCDHENAAFK 0.051 0.074 0.022 0.018 2.46 3.60 GTF2I P78347 YCVEEEEK 0.055 0.069 0.077 0.084 1.75 2.23 YWHAB P31946 YDDMAAAMK 0.041 0.047 0.011 0.041 2.76 2.37 YWHAZ P63104 YDDMAACMK 0.054 0.049 0.022 0.039 2.77 2.47 YWHAQ P27348 YDDMATCMK 0.042 0.045 0.023 0.035 2.55 2.39 RBMX P38159 YDDYSSSR 0.036 0.042 0.030 0.028 2.50 3.27 CSE1L P55060 YDEEFQR 0.067 0.036 0.029 0.031 4.02 2.50 YWHAE P62258 YDEM~VESMK 0.042 0.042 0.027 0.027 2.57 2.37 PDCD6IP Q8WUM4 YDEYVNVK 0.081 0.062 0.041 0.042 2.87 2.66 AKAP8L Q9ULX6 YDSYESCDSR 0.104 0.097 0.037 0.046 3.85 3.80 KRT2 P35908 YEDEINK 0.088 0.109 0.024 0.029 4.58 4.90 SFN P31947 YEDMAAFMK 0.076 0.080 0.055 0.051 2.63 2.74 SLC25A12 O75746 YEGFFGLYR 0.033 0.036 0.024 0.017 2.50 3.40 ILF3 Q12906 YELISETGGSHDK 0.038 0.036 0.025 0.027 2.41 2.74 ATP1A1 P05023 YEPAAVSEQGDK 0.056 0.055 0.023 0.037 2.93 2.80 HSP90AB2P Q58FF8 YESLTDPSK 0.090 0.058 0.017 0.032 4.59 2.92 KRT18 P05783 YETELAM~R 0.086 0.084 0.026 0.033 4.29 3.80 KRT18 P05783 YETELAMR 0.084 0.107 0.030 0.031 4.05 4.58 SLC25A24 Q6NUK1 YETLFQALDR 0.023 0.049 0.019 0.022 2.11 3.20 EEF2 P13639 YEWDVAEAR 0.141 0.080 0.020 0.041 6.06 3.25 IDH2 P48735 YFDLGLPNR 0.041 0.022 0.034 0.024 2.53 3.13 ARPP19 P56211 YFDSGDYNMAK 0.101 0.057 0.030 0.055 4.07 2.26 SF3B3 Q15393 YFDTVPVAAAMCVLK 0.041 0.057 0.023 0.025 2.62 3.38 UBA1 P22314 YFLVGAGAIGCELLK 0.055 0.041 0.012 0.024 3.59 2.54 USMG5 Q96IX5 YFNSYTLTGR 0.040 0.044 0.048 0.060 1.77 2.22 SLC25A4 P12235 YFPTQALNFAFK 0.037 0.046 0.021 0.023 2.83 3.45 YGDSEFTVQSTTGHCVHM HNRNPF P52597 0.054 0.070 0.033 0.034 2.84 3.18 R CTSA P10619 YGDSGEQIAGFVK 0.027 0.058 0.042 0.039 1.68 2.67

177

Appendix 2. Peptide synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

SFPQ P23246 YGEPGEVFINK 0.048 0.065 0.031 0.032 2.66 3.65 HDAC1 Q13547 YGEYFPGTGDLR 0.095 0.105 0.038 0.039 3.30 3.60 FLNA P21333 YGGDEIPFSPYR 0.056 0.092 0.019 0.021 2.21 3.43 CTTN Q14247 YGLFPANYVELR 0.082 0.077 0.024 0.029 3.56 3.62 ATP5F1 P24539 YGPFVADFADK 0.021 0.021 0.024 0.016 2.31 3.26 NARS O43776 YGTCPHGGYGLGLER 0.054 0.052 0.031 0.029 3.16 2.78 PDIA3 P30101 YGVSGYPTLK 0.048 0.052 0.031 0.019 2.39 3.02 HSP90AB1 P08238 YHTSQSGDEMTSLSEYVSR 0.094 0.062 0.022 0.032 4.77 3.22 CNDP2 Q96KP4 YIDENQDR 0.032 0.023 0.018 0.034 2.81 2.04 HSP90AB2P Q58FF8 YIDQEELNK 0.075 0.054 0.018 0.029 4.21 2.80 RPL15 P61313 YIQELWR 0.058 0.057 0.020 0.035 3.54 3.05 ENO1 P06733 YISPDQLADLYK 0.053 0.041 0.025 0.032 2.88 2.39 PDCD6 O75340 YITDWQNVFR 0.071 0.049 0.040 0.033 3.54 2.81 TMED5 Q9Y3A6 YITGTDILDMK 0.036 0.052 0.039 0.028 1.77 2.56 RPL26 P61254 YKEETIEK 0.058 0.056 0.020 0.032 2.89 2.86 P4HB P07237 YKPESEELTAER 0.033 0.047 0.024 0.023 2.05 2.99 YWHAE P62258 YLAEFATGNDR 0.055 0.061 0.020 0.030 3.42 2.77 YWHAZ P63104 YLAEVAAGDDK 0.072 0.057 0.020 0.031 3.50 2.76 YWHAQ P27348 YLAEVACGDDR 0.052 0.052 0.020 0.036 3.22 2.83 YWHAH Q04917 YLAEVASGEK 0.082 0.049 0.014 0.042 4.04 2.73 SFN P31947 YLAEVATGDDK 0.142 0.142 0.014 0.024 5.04 4.74 YWHAG P61981 YLAEVATGEK 0.059 0.047 0.014 0.028 3.25 2.54 MAT2A P31153 YLDEDTIYHLQPSGR 0.140 0.092 0.050 0.056 4.69 3.44 RAC1 P63000 YLECSALTQR 0.050 0.052 0.040 0.051 2.30 2.27 EPHX1 P07099 YLEDGGLER 0.023 0.029 0.021 0.022 2.09 2.77 EPPK1 P58107 YLEGTSCIAGVLVPAK 0.071 0.054 0.024 0.032 3.16 2.41 IPO7 O95373 YLEMIYSMCK 0.084 0.059 0.028 0.060 3.21 2.20 MRPS18B Q9Y676 YLESEEYQER 0.038 0.052 0.028 0.037 3.06 3.47 NUCB1 Q02818 YLESLGEEQR 0.059 0.039 0.087 0.078 1.83 2.21 CAPN1 P07384 YLGQDYEQLR 0.039 0.072 0.020 0.036 2.89 2.00 YWHAQ P27348 YLIANATNPESK 0.063 0.077 0.036 0.026 2.56 2.93 SLC25A13 Q9UJS0 YLNIFGESQPNPK 0.033 0.043 0.029 0.019 2.23 3.28 CAPN2 P17655 YLNQDYEALR 0.054 0.157 0.023 0.041 3.23 5.30 ADAR P55265 YLNTNPVGGLLEYAR 0.045 0.067 0.041 0.035 2.27 3.03 YWHAB P31946 YLSEVASGDNK 0.048 0.042 0.013 0.020 2.90 2.37 DDX3X O00571 YLVLDEADR 0.098 0.091 0.068 0.057 3.05 3.00 TUBA1A Q71U36 YMACCLLYR 0.043 0.040 0.026 0.038 2.73 2.44 BCAP31 P51572 YMEENDQLK 0.039 0.055 0.021 0.020 2.23 3.09 HSPH1 Q92598 YNHIDESEMK 0.176 0.095 0.028 0.036 5.87 3.42 HNRNPU Q00839 YNILGTNTIMDK 0.045 0.059 0.028 0.031 2.62 3.19 TXNDC5 Q8NBS9 YNSMEDAK 0.048 0.052 0.021 0.023 2.74 2.96 PRKDC P78527 YPEETLSLMTK 0.028 0.042 0.025 0.018 2.70 2.96 PPP1CA P62136 YPENFFLLR 0.076 0.079 0.047 0.051 2.86 3.09 ACTA2 P62736 YPIEHGIITNWDDM~EK 0.061 0.095 0.018 0.031 3.03 3.75 ACTA2 P62736 YPIEHGIITNWDDMEK 0.054 0.093 0.030 0.037 2.47 3.42 ACTB P60709 YPIEHGIVTNWDDM~EK 0.063 0.110 0.018 0.030 2.86 3.87 ACTB P60709 YPIEHGIVTNWDDMEK 0.063 0.119 0.024 0.030 2.83 3.95 DHX9 Q08211 YPSPFFVFGEK 0.051 0.057 0.028 0.031 3.00 3.00 Q6NVV1 YQAVTATLEEK 0.057 0.054 0.018 0.023 3.23 3.10 IQGAP1 P46940 YQELINDIAR 0.048 0.052 0.028 0.030 2.48 2.80 CAPRIN1 Q14444 YQEVTNNLEFAK 0.084 0.082 0.047 0.051 2.65 2.90 VDAC1 P21796 YQIDPDACFSAK 0.028 0.033 0.022 0.020 2.50 3.15 VDAC2 P45880 YQLDPTASISAK 0.041 0.049 0.022 0.018 2.75 3.80 S100P P25815 YSGSEGSTQTLTK 0.032 0.020 0.014 0.037 1.88 1.59 FASN P49327 YSGTLNLDR 0.082 0.041 0.025 0.041 4.36 2.45 RPL17 P18621 YSLDPENPTK 0.059 0.063 0.013 0.024 3.89 3.33 PGK1 P00558 YSLEPVAVELK 0.051 0.039 0.010 0.032 3.46 2.49

178

Appendix 2. Peptide synthesis rate, degradation rate constant and relative abundance at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

GLUD1 P00367 YSTDVSVDEVK 0.047 0.031 0.020 0.026 2.98 2.96 RPL27 P61353 YSVDIPLDK 0.065 0.056 0.021 0.023 3.49 3.15 ACTR3 P61158 YSYVCPDLVK 0.050 0.060 0.020 0.018 2.71 3.16 GNAS Q5JWF2 YTTPEDATPEPGEDPR 0.097 0.090 0.036 0.040 4.21 4.21 GNB2L1 P63244 YTVQDESHSEWVSCVR 0.087 0.083 0.032 0.034 3.72 3.50 RPLP2 P05387 YVASYLLAALGGNSSPSAK 0.077 0.075 0.023 0.026 3.72 3.64 RPSA P08865 YVDIAIPCNNK 0.047 0.059 0.042 0.028 2.52 3.07 GNB2L1 P63244 YWLCAATGPSIK 0.083 0.077 0.022 0.038 3.76 3.24 COPZ1 P61923 YYDDTYPSVK 0.050 0.037 0.014 0.031 2.85 2.33 PDCD6IP Q8WUM4 YYDQICSIEPK 0.067 0.058 0.031 0.053 3.02 2.21 HNRNPL P14866 YYGGGSEGGR 0.049 0.080 0.032 0.024 2.86 3.53 HSP90AA1 P07900 YYTSASGDEMVSLK 0.072 0.044 0.016 0.029 4.13 2.75

179

Appendix 3. Protein synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells.

*: Ratio = SKBR3_shVPS4B vs. SKBR3

Synthesis Rate Degradation Rate Constant Relative Abundance Ratio* of Ratio* of Ratio* of UniProt # of Degradation Gene Name Synthesis Relative Accession peptides Rate Rate Abundance SKBR3_ SKBR3_ SKBR3_ Constant SKBR3 SKBR3 SKBR3 shVPS4B shVPS4B shVPS4B

AARS P49588 1 0.05 0.179 0.03 0.045 2.8 1.88 3.58 1.5 0.67 ABCD3 P28288 1 0.028 0.024 0.015 0.017 2.53 3.1 0.86 1.13 1.23 ABCF1 Q8NE71 1 0.088 0.067 0.023 0.04 4.01 3.1 0.76 1.74 0.77 ACAA1 P09110 4 0.03 ± 0.003 0.027 ± 0.005 0.023 ± 0.003 0.019 ± 0.001 2.82 ± 0.24 3.34 ± 0.15 0.9 0.83 1.18 ACADM P11310 2 0.033 ± 0.003 0.016 ± 0 0.026 ± 0.006 0.026 ± 0.002 2.58 ± 0.3 2.96 ± 0.15 0.48 1 1.15 ACADVL P49748 8 0.03 ± 0.005 0.037 ± 0.004 0.028 ± 0.004 0.028 ± 0.002 2.03 ± 0.13 2.64 ± 0.16 1.23 1 1.3

180 ACAT1 P24752 3 0.031 ± 0.001 0.035 ± 0.004 0.025 ± 0.002 0.022 ± 0.002 2.83 ± 0.27 3.6 ± 0.33 1.13 0.88 1.27 ACBD3 Q9H3P7 1 0.033 0.066 0.068 0.032 1.63 2.88 2 0.47 1.77 ACLY P53396 4 0.12 ± 0.032 0.103 ± 0.007 0.04 ± 0.016 0.041 ± 0.013 4.37 ± 1.14 3.81 ± 0.43 0.86 1.03 0.87 ACO2 Q99798 2 0.034 ± 0.004 0.027 ± 0.006 0.019 ± 0.004 0.025 ± 0.002 3.31 ± 0.12 3.44 ± 0.04 0.79 1.32 1.04 ACOT7 O00154 1 0.062 0.036 0.016 0.026 3.2 2.79 0.58 1.63 0.87 ACTA2 P62736 10 0.059 ± 0.009 0.089 ± 0.008 0.024 ± 0.004 0.03 ± 0.004 2.74 ± 0.28 3.48 ± 0.29 1.51 1.25 1.27 ACTB P60709 6 0.065 ± 0.004 0.106 ± 0.015 0.024 ± 0.003 0.028 ± 0.003 3.01 ± 0.18 4.04 ± 0.4 1.63 1.17 1.34 ACTN1 P12814 9 0.044 ± 0.005 0.064 ± 0.013 0.028 ± 0.005 0.026 ± 0.004 2.34 ± 0.21 3.13 ± 0.22 1.45 0.93 1.34 ACTN4 O43707 10 0.045 ± 0.007 0.053 ± 0.008 0.027 ± 0.003 0.024 ± 0.002 2.52 ± 0.32 3.03 ± 0.21 1.18 0.89 1.2 ACTR2 P61160 2 0.058 ± 0.001 0.056 ± 0.006 0.015 ± 0.003 0.031 ± 0.002 3.15 ± 0.23 3.07 ± 0.09 0.97 2.07 0.98 ACTR3 P61158 3 0.045 ± 0.009 0.053 ± 0.006 0.025 ± 0.007 0.025 ± 0.005 2.63 ± 0.56 2.96 ± 0.3 1.18 1 1.13 ADAR P55265 1 0.045 0.067 0.041 0.035 2.27 3.03 1.49 0.85 1.33 AGR2 O95994 1 0.089 0.055 0.022 0.021 4.54 2.98 0.62 0.95 0.66 AHCY P23526 1 0.041 0.054 0.025 0.035 3.17 2.6 1.32 1.4 0.82 AHNAK Q09666 22 0.087 ± 0.021 0.138 ± 0.069 0.039 ± 0.012 0.041 ± 0.011 3.32 ± 0.84 4.54 ± 0.96 1.59 1.05 1.37 AIFM1 O95831 2 0.034 ± 0.005 0.03 ± 0.002 0.024 ± 0 0.017 ± 0.002 2.9 ± 0.08 3.46 ± 0.03 0.88 0.71 1.2 AIMP1 Q12904 1 0.032 0.048 0.023 0.017 2.51 2.77 1.5 0.74 1.1 AKAP8L Q9ULX6 1 0.104 0.097 0.037 0.046 3.85 3.8 0.93 1.24 0.99 AKR1A1 P14550 1 0.03 0.023 0.025 0.029 2.83 2.3 0.77 1.16 0.81 ALDH2 P05091 4 0.02 ± 0.003 0.017 ± 0.004 0.025 ± 0.003 0.02 ± 0.002 2.25 ± 0.07 2.7 ± 0.07 0.85 0.8 1.2 ALDH3A2 P51648 1 0.042 0.05 0.035 0.033 2.53 2.94 1.19 0.94 1.16 ALDOA P04075 6 0.085 ± 0.029 0.07 ± 0.002 0.021 ± 0.005 0.034 ± 0.004 4.21 ± 1.09 3.22 ± 0.3 0.82 1.62 0.77

180

Appendix 3. Protein synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

ALDOC P09972 1 0.12 0.076 0.02 0.032 5.45 3.64 0.63 1.6 0.67 ANP32B Q92688 1 0.035 0.026 0.016 0.03 2.88 2.17 0.74 1.88 0.75 ANXA11 P50995 2 0.049 ± 0.008 0.048 ± 0.002 0.032 ± 0.007 0.043 ± 0 2.26 ± 0.13 2.18 ± 0.08 0.98 1.34 0.97 ANXA2 P07355 11 0.051 ± 0.003 0.1 ± 0.014 0.027 ± 0.004 0.024 ± 0.007 2.11 ± 0.16 3.83 ± 0.55 1.96 0.89 1.82 ANXA4 P09525 1 0.032 0.026 0.014 0.033 2.39 2.05 0.81 2.36 0.86 ANXA5 P08758 3 0.073 ± 0.014 0.087 ± 0.04 0.023 ± 0.008 0.042 ± 0.005 3.47 ± 0.59 3.49 ± 1.13 1.19 1.83 1.01 ANXA6 A6NN80 1 0.1 0.028 0.049 0.023 5.36 4.31 0.28 0.47 0.8 ANXA6 P08133 3 0.069 ± 0.018 0.037 ± 0.003 0.029 ± 0.004 0.037 ± 0.002 5.6 ± 1.19 3.89 ± 0.38 0.54 1.28 0.69 AP1B1 Q10567 1 0.045 0.048 0.024 0.019 2.77 2.86 1.07 0.79 1.03 AP3D1 O14617 1 0.09 0.057 0.032 0.031 3.78 2.85 0.63 0.97 0.75 API5 Q9BZZ5 1 0.026 0.048 0.017 0.031 2.58 2.55 1.85 1.82 0.99 APMAP Q9HDC9 1 0.029 0.045 0.025 0.022 2.24 3.15 1.55 0.88 1.41 ARCN1 P48444 4 0.053 ± 0.009 0.046 ± 0.005 0.026 ± 0.004 0.028 ± 0.001 2.6 ± 0.23 2.31 ± 0.07 0.87 1.08 0.89 ARF1 P84077 4 0.063 ± 0.011 0.055 ± 0.005 0.022 ± 0.004 0.035 ± 0.004 3.29 ± 0.59 2.86 ± 0.26 0.87 1.59 0.87 ARF4 P18085 3 0.068 ± 0.008 0.072 ± 0.007 0.031 ± 0.011 0.037 ± 0.004 2.93 ± 0.38 2.9 ± 0.31 1.06 1.19 0.99 181 ARF6 P62330 1 0.066 0.07 0.027 0.038 3.16 3.2 1.06 1.41 1.01 ARHGDIA P52565 1 0.097 0.05 0.021 0.029 4.46 2.8 0.52 1.38 0.63 ARL8A Q96BM9 1 0.074 0.103 0.042 0.034 2.7 3.91 1.39 0.81 1.45 ARPC2 O15144 1 0.04 0.054 0.017 0.025 2.76 3.08 1.35 1.47 1.12 ARPC3 O15145 1 0.057 0.061 0.03 0.029 3.01 3.24 1.07 0.97 1.08 ARPP19 P56211 1 0.101 0.057 0.03 0.055 4.07 2.26 0.56 1.83 0.56 ASPH Q12797 1 0.094 0.077 0.035 0.021 3.54 3.36 0.82 0.6 0.95 ASS1 P00966 1 0.084 0.026 0.018 0.032 4.57 2.43 0.31 1.78 0.53 ATAD3A Q9NVI7 1 0.049 0.058 0.036 0.024 2.86 3.81 1.18 0.67 1.33 ATL3 Q6DD88 2 0.032 ± 0.001 0.052 ± 0.005 0.029 ± 0.004 0.028 ± 0 2.16 ± 0.26 3.06 ± 0.23 1.63 0.97 1.41 ATP1A1 P05023 6 0.049 ± 0.01 0.051 ± 0.011 0.035 ± 0.013 0.037 ± 0.003 2.62 ± 0.46 2.94 ± 0.31 1.04 1.06 1.12 ATP1B1 P05026 2 0.035 ± 0.001 0.059 ± 0.001 0.03 ± 0 0.036 ± 0.004 2.13 ± 0.03 2.66 ± 0.15 1.69 1.2 1.25 ATP2A1 O14983 1 0.052 0.071 0.021 0.022 2.62 3.16 1.37 1.05 1.21 ATP2A2 P16615 3 0.053 ± 0.009 0.066 ± 0.006 0.026 ± 0.005 0.027 ± 0 2.72 ± 0.03 3.26 ± 0.11 1.25 1.04 1.2 ATP5A1 P25705 7 0.026 ± 0.002 0.029 ± 0.004 0.024 ± 0.003 0.021 ± 0.002 2.64 ± 0.22 3.45 ± 0.2 1.12 0.88 1.31 ATP5B P06576 10 0.029 ± 0.005 0.031 ± 0.004 0.023 ± 0.003 0.023 ± 0.002 2.73 ± 0.3 3.35 ± 0.29 1.07 1 1.23 ATP5F1 P24539 1 0.021 0.021 0.024 0.017 2.31 3.26 1 0.71 1.41 ATP5J2 P56134 3 0.027 ± 0.003 0.031 ± 0.007 0.032 ± 0.004 0.023 ± 0.004 2.52 ± 0.25 3.44 ± 0.09 1.15 0.72 1.36 ATP5O P48047 1 0.021 0.022 0.023 0.016 2.29 3.07 1.05 0.7 1.34 BANF1 O75531 1 0.033 0.031 0.021 0.028 2.36 2.4 0.94 1.33 1.02 BAT1 Q13838 1 0.032 0.041 0.022 0.025 2.47 2.88 1.28 1.14 1.17 BCAP31 P51572 2 0.042 ± 0.002 0.051 ± 0.004 0.033 ± 0.011 0.022 ± 0.003 2.01 ± 0.22 2.94 ± 0.16 1.21 0.67 1.46

181

Appendix 3. Protein synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

BCLAF1 Q9NYF8 1 0.04 0.059 0.031 0.037 2.31 3 1.48 1.19 1.3 BLVRB P30043 1 0.026 0.029 0.016 0.033 3.19 2.29 1.12 2.06 0.72 BOP1 Q14137 1 0.103 0.039 0.034 0.036 4.47 3.7 0.38 1.06 0.83 C10orf58 Q9BRX8 3 0.049 ± 0.003 0.051 ± 0.006 0.026 ± 0 0.029 ± 0.002 2.51 ± 0.04 2.88 ± 0.16 1.04 1.12 1.15 C11orf59 Q6IAA8 1 0.055 0.053 0.036 0.046 2.43 2.37 0.96 1.28 0.98 C14orf166 Q9Y224 2 0.059 ± 0.002 0.056 ± 0.004 0.026 ± 0.004 0.031 ± 0.003 2.95 ± 0.27 2.81 ± 0.3 0.95 1.19 0.95 C17orf25 D3DTG9 1 0.044 0.023 0.022 0.035 3.09 1.93 0.52 1.59 0.62 C1QBP Q07021 1 0.053 0.055 0.026 0.017 3.61 3.83 1.04 0.65 1.06 C6orf115 Q9P1F3 1 0.039 0.035 0.028 0.034 2.16 2.12 0.9 1.21 0.98 C8orf55 Q8WUY1 1 0.013 0.03 0.021 0.023 2.99 3.3 2.31 1.1 1.1 CACYBP Q9HB71 4 0.095 ± 0.009 0.061 ± 0.005 0.027 ± 0.008 0.034 ± 0.007 4.25 ± 0.69 2.99 ± 0.12 0.64 1.26 0.7 CALD1 Q05682 1 0.038 0.067 0.032 0.035 1.69 2.64 1.76 1.09 1.56 CALM1 P62158 3 0.06 ± 0.003 0.048 ± 0.003 0.024 ± 0.001 0.03 ± 0.002 3.21 ± 0.26 2.74 ± 0.11 0.8 1.25 0.85 CALR P27797 4 0.042 ± 0.004 0.051 ± 0.003 0.024 ± 0.004 0.02 ± 0.006 2.76 ± 0.16 3.36 ± 0.09 1.21 0.83 1.22 CALU O43852 1 0.039 0.055 0.024 0.026 2.09 2.89 1.41 1.08 1.38 CAMK2B Q13554 1 0.04 0.042 0.021 0.029 2.2 2.63 1.05 1.38 1.2 182 CAND1 Q86VP6 1 0.048 0.04 0.036 0.029 2.87 2.98 0.83 0.81 1.04 CANX P27824 7 0.052 ± 0.022 0.057 ± 0.008 0.026 ± 0.006 0.024 ± 0.004 2.81 ± 0.58 3.48 ± 0.54 1.1 0.92 1.24 CAP1 Q01518 2 0.07 ± 0.002 0.063 ± 0.001 0.015 ± 0.005 0.031 ± 0 3.61 ± 0.04 2.95 ± 0.06 0.9 2.07 0.82 CAPN1 P07384 4 0.04 ± 0.003 0.039 ± 0.019 0.017 ± 0.003 0.036 ± 0.001 2.81 ± 0.17 2.03 ± 0.03 0.98 2.12 0.72 CAPN2 P17655 2 0.067 ± 0.013 0.112 ± 0.045 0.016 ± 0.007 0.038 ± 0.003 4 ± 0.77 4.33 ± 0.98 1.67 2.38 1.08 CAPNS1 P04632 2 0.056 ± 0.012 0.039 ± 0.002 0.025 ± 0.001 0.035 ± 0.001 3.2 ± 0.34 2.5 ± 0.12 0.7 1.4 0.78 CAPRIN1 Q14444 1 0.084 0.082 0.047 0.051 2.65 2.9 0.98 1.09 1.09 CAPZA1 P52907 2 0.051 ± 0.015 0.055 ± 0.001 0.024 ± 0.001 0.031 ± 0 3.05 ± 0.42 2.9 ± 0.03 1.08 1.29 0.95 CAPZA2 P47755 1 0.062 0.058 0.018 0.026 3.05 3.02 0.94 1.44 0.99 CAPZB P47756 3 0.047 ± 0.005 0.054 ± 0 0.024 ± 0.004 0.029 ± 0.008 2.58 ± 0.15 2.79 ± 0.03 1.15 1.21 1.08 CAT P04040 1 0.034 0.044 0.033 0.032 2.19 3.26 1.29 0.97 1.49 CBX3 Q13185 2 0.06 ± 0.008 0.054 ± 0.006 0.041 ± 0.005 0.044 ± 0.015 2.35 ± 0.16 2.5 ± 0.48 0.9 1.07 1.07 CCDC47 Q96A33 2 0.113 ± 0 0.117 ± 0.006 0.037 ± 0.005 0.039 ± 0.004 4.1 ± 0.09 4.49 ± 0 1.04 1.05 1.1 CCDC6 Q16204 1 0.06 0.046 0.029 0.037 3.09 2.8 0.77 1.28 0.91 CCT2 P78371 3 0.08 ± 0.002 0.061 ± 0.003 0.02 ± 0.001 0.031 ± 0.002 4.2 ± 0.26 3.21 ± 0.16 0.76 1.55 0.76 CCT3 P49368 6 0.064 ± 0.012 0.06 ± 0.011 0.033 ± 0.006 0.037 ± 0.003 3.11 ± 0.45 2.92 ± 0.23 0.94 1.12 0.94 CCT4 P50991 3 0.066 ± 0.008 0.055 ± 0.005 0.028 ± 0.006 0.032 ± 0.003 3.62 ± 0.55 3.05 ± 0.12 0.83 1.14 0.84 CCT5 P48643 1 0.091 0.07 0.024 0.031 3.76 2.91 0.77 1.29 0.77 CCT6A P40227 3 0.066 ± 0.011 0.053 ± 0.002 0.027 ± 0.008 0.029 ± 0.01 3.34 ± 0.58 2.81 ± 0.16 0.8 1.07 0.84 CCT6B Q92526 1 0.065 0.061 0.02 0.037 3.61 2.79 0.94 1.85 0.77 CCT7 Q99832 2 0.08 ± 0.005 0.056 ± 0.001 0.019 ± 0.003 0.033 ± 0 4.62 ± 0.01 3.25 ± 0.02 0.7 1.74 0.7 CCT8 P50990 5 0.062 ± 0.006 0.059 ± 0.01 0.025 ± 0.007 0.033 ± 0.002 3.58 ± 0.48 3.05 ± 0.23 0.95 1.32 0.85

182

Appendix 3. Protein synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

CD59 P13987 1 0.08 0.127 0.038 0.034 2.61 4.35 1.59 0.89 1.67 CD81 P60033 1 0.067 0.105 0.055 0.091 2.18 2.97 1.57 1.65 1.36 CDC37 Q16543 2 0.057 ± 0.007 0.068 ± 0.005 0.048 ± 0.002 0.039 ± 0.002 2.47 ± 0.25 2.81 ± 0.42 1.19 0.81 1.14 CDC73 Q6P1J9 1 0.073 0.077 0.069 0.087 2.51 2.67 1.05 1.26 1.06 CFL1 P23528 3 0.059 ± 0.005 0.052 ± 0.002 0.018 ± 0.001 0.039 ± 0.002 3.11 ± 0.15 2.37 ± 0.03 0.88 2.17 0.76 CHD3 Q12873 1 0.058 0.04 0.05 0.052 2.3 2.82 0.69 1.04 1.23 CKMT1A P12532 3 0.02 ± 0.002 0.025 ± 0.002 0.022 ± 0.002 0.02 ± 0 2.32 ± 0.17 3.49 ± 0.15 1.25 0.91 1.51 CKMT2 P17540 1 0.02 0.023 0.025 0.025 2.57 3.64 1.15 1 1.42 CLIC1 O00299 1 0.115 0.077 0.018 0.043 4.77 3.13 0.67 2.39 0.66 CLTB P09497 1 0.062 0.117 0.054 0.059 2.23 3.55 1.89 1.09 1.59 CLTC Q00610 10 0.041 ± 0.004 0.05 ± 0.025 0.028 ± 0.003 0.026 ± 0.002 2.6 ± 0.2 2.72 ± 0.18 1.22 0.93 1.04 CMPK1 P30085 1 0.035 0.036 0.018 0.029 2.81 2.24 1.03 1.61 0.8 CNDP2 Q96KP4 3 0.033 ± 0.003 0.024 ± 0.002 0.016 ± 0.002 0.03 ± 0.004 2.97 ± 0.33 2.16 ± 0.21 0.73 1.88 0.73 CNN3 Q15417 1 0.12 0.128 0.054 0.062 3.55 3.73 1.07 1.15 1.05 CNP P09543 1 0.011 0.026 0.015 0.025 1.05 2.02 2.36 1.67 1.92 COPA P53621 5 0.046 ± 0.003 0.044 ± 0.003 0.024 ± 0.004 0.029 ± 0.003 2.66 ± 0.27 2.49 ± 0.11 0.96 1.21 0.94 183 COPB2 P35606 2 0.051 ± 0.001 0.048 ± 0.005 0.031 ± 0.004 0.03 ± 0.001 2.58 ± 0.06 2.52 ± 0.04 0.94 0.97 0.98 COPG2 Q9UBF2 1 0.046 0.043 0.027 0.03 2.58 2.48 0.93 1.11 0.96 COPZ1 P61923 1 0.05 0.037 0.014 0.031 2.85 2.33 0.74 2.21 0.82 CORO1B Q9BR76 2 0.054 ± 0.003 0.049 ± 0.002 0.031 ± 0.005 0.032 ± 0.001 2.92 ± 0.08 3.13 ± 0.17 0.91 1.03 1.07 COX4I1 P13073 1 0.034 0.053 0.014 0.051 2.98 2.66 1.56 3.64 0.89 COX5A P20674 1 0.066 0.095 0.025 0.031 3.25 4.12 1.44 1.24 1.27 CPNE2 Q96FN4 1 0.078 0.052 0.019 0.042 4.6 2.78 0.67 2.21 0.6 CPNE3 O75131 3 0.064 ± 0.022 0.042 ± 0.005 0.026 ± 0.004 0.036 ± 0.003 3.42 ± 0.61 2.33 ± 0.18 0.66 1.38 0.68 CPSF1 Q10570 1 0.048 0.052 0.031 0.02 2.95 3.63 1.08 0.65 1.23 CS O75390 3 0.043 ± 0.003 0.048 ± 0.005 0.021 ± 0.002 0.019 ± 0.002 3.34 ± 0.12 3.86 ± 0.26 1.12 0.9 1.16 CSE1L P55060 2 0.057 ± 0.01 0.033 ± 0.003 0.033 ± 0.004 0.029 ± 0.002 3.39 ± 0.64 2.42 ± 0.08 0.58 0.88 0.71 CSNK2A1 P68400 2 0.057 ± 0.005 0.074 ± 0.014 0.039 ± 0.005 0.041 ± 0.002 2.66 ± 0.36 3.11 ± 0.33 1.3 1.05 1.17 CSRP1 P21291 1 0.04 0.06 0.014 0.035 2.32 2.34 1.5 2.5 1.01 CTSA P10619 3 0.033 ± 0.005 0.051 ± 0.005 0.041 ± 0.003 0.039 ± 0.002 1.91 ± 0.16 2.76 ± 0.21 1.55 0.95 1.45 CTSD P07339 7 0.04 ± 0.005 0.043 ± 0.005 0.037 ± 0.004 0.045 ± 0.004 1.99 ± 0.08 2.35 ± 0.05 1.08 1.22 1.18 CTTN Q14247 1 0.082 0.077 0.024 0.029 3.56 3.62 0.94 1.21 1.02 CUTA O60888 1 0.035 0.023 0.019 0.035 3.39 2.33 0.66 1.84 0.69 DAD1 P61803 1 0.034 0.057 0.04 0.028 2.11 3.36 1.68 0.7 1.59 DARS2 1 0.022 0.025 0.022 0.018 2.45 3.27 1.14 0.82 1.33 DCI P42126 1 0.024 0.023 0.026 0.017 2.3 3.32 0.96 0.65 1.44 DDOST P39656 2 0.036 ± 0.006 0.054 ± 0.013 0.028 ± 0.006 0.023 ± 0.002 2.59 ± 0.18 3.29 ± 0.08 1.5 0.82 1.27 DDT P30046 1 0.043 0.026 0.017 0.033 3.61 2.37 0.6 1.94 0.66

183

Appendix 3. Protein synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

DDX1 Q92499 1 0.05 0.05 0.026 0.025 2.96 2.97 1 0.96 1 DDX17 Q92841 3 0.082 ± 0.018 0.095 ± 0.036 0.045 ± 0.002 0.049 ± 0.006 3.16 ± 0.53 3.83 ± 0.59 1.16 1.09 1.21 DDX21 Q9NR30 7 0.105 ± 0.025 0.161 ± 0.038 0.037 ± 0.008 0.044 ± 0.009 3.91 ± 0.83 5.04 ± 0.99 1.53 1.19 1.29 DDX39 O00148 3 0.041 ± 0.009 0.052 ± 0.01 0.028 ± 0.006 0.03 ± 0.006 2.74 ± 0.13 3.24 ± 0.12 1.27 1.07 1.18 DDX3X O00571 4 0.093 ± 0.009 0.092 ± 0.03 0.056 ± 0.008 0.051 ± 0.006 3.06 ± 0.2 3.33 ± 0.53 0.99 0.91 1.09 DDX5 P17844 2 0.103 ± 0.006 0.122 ± 0.001 0.06 ± 0.012 0.075 ± 0 3.19 ± 0.3 3.82 ± 0.04 1.18 1.25 1.2 DDX6 P26196 1 0.059 0.071 0.058 0.069 2.33 2.46 1.2 1.19 1.06 DECR1 Q16698 1 0.027 0.026 0.025 0.015 2.26 3.09 0.96 0.6 1.37 DEK P35659 1 0.034 0.031 0.03 0.03 2.32 2.75 0.91 1 1.19 DERL1 Q9BUN8 1 0.068 0.089 0.031 0.028 3.3 4.08 1.31 0.9 1.24 DES P17661 2 0.059 ± 0.009 0.094 ± 0.004 0.029 ± 0.003 0.026 ± 0.002 2.91 ± 0.77 4.1 ± 0.36 1.59 0.9 1.41 DHCR24 Q15392 1 0.056 0.064 0.031 0.033 2.79 3.31 1.14 1.06 1.19 DHRS2 Q13268 6 0.013 ± 0.001 0.017 ± 0.001 0.022 ± 0.003 0.022 ± 0.003 2.07 ± 0.18 2.96 ± 0.23 1.31 1 1.43 DHRS7 Q9Y394 1 0.074 0.081 0.032 0.041 2.79 3.05 1.09 1.28 1.09 DHX15 O43143 3 0.069 ± 0.009 0.065 ± 0.005 0.029 ± 0.002 0.036 ± 0.004 3.41 ± 0.37 3.19 ± 0.23 0.94 1.24 0.94 DHX29 Q7Z478 1 0.09 0.072 0.044 0.037 3.01 3.29 0.8 0.84 1.09

184 DHX9 Q08211 6 0.047 ± 0.005 0.052 ± 0.005 0.029 ± 0.004 0.031 ± 0.004 2.87 ± 0.27 3.18 ± 0.21 1.11 1.07 1.11 DIAPH1 O60610 2 0.111 ± 0.038 0.088 ± 0.001 0.033 ± 0.009 0.039 ± 0 4.05 ± 1 3.29 ± 0.1 0.79 1.18 0.81 DKC1 O60832 1 0.039 0.05 0.027 0.024 2.55 2.99 1.28 0.89 1.17 DLAT P10515 1 0.048 0.041 0.024 0.02 3.24 3.83 0.85 0.83 1.18 DLD P09622 3 0.041 ± 0.009 0.036 ± 0.008 0.028 ± 0.006 0.031 ± 0.007 2.69 ± 0.53 3.25 ± 0.45 0.88 1.11 1.21 DNAJA1 P31689 1 0.244 0.149 0.052 0.073 6.84 4.5 0.61 1.4 0.66 DNAJA2 O60884 1 0.058 0.066 0.041 0.054 2.48 2.31 1.14 1.32 0.93 DNAJB1 P25685 2 0.059 ± 0 0.065 ± 0.001 0.049 ± 0.002 0.049 ± 0.005 2.49 ± 0.01 2.74 ± 0.07 1.1 1 1.1 DNAJC3 Q13217 1 0.073 0.099 0.055 0.037 2.56 3.8 1.36 0.67 1.48 DRG1 Q9Y295 1 0.06 0.074 0.058 0.041 2.23 2.89 1.23 0.71 1.3 DSG2 Q14126 1 0.045 0.128 0.043 0.061 2.2 4.21 2.84 1.42 1.91 DSTN P60981 2 0.062 ± 0.003 0.041 ± 0.005 0.024 ± 0.008 0.04 ± 0.005 2.85 ± 0.06 2.17 ± 0.01 0.66 1.67 0.76 DYNC1H1 Q14204 2 0.065 ± 0.004 0.054 ± 0 0.013 ± 0.001 0.03 ± 0 3.8 ± 0.13 2.71 ± 0.03 0.83 2.31 0.71 DYNC1I2 Q13409 1 0.069 0.056 0.014 0.028 3.55 3 0.81 2 0.85 ECH1 Q13011 1 0.021 0.028 0.032 0.024 2.35 3.02 1.33 0.75 1.29 ECHS1 P30084 2 0.023 ± 0 0.036 ± 0.001 0.021 ± 0.002 0.018 ± 0.002 2.6 ± 0.11 3.64 ± 0.27 1.57 0.86 1.4 EEF1A1 P68104 6 0.104 ± 0.018 0.071 ± 0.01 0.019 ± 0.006 0.036 ± 0.007 4.73 ± 0.58 3.08 ± 0.52 0.68 1.89 0.65 EEF1B2 P24534 1 0.083 0.065 0.028 0.034 3.73 2.9 0.78 1.21 0.78 EEF1D P29692 2 0.076 ± 0.028 0.072 ± 0 0.034 ± 0.011 0.029 ± 0.001 3.19 ± 0.87 3.25 ± 0.01 0.95 0.85 1.02 EEF1G P26641 4 0.066 ± 0.009 0.053 ± 0.009 0.028 ± 0.006 0.028 ± 0.007 3.27 ± 0.49 2.88 ± 0.18 0.8 1 0.88 EEF2 P13639 6 0.115 ± 0.02 0.076 ± 0.007 0.022 ± 0.004 0.038 ± 0.002 4.92 ± 0.77 3.04 ± 0.17 0.66 1.73 0.62 EHD1 Q9H4M9 2 0.081 ± 0.01 0.086 ± 0.004 0.027 ± 0.018 0.042 ± 0.003 3.37 ± 0.81 3.2 ± 0.18 1.06 1.56 0.95

184

Appendix 3. Protein synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

EIF1AX P47813 1 0.066 0.063 0.038 0.035 3.04 2.69 0.95 0.92 0.88 EIF1B O60739 1 0.203 0.155 0.054 0.116 5.74 3.82 0.76 2.15 0.67 EIF2S1 P05198 1 0.083 0.066 0.028 0.033 3.89 3.15 0.8 1.18 0.81 EIF3A Q14152 2 0.115 ± 0.008 0.088 ± 0.002 0.027 ± 0.004 0.033 ± 0 4.51 ± 0.33 3.53 ± 0.19 0.77 1.22 0.78 EIF3B P55884 3 0.111 ± 0.024 0.081 ± 0.007 0.034 ± 0.006 0.037 ± 0.004 3.95 ± 0.72 3.16 ± 0.24 0.73 1.09 0.8 EIF3D O15371 1 0.08 0.082 0.033 0.034 3.43 3.14 1.03 1.03 0.92 EIF3G O75821 1 0.052 0.076 0.041 0.031 2.48 3.04 1.46 0.76 1.23 EIF3I Q13347 1 0.117 0.102 0.05 0.035 3.83 3.8 0.87 0.7 0.99 EIF3M Q7L2H7 1 0.081 0.062 0.022 0.045 3.64 2.68 0.77 2.05 0.74 EIF4A1 P60842 6 0.087 ± 0.012 0.076 ± 0.008 0.029 ± 0.013 0.035 ± 0.006 3.54 ± 0.53 2.98 ± 0.14 0.87 1.21 0.84 EIF4G1 Q04637 5 0.093 ± 0.029 0.084 ± 0.011 0.053 ± 0.012 0.058 ± 0.007 3.2 ± 0.91 2.79 ± 0.41 0.9 1.09 0.87 EIF4G2 P78344 1 0.134 0.125 0.045 0.048 4.03 3.83 0.93 1.07 0.95 EIF4H Q15056 1 0.099 0.072 0.032 0.044 3.91 2.9 0.73 1.38 0.74 EIF5A P63241 2 0.083 ± 0.008 0.051 ± 0.006 0.018 ± 0.006 0.038 ± 0.001 3.92 ± 0.08 2.37 ± 0.17 0.61 2.11 0.6 EIF5B O60841 1 0.117 0.079 0.023 0.03 4.65 3.35 0.68 1.3 0.72 EIF6 P56537 1 0.07 0.079 0.03 0.039 3.12 2.93 1.13 1.3 0.94 185 ELAVL1 Q15717 1 0.053 0.057 0.033 0.035 2.51 2.88 1.08 1.06 1.15 ENO1 P06733 9 0.056 ± 0.006 0.04 ± 0.003 0.016 ± 0.003 0.03 ± 0.004 3.49 ± 0.44 2.52 ± 0.15 0.71 1.88 0.72 EPB41L1 Q9H4G0 1 0.028 0.043 0.034 0.038 1.82 2.85 1.54 1.12 1.57 EPHX1 P07099 2 0.022 ± 0.001 0.029 ± 0 0.026 ± 0.005 0.022 ± 0 1.83 ± 0.27 2.6 ± 0.17 1.32 0.85 1.42 EPPK1 P58107 9 0.065 ± 0.008 0.071 ± 0.011 0.027 ± 0.006 0.028 ± 0.004 3.05 ± 0.37 3.12 ± 0.45 1.09 1.04 1.02 EPRS P07814 3 0.046 ± 0.011 0.037 ± 0.004 0.02 ± 0.002 0.03 ± 0.007 2.77 ± 0.15 2.47 ± 0.3 0.8 1.5 0.89 ERBB2 P04626 2 0.052 ± 0.002 0.051 ± 0.002 0.056 ± 0.001 0.058 ± 0.001 2.12 ± 0.01 2.16 ± 0.05 0.98 1.04 1.02 ERP29 P30040 3 0.042 ± 0.006 0.041 ± 0.005 0.031 ± 0.005 0.021 ± 0.002 2.98 ± 0.29 3.44 ± 0.04 0.98 0.68 1.15 ETFA P13804 2 0.034 ± 0.002 0.044 ± 0.004 0.029 ± 0 0.03 ± 0.003 2.41 ± 0.09 3.4 ± 0.1 1.29 1.03 1.41 ETHE1 O95571 1 0.03 0.042 0.021 0.023 2.69 3.64 1.4 1.1 1.35 EWSR1 Q01844 2 0.047 ± 0.008 0.049 ± 0.015 0.028 ± 0 0.024 ± 0.003 2.61 ± 0.27 3.04 ± 0.22 1.04 0.86 1.17 EZR P15311 6 0.078 ± 0.013 0.071 ± 0.009 0.034 ± 0.008 0.041 ± 0.003 3.25 ± 0.43 2.85 ± 0.18 0.91 1.21 0.88 FAM114A2 Q9NRY5 1 0.04 0.039 0.022 0.03 2.62 2.43 0.98 1.36 0.93 FAM3C Q92520 1 0.04 0.07 0.041 0.037 1.87 2.7 1.75 0.9 1.44 FAM82B Q96DB5 2 0.02 ± 0.003 0.031 ± 0.003 0.024 ± 0.002 0.025 ± 0.003 2.1 ± 0.21 2.86 ± 0.29 1.55 1.04 1.37 FASN P49327 30 0.105 ± 0.032 0.047 ± 0.005 0.023 ± 0.01 0.039 ± 0.003 4.96 ± 1.47 2.61 ± 0.27 0.45 1.7 0.53 FH P07954 1 0.03 0.027 0.019 0.02 2.84 3.46 0.9 1.05 1.22 FIS1 Q9Y3D6 1 0.039 0.048 0.029 0.037 2.37 2.79 1.23 1.28 1.18 FKBP4 Q02790 2 0.075 ± 0.028 0.058 ± 0.002 0.021 ± 0.003 0.022 ± 0.001 4.23 ± 1 3.16 ± 0.11 0.77 1.05 0.75 FLII Q13045 1 0.071 0.086 0.057 0.048 2.58 3.1 1.21 0.84 1.2 FLNA P21333 5 0.048 ± 0.005 0.088 ± 0.009 0.023 ± 0.004 0.024 ± 0.003 1.96 ± 0.3 3.16 ± 0.26 1.83 1.04 1.61 FLNB B2ZZ83 1 0.055 0.072 0.033 0.044 2.28 3 1.31 1.33 1.32

185

Appendix 3. Protein synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

FLNB O75369 12 0.063 ± 0.013 0.1 ± 0.018 0.031 ± 0.01 0.033 ± 0.006 2.88 ± 0.54 3.8 ± 0.63 1.59 1.06 1.32 FUBP1 Q96AE4 2 0.064 ± 0.003 0.087 ± 0.001 0.052 ± 0.005 0.032 ± 0.002 2.49 ± 0.21 3.6 ± 0.34 1.36 0.62 1.45 FUS P35637 2 0.053 ± 0.006 0.052 ± 0.001 0.038 ± 0.002 0.034 ± 0.005 2.54 ± 0.29 2.95 ± 0.18 0.98 0.89 1.16 G3BP1 Q13283 2 0.077 ± 0.003 0.085 ± 0 0.035 ± 0.008 0.032 ± 0.001 3.33 ± 0.24 3.68 ± 0.14 1.1 0.91 1.11 G6PD P11413 3 0.056 ± 0.003 0.028 ± 0.002 0.014 ± 0.001 0.03 ± 0.002 3.7 ± 0.21 2.46 ± 0.09 0.5 2.14 0.67 GANAB Q14697 2 0.036 ± 0.001 0.04 ± 0.001 0.022 ± 0.004 0.026 ± 0.003 2.66 ± 0.03 3.15 ± 0.01 1.11 1.18 1.19 GAPDH P04406 5 0.053 ± 0.005 0.036 ± 0.003 0.014 ± 0.004 0.03 ± 0.001 3.85 ± 0.26 2.56 ± 0.12 0.68 2.14 0.66 GAR1 Q9NY12 1 0.065 0.065 0.029 0.03 3.17 3.42 1 1.03 1.08 GARS P41250 1 0.061 0.063 0.026 0.038 3.17 2.61 1.03 1.46 0.82 GART P22102 1 0.073 0.034 0.016 0.034 4.11 2.59 0.47 2.13 0.63 GDI1 P31150 1 0.051 0.037 0.023 0.033 2.8 2.27 0.73 1.43 0.81 GDI2 P50395 4 0.048 ± 0.007 0.035 ± 0.002 0.018 ± 0.003 0.034 ± 0.001 2.96 ± 0.22 2.28 ± 0.1 0.73 1.89 0.77 GGCT O75223 2 0.028 ± 0.006 0.017 ± 0.003 0.01 ± 0.002 0.028 ± 0.006 3.51 ± 0.06 1.95 ± 0.04 0.61 2.8 0.55 GLB1 P16278 1 0.011 0.017 0.021 0.016 1.73 2.65 1.55 0.76 1.53 GLUD1 P00367 1 0.047 0.031 0.02 0.026 2.98 2.96 0.66 1.3 0.99 GNAS Q5JWF2 2 0.08 ± 0.016 0.078 ± 0.012 0.035 ± 0.002 0.037 ± 0.003 3.57 ± 0.64 3.77 ± 0.44 0.98 1.06 1.06 186 GNB1 P62873 1 0.045 0.081 0.047 0.054 1.96 2.91 1.8 1.15 1.48 GNB2 P62879 1 0.068 0.089 0.04 0.044 2.88 3.64 1.31 1.1 1.26 GNB2L1 P63244 6 0.087 ± 0.013 0.078 ± 0.01 0.034 ± 0.009 0.038 ± 0.005 3.55 ± 0.55 3.25 ± 0.37 0.9 1.12 0.92 GNG12 Q9UBI6 1 0.064 0.09 0.033 0.03 2.99 4.19 1.41 0.91 1.4 GOT2 P00505 5 0.035 ± 0.003 0.037 ± 0.004 0.023 ± 0.004 0.019 ± 0.003 3.05 ± 0.18 3.52 ± 0.2 1.06 0.83 1.15 GPD2 P43304 1 0.028 0.031 0.019 0.017 2.59 3.15 1.11 0.89 1.22 GPI P06744 4 0.014 ± 0.002 0.029 ± 0.002 0.016 ± 0.003 0.032 ± 0.003 1.12 ± 0.06 2.16 ± 0.09 2.07 2 1.93 GRB7 Q14451 2 0.082 ± 0.025 0.08 ± 0.006 0.048 ± 0.011 0.057 ± 0.004 3.02 ± 0.76 2.92 ± 0.23 0.98 1.19 0.97 GSN P06396 1 0.028 0.033 0.017 0.023 1.9 1.88 1.18 1.35 0.99 GSTK1 Q9Y2Q3 2 0.018 ± 0 0.03 ± 0.001 0.027 ± 0.002 0.016 ± 0.001 1.82 ± 0.11 2.79 ± 0.08 1.67 0.59 1.53 GSTM3 P21266 2 0.033 ± 0.002 0.019 ± 0.001 0.01 ± 0.002 0.032 ± 0.001 3.05 ± 0.14 2.07 ± 0 0.58 3.2 0.68 GTF2I P78347 2 0.053 ± 0.002 0.062 ± 0.008 0.055 ± 0.022 0.087 ± 0.003 2.05 ± 0.3 2.02 ± 0.21 1.17 1.58 0.99 H2AFV Q71UI9 1 0.033 0.032 0.033 0.03 2.36 2.99 0.97 0.91 1.27 H2AFY O75367 1 0.027 0.036 0.023 0.027 2.07 2.88 1.33 1.17 1.39 HADH Q16836 2 0.014 ± 0.001 0.02 ± 0.001 0.045 ± 0.026 0.023 ± 0.002 1.76 ± 0.99 3.05 ± 0.19 1.43 0.51 1.74 HADHA P40939 4 0.029 ± 0.004 0.034 ± 0.007 0.025 ± 0.003 0.023 ± 0.001 2.18 ± 0.2 3.3 ± 0.29 1.17 0.92 1.51 HADHB P55084 5 0.02 ± 0.002 0.028 ± 0.004 0.021 ± 0.004 0.02 ± 0.003 2.05 ± 0.09 3.18 ± 0.1 1.4 0.95 1.55 HDAC1 Q13547 1 0.095 0.105 0.038 0.039 3.3 3.6 1.11 1.03 1.09 HDGF P51858 1 0.035 0.032 0.025 0.026 2.44 2.25 0.91 1.04 0.92 HEXB P07686 1 0.037 0.042 0.028 0.012 2.69 3.37 1.14 0.43 1.25 HGS O14964 1 0.089 0.094 0.041 0.059 3.17 3.04 1.06 1.44 0.96 HIBADH P31937 1 0.021 0.022 0.025 0.021 2.42 2.9 1.05 0.84 1.2

186

Appendix 3. Protein synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

HIST1H1A Q02539 1 0.028 0.018 0.026 0.032 2.18 2.34 0.64 1.23 1.07 HIST1H1B P16401 2 0.018 ± 0 0.011 ± 0.001 0.02 ± 0.001 0.023 ± 0.004 2.49 ± 0.16 2.82 ± 0.22 0.61 1.15 1.13 HIST1H1C P16403 2 0.022 ± 0.004 0.019 ± 0 0.023 ± 0.004 0.029 ± 0.002 2.29 ± 0.23 2.62 ± 0.11 0.86 1.26 1.14 HIST1H2AA Q96QV6 3 0.022 ± 0.001 0.018 ± 0.001 0.026 ± 0.003 0.026 ± 0.002 2.41 ± 0.29 2.95 ± 0.25 0.82 1 1.23 HIST1H2AB P04908 1 0.022 0.02 0.024 0.021 2.66 3.29 0.91 0.88 1.24 HIST1H2BC P62807 1 0.013 0.013 0.017 0.018 1.99 2.66 1 1.06 1.34 HIST1H4A P62805 8 0.018 ± 0.002 0.015 ± 0.006 0.025 ± 0.006 0.02 ± 0.003 2.2 ± 0.22 2.8 ± 0.24 0.83 0.8 1.27 HIST3H3 Q16695 1 0.008 0.013 0.021 0.022 2.35 3.2 1.63 1.05 1.36 HK1 P19367 4 0.04 ± 0.003 0.05 ± 0.005 0.024 ± 0.002 0.025 ± 0.005 2.81 ± 0.21 3.67 ± 0.38 1.25 1.04 1.31 HK2 P52789 2 0.108 ± 0.015 0.108 ± 0.03 0.04 ± 0.001 0.055 ± 0.005 4.32 ± 0.72 4.27 ± 1.11 1 1.38 0.99 HKDC1 Q2TB90 1 0.036 0.044 0.03 0.024 2.54 3.6 1.22 0.8 1.42 HMGB1 P09429 1 0.032 0.028 0.017 0.026 2.81 2.17 0.88 1.53 0.77 HMGB1L1 B2RPK0 2 0.031 ± 0.003 0.022 ± 0.001 0.018 ± 0.002 0.033 ± 0.005 2.51 ± 0.26 1.91 ± 0.22 0.71 1.83 0.76 HMGN1 P05114 1 0.056 0.052 0.034 0.04 3.04 2.69 0.93 1.18 0.88 HNRNPA1 P09651 1 0.059 0.064 0.031 0.026 3.2 3.87 1.08 0.84 1.21 HNRNPA1L2 Q32P51 4 0.052 ± 0.003 0.059 ± 0.006 0.031 ± 0.002 0.032 ± 0.008 2.79 ± 0.17 3.22 ± 0.5 1.13 1.03 1.15 187 HNRNPA2B P22626 6 0.045 ± 0.005 0.051 ± 0.01 0.035 ± 0.005 0.028 ± 0.005 2.56 ± 0.22 3.4 ± 0.43 1.13 0.8 1.33 1 HNRNPA3 P51991 3 0.043 ± 0.006 0.07 ± 0.012 0.035 ± 0.003 0.032 ± 0.001 2.49 ± 0.2 3.28 ± 0.16 1.63 0.91 1.32 HNRNPAB Q99729 1 0.045 0.052 0.033 0.032 2.53 3.04 1.16 0.97 1.2 HNRNPC P07910 1 0.045 0.051 0.033 0.028 3.01 3.53 1.13 0.85 1.17 HNRNPCL1 O60812 4 0.04 ± 0.003 0.045 ± 0.003 0.035 ± 0.004 0.03 ± 0.002 2.43 ± 0.2 3.1 ± 0.15 1.13 0.86 1.28 HNRNPD Q14103 2 0.045 ± 0.006 0.054 ± 0.01 0.026 ± 0.005 0.024 ± 0.002 2.67 ± 0.1 3.3 ± 0.08 1.2 0.92 1.23 HNRNPF P52597 2 0.053 ± 0.001 0.064 ± 0.006 0.031 ± 0.002 0.032 ± 0.002 2.89 ± 0.05 3.26 ± 0.08 1.21 1.03 1.13 HNRNPH1 P31943 2 0.063 ± 0.007 0.072 ± 0.009 0.033 ± 0.002 0.031 ± 0.001 2.93 ± 0.17 3.52 ± 0.25 1.14 0.94 1.2 HNRNPK P61978 10 0.055 ± 0.006 0.064 ± 0.006 0.04 ± 0.004 0.037 ± 0.004 2.64 ± 0.27 3.2 ± 0.27 1.16 0.93 1.21 HNRNPL P14866 4 0.048 ± 0.004 0.059 ± 0.013 0.028 ± 0.004 0.028 ± 0.004 2.79 ± 0.08 3.25 ± 0.26 1.23 1 1.17 HNRNPM P52272 3 0.033 ± 0.01 0.05 ± 0.001 0.025 ± 0.003 0.028 ± 0.001 2.36 ± 0.27 3.04 ± 0.27 1.52 1.12 1.29 HNRNPR O43390 2 0.054 ± 0.008 0.058 ± 0.008 0.029 ± 0.001 0.024 ± 0.006 3.07 ± 0.53 3.52 ± 0.44 1.07 0.83 1.15 HNRNPU Q00839 7 0.052 ± 0.007 0.06 ± 0.006 0.032 ± 0.006 0.028 ± 0.006 2.73 ± 0.46 3.53 ± 0.48 1.15 0.88 1.29 HNRNPUL2 Q1KMD3 2 0.036 ± 0.002 0.04 ± 0.002 0.037 ± 0.003 0.032 ± 0.005 2.28 ± 0.02 2.87 ± 0.12 1.11 0.86 1.26 HNRPAB D3DWP7 1 0.053 0.061 0.028 0.032 2.95 3.41 1.15 1.14 1.16 HNRPDL O14979 3 0.046 ± 0.008 0.064 ± 0.011 0.027 ± 0.005 0.033 ± 0.003 2.57 ± 0.18 3.33 ± 0.3 1.39 1.22 1.3 HSD17B10 Q99714 1 0.038 0.05 0.018 0.019 3.36 4.24 1.32 1.06 1.26 HSD17B4 P51659 1 0.036 0.039 0.032 0.028 2.23 3.06 1.08 0.88 1.37 HSP90AA1 P07900 8 0.072 ± 0.012 0.047 ± 0.004 0.023 ± 0.006 0.03 ± 0.002 3.97 ± 0.55 2.91 ± 0.25 0.65 1.3 0.73 HSP90AA2 Q14568 1 0.068 0.044 0.018 0.03 3.88 2.69 0.65 1.67 0.69 HSP90AA5P Q58FG0 2 0.073 ± 0.009 0.047 ± 0.003 0.018 ± 0.001 0.031 ± 0.001 4.14 ± 0.29 2.77 ± 0.13 0.64 1.72 0.67

187

Appendix 3. Protein synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

HSP90AB1 P08238 3 0.105 ± 0.008 0.067 ± 0.004 0.018 ± 0.003 0.031 ± 0.001 5.31 ± 0.44 3.42 ± 0.18 0.64 1.72 0.64 HSP90AB2P Q58FF8 5 0.073 ± 0.012 0.053 ± 0.004 0.019 ± 0.003 0.029 ± 0.003 3.96 ± 0.43 2.86 ± 0.07 0.73 1.53 0.72 HSP90AB3P Q58FF7 5 0.067 ± 0.021 0.058 ± 0.006 0.03 ± 0.006 0.033 ± 0.002 3.58 ± 0.94 2.89 ± 0.21 0.87 1.1 0.81 HSP90B1 P14625 7 0.053 ± 0.012 0.056 ± 0.004 0.027 ± 0.006 0.024 ± 0.003 3.15 ± 0.33 3.42 ± 0.37 1.06 0.89 1.09 HSP90B2P Q58FF3 1 0.037 0.051 0.029 0.032 2.48 2.88 1.38 1.1 1.16 HSPA1A P08107 6 0.091 ± 0.018 0.053 ± 0.004 0.037 ± 0.003 0.033 ± 0.005 3.28 ± 0.5 2.74 ± 0.2 0.58 0.89 0.84 HSPA1L P34931 5 0.096 ± 0.008 0.06 ± 0.015 0.029 ± 0.003 0.034 ± 0.001 3.72 ± 0.34 2.91 ± 0.49 0.63 1.17 0.78 HSPA2 P54652 4 0.104 ± 0.019 0.092 ± 0.008 0.031 ± 0.004 0.036 ± 0.004 4.26 ± 0.73 3.81 ± 0.42 0.88 1.16 0.89 HSPA4 P34932 3 0.069 ± 0.013 0.052 ± 0.001 0.023 ± 0.002 0.034 ± 0.004 3.36 ± 0.64 2.7 ± 0.17 0.75 1.48 0.8 HSPA5 P11021 11 0.059 ± 0.018 0.067 ± 0.01 0.027 ± 0.006 0.022 ± 0.004 3.07 ± 0.65 3.66 ± 0.3 1.14 0.81 1.19 HSPA8 P11142 13 0.1 ± 0.024 0.089 ± 0.012 0.029 ± 0.012 0.035 ± 0.005 4.08 ± 0.94 3.59 ± 0.45 0.89 1.21 0.88 HSPA9 P38646 12 0.066 ± 0.013 0.059 ± 0.009 0.031 ± 0.008 0.031 ± 0.012 3.85 ± 0.53 3.82 ± 0.66 0.89 1 0.99 HSPB1 P04792 3 0.097 ± 0.011 0.044 ± 0.002 0.017 ± 0.002 0.033 ± 0.002 4.6 ± 0.38 2.34 ± 0.13 0.45 1.94 0.51 HSPD1 P10809 10 0.054 ± 0.004 0.051 ± 0.003 0.022 ± 0.004 0.02 ± 0.002 3.78 ± 0.29 3.97 ± 0.24 0.94 0.91 1.05 HSPE1 P61604 1 0.04 0.042 0.023 0.016 2.94 3.45 1.05 0.7 1.17 HSPH1 Q92598 2 0.146 ± 0.03 0.107 ± 0.013 0.029 ± 0.001 0.034 ± 0.002 5.3 ± 0.58 3.87 ± 0.45 0.73 1.17 0.73 188 HYOU1 Q9Y4L1 3 0.049 ± 0.004 0.064 ± 0.006 0.024 ± 0.005 0.023 ± 0.001 2.96 ± 0.11 3.43 ± 0.16 1.31 0.96 1.16 IARS2 Q9NSE4 2 0.03 ± 0.002 0.035 ± 0.004 0.021 ± 0.007 0.023 ± 0.002 2.67 ± 0.22 3.25 ± 0.24 1.17 1.1 1.22 IDH2 P48735 4 0.027 ± 0.009 0.023 ± 0.003 0.026 ± 0.005 0.018 ± 0.005 2.08 ± 0.27 2.81 ± 0.19 0.85 0.69 1.35 IDH3A P50213 1 0.019 0.036 0.034 0.021 2.31 3.66 1.89 0.62 1.58 IDH3B O43837 1 0.04 0.041 0.05 0.029 1.99 3.32 1.03 0.58 1.67 ILF2 Q12905 3 0.044 ± 0.004 0.04 ± 0.002 0.025 ± 0.002 0.023 ± 0.002 2.47 ± 0.06 3 ± 0.05 0.91 0.92 1.21 ILF3 Q12906 5 0.037 ± 0.005 0.043 ± 0.008 0.028 ± 0.005 0.026 ± 0.003 2.35 ± 0.4 2.89 ± 0.42 1.16 0.93 1.23 IPO7 O95373 2 0.11 ± 0.026 0.074 ± 0.015 0.024 ± 0.003 0.042 ± 0.018 4.36 ± 1.15 2.84 ± 0.64 0.67 1.75 0.65 IQGAP1 P46940 6 0.054 ± 0.008 0.057 ± 0.006 0.024 ± 0.002 0.032 ± 0.003 2.54 ± 0.26 2.65 ± 0.21 1.06 1.33 1.04 IVD P26440 1 0.027 0.033 0.016 0.017 2.92 3.52 1.22 1.06 1.21 IVL P07476 1 0.043 0.049 0.013 0.03 1.68 1.95 1.14 2.31 1.16 JUP P14923 1 0.116 0.151 0.074 0.081 3.28 4.24 1.3 1.09 1.29 KDM1A O60341 1 0.04 0.041 0.029 0.054 2.19 2.39 1.03 1.86 1.09 KHDRBS1 Q07666 1 0.04 0.057 0.041 0.026 1.88 2.85 1.43 0.63 1.52 KIF5A Q12840 1 0.055 0.06 0.026 0.037 2.93 2.46 1.09 1.42 0.84 KPNB1 Q14974 3 0.049 ± 0.008 0.05 ± 0.001 0.028 ± 0.009 0.03 ± 0.007 3 ± 0.45 2.83 ± 0.23 1.02 1.07 0.94 KRT1 P04264 1 0.085 0.1 0.033 0.03 3.68 4.23 1.18 0.91 1.15 KRT10 P13645 1 0.069 0.089 0.025 0.03 3.68 4.12 1.29 1.2 1.12 KRT13 P13646 1 0.049 0.084 0.028 0.026 2.96 4.1 1.71 0.93 1.39 KRT14 P02533 2 0.046 ± 0.002 0.08 ± 0.006 0.029 ± 0.004 0.023 ± 0.004 2.73 ± 0.2 4.18 ± 0.1 1.74 0.79 1.53 KRT16 P08779 2 0.046 ± 0.001 0.077 ± 0.007 0.028 ± 0.003 0.028 ± 0.004 2.57 ± 0.08 3.28 ± 0.11 1.67 1 1.28 KRT17 Q04695 2 0.046 ± 0.001 0.081 ± 0.004 0.027 ± 0.002 0.026 ± 0 2.71 ± 0.33 3.81 ± 0 1.76 0.96 1.41

188

Appendix 3. Protein synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

KRT18 P05783 15 0.09 ± 0.017 0.103 ± 0.012 0.031 ± 0.008 0.03 ± 0.008 4.12 ± 0.64 4.5 ± 0.58 1.14 0.97 1.09 KRT19 P08727 11 0.047 ± 0.005 0.084 ± 0.005 0.027 ± 0.002 0.027 ± 0.001 2.81 ± 0.16 3.99 ± 0.2 1.79 1 1.42 KRT2 P35908 2 0.073 ± 0.016 0.102 ± 0.007 0.023 ± 0.001 0.029 ± 0.001 3.88 ± 0.71 4.4 ± 0.51 1.4 1.26 1.13 KRT3 P12035 1 0.038 0.026 0.022 0.031 2.8 2.34 0.68 1.41 0.84 KRT4 P19013 4 0.035 ± 0.006 0.045 ± 0.021 0.029 ± 0.003 0.032 ± 0.002 2.49 ± 0.31 2.74 ± 0.32 1.29 1.1 1.1 KRT6A P02538 2 0.079 ± 0.002 0.098 ± 0.003 0.021 ± 0.003 0.03 ± 0 4.15 ± 0.1 4.38 ± 0.04 1.24 1.43 1.05 KRT7 P08729 18 0.044 ± 0.004 0.084 ± 0.007 0.027 ± 0.002 0.029 ± 0.004 2.16 ± 0.16 3.44 ± 0.3 1.91 1.07 1.59 KRT75 O95678 1 0.076 0.106 0.033 0.03 3.97 4.97 1.39 0.91 1.25 KRT79 Q5XKE5 1 0.041 0.088 0.025 0.025 2.08 3.48 2.15 1 1.67 KRT8 P05787 21 0.073 ± 0.012 0.092 ± 0.013 0.027 ± 0.004 0.03 ± 0.004 3.83 ± 0.42 4.23 ± 0.38 1.26 1.11 1.1 KTN1 Q86UP2 2 0.057 ± 0.001 0.068 ± 0.008 0.038 ± 0.001 0.032 ± 0.004 2.49 ± 0.09 3.4 ± 0.39 1.19 0.84 1.36 LACTB2 Q53H82 1 0.031 0.031 0.033 0.032 1.76 2.16 1 0.97 1.23 LAD1 O00515 1 0.073 0.08 0.05 0.055 2.54 2.66 1.1 1.1 1.05 LARP7 Q4G0J3 1 0.029 0.033 0.027 0.023 2.18 3.18 1.14 0.85 1.46 LCP1 P13796 2 0.077 ± 0.008 0.084 ± 0.004 0.029 ± 0.006 0.039 ± 0.007 3.11 ± 0.34 3.13 ± 0.25 1.09 1.34 1.01 LDHA P00338 3 0.105 ± 0.008 0.085 ± 0.003 0.016 ± 0.001 0.033 ± 0.002 5.11 ± 0.3 3.62 ± 0.17 0.81 2.06 0.71 189 LDHAL6A Q6ZMR3 1 0.115 0.075 0.012 0.036 6.07 3.56 0.65 3 0.59 LDHB P07195 2 0.09 ± 0.001 0.042 ± 0.002 0.019 ± 0.002 0.029 ± 0.004 4.98 ± 0.17 2.72 ± 0.07 0.47 1.53 0.55 LGALS3BP Q08380 4 0.021 ± 0.001 0.026 ± 0.002 0.028 ± 0.004 0.037 ± 0.006 1.36 ± 0.08 2.15 ± 0.13 1.24 1.32 1.58 LMAN2 Q12907 1 0.039 0.046 0.032 0.029 2.51 3.1 1.18 0.91 1.24 LMNA P02545 15 0.037 ± 0.004 0.054 ± 0.006 0.028 ± 0.004 0.026 ± 0.004 2.15 ± 0.18 3.35 ± 0.21 1.46 0.93 1.56 LMNB1 P20700 1 0.02 0.051 0.019 0.022 2.72 3.29 2.55 1.16 1.21 LONP1 P36776 1 0.045 0.04 0.031 0.021 2.47 2.99 0.89 0.68 1.21 LRRC59 Q96AG4 1 0.065 0.062 0.038 0.03 2.7 3.24 0.95 0.79 1.2 LRRFIP1 Q32MZ4 2 0.078 ± 0.006 0.084 ± 0.008 0.028 ± 0.001 0.04 ± 0.001 3.42 ± 0.22 2.98 ± 0.21 1.08 1.43 0.87 MAGOHB Q96A72 1 0.038 0.041 0.036 0.031 2.15 2.65 1.08 0.86 1.23 MAPRE1 Q15691 1 0.063 0.064 0.023 0.047 2.97 2.51 1.02 2.04 0.85 MARS P56192 2 0.056 ± 0.002 0.056 ± 0.003 0.026 ± 0.002 0.029 ± 0.001 2.93 ± 0.21 2.87 ± 0.04 1 1.12 0.98 MAT2A P31153 2 0.123 ± 0.017 0.088 ± 0.005 0.046 ± 0.004 0.052 ± 0.003 4.19 ± 0.51 3.17 ± 0.27 0.72 1.13 0.76 MATR3 P43243 3 0.07 ± 0.022 0.071 ± 0.011 0.034 ± 0.005 0.036 ± 0.002 3.21 ± 0.65 3.67 ± 0.26 1.01 1.06 1.14 MDH1 P40925 1 0.034 0.025 0.019 0.033 3.66 2.58 0.74 1.74 0.7 MDH2 P40926 13 0.029 ± 0.002 0.034 ± 0.003 0.022 ± 0.003 0.021 ± 0.003 2.66 ± 0.2 3.44 ± 0.19 1.17 0.95 1.29 MFAP1 P55081 1 0.082 0.082 0.061 0.059 2.7 3.29 1 0.97 1.22 MFSD10 Q14728 1 0.029 0.044 0.029 0.021 2.07 3.12 1.52 0.72 1.51 MGST1 P10620 1 0.052 0.044 0.02 0.021 3.39 2.99 0.85 1.05 0.88 MIF P14174 1 0.049 0.027 0.013 0.038 3.91 2.19 0.55 2.92 0.56 MOGS Q13724 1 0.051 0.055 0.03 0.023 2.71 3.54 1.08 0.77 1.31 MRPL38 Q96DV4 1 0.041 0.046 0.028 0.023 3.04 3.35 1.12 0.82 1.1

189

Appendix 3. Protein synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

MRPL49 Q13405 1 0.023 0.03 0.023 0.025 2.64 3.36 1.3 1.09 1.27 MRPS18B Q9Y676 1 0.038 0.052 0.028 0.037 3.06 3.47 1.37 1.32 1.13 MRPS7 Q9Y2R9 1 0.042 0.063 0.033 0.03 2.39 3.25 1.5 0.91 1.36 MRPS9 P82933 1 0.04 0.05 0.025 0.026 3.01 3.41 1.25 1.04 1.13 MT‐ATP6 P00846 1 0.018 0.026 0.019 0.014 2.26 3.2 1.44 0.74 1.42 MTHFD1 P11586 1 0.046 0.024 0.022 0.027 3.19 2.47 0.52 1.23 0.77 MVP Q14764 2 0.052 ± 0.01 0.068 ± 0.004 0.022 ± 0.008 0.036 ± 0.005 2.22 ± 0.04 2.61 ± 0.16 1.31 1.64 1.18 MYBBP1A Q9BQG0 3 0.048 ± 0.001 0.045 ± 0.009 0.039 ± 0.003 0.029 ± 0.003 2.56 ± 0.04 2.69 ± 0.09 0.94 0.74 1.05 MYH10 P35580 6 0.055 ± 0.013 0.06 ± 0.016 0.023 ± 0.003 0.029 ± 0.006 2.68 ± 0.53 2.56 ± 0.27 1.09 1.26 0.95 MYH11 P35749 1 0.068 0.071 0.02 0.03 3.12 2.84 1.04 1.5 0.91 MYH9 P35579 13 0.067 ± 0.011 0.063 ± 0.005 0.024 ± 0.007 0.034 ± 0.007 2.93 ± 0.46 2.63 ± 0.23 0.94 1.42 0.9 MYL12A P19105 2 0.067 ± 0.011 0.085 ± 0.01 0.029 ± 0.007 0.037 ± 0.002 3.02 ± 0.56 3.01 ± 0.03 1.27 1.28 1 MYL6 P60660 1 0.067 0.077 0.032 0.042 2.84 2.92 1.15 1.31 1.03 MYL6B P14649 1 0.125 0.105 0.037 0.043 4.19 3.31 0.84 1.16 0.79 MYO1C O00159 2 0.04 ± 0.003 0.052 ± 0.005 0.026 ± 0.005 0.031 ± 0.001 2.54 ± 0.37 2.78 ± 0.19 1.3 1.19 1.09 MYO6 Q9UM54 2 0.063 ± 0 0.068 ± 0.009 0.03 ± 0.004 0.036 ± 0.006 2.64 ± 0.17 2.66 ± 0.39 1.08 1.2 1.01 190 NACA Q13765 2 0.089 ± 0.001 0.074 ± 0.001 0.02 ± 0.002 0.039 ± 0.001 4.04 ± 0.11 3.14 ± 0.01 0.83 1.95 0.78 NANS Q9NR45 1 0.033 0.02 0.017 0.027 2.34 1.97 0.61 1.59 0.84 NAP1L1 P55209 3 0.11 ± 0.016 0.077 ± 0.012 0.019 ± 0.005 0.036 ± 0.004 4.73 ± 0.37 3.29 ± 0.34 0.7 1.89 0.7 NAP1L4 Q99733 1 0.105 0.036 0.039 0.026 3.18 2.61 0.34 0.67 0.82 NAPA P54920 3 0.051 ± 0.008 0.043 ± 0.007 0.045 ± 0.004 0.05 ± 0.004 2.48 ± 0.35 2.4 ± 0.17 0.84 1.11 0.97 NARS O43776 1 0.054 0.052 0.031 0.029 3.16 2.78 0.96 0.94 0.88 NASP P49321 1 0.056 0.024 0.035 0.045 3.01 2.23 0.43 1.29 0.74 NCL P19338 7 0.058 ± 0.008 0.052 ± 0.005 0.029 ± 0.004 0.026 ± 0.003 3.42 ± 0.37 3.25 ± 0.21 0.9 0.9 0.95 NDUFA8 P51970 1 0.025 0.038 0.02 0.025 2.35 3.21 1.52 1.25 1.37 NDUFB7 P17568 1 0.03 0.032 0.027 0.028 2.69 3.43 1.07 1.04 1.28 NDUFV2 P19404 1 0.042 0.058 0.028 0.039 2.37 2.88 1.38 1.39 1.22 NHP2L1 P55769 1 0.042 0.054 0.022 0.021 2.88 3.15 1.29 0.95 1.09 NMNAT1 Q9HAN9 1 0.025 0.031 0.029 0.018 2.34 3.14 1.24 0.62 1.34 NOLC1 Q14978 1 0.094 0.113 0.05 0.062 3.18 4.21 1.2 1.24 1.32 NOP58 Q9Y2X3 3 0.04 ± 0.007 0.052 ± 0.007 0.03 ± 0.008 0.024 ± 0.006 2.5 ± 0.16 3.32 ± 0.41 1.3 0.8 1.33 NPM1 P06748 1 0.047 0.073 0.03 0.035 2.73 3.54 1.55 1.17 1.3 NSF P46459 1 0.036 0.039 0.019 0.026 2.7 2.89 1.08 1.37 1.07 NSUN2 Q08J23 1 0.095 0.075 0.033 0.035 4.28 3.46 0.79 1.06 0.81 NUCB1 Q02818 1 0.059 0.039 0.087 0.078 1.83 2.21 0.66 0.9 1.21 NUP133 Q8WUM0 1 0.042 0.053 0.033 0.033 2.75 3.31 1.26 1 1.2 OAT P04181 1 0.07 0.073 0.063 0.063 2.3 2.66 1.04 1 1.16 OSTC Q9NRP0 1 0.045 0.053 0.03 0.028 2.68 3.29 1.18 0.93 1.23

190

Appendix 3. Protein synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

OXSR1 O95747 1 0.066 0.048 0.018 0.034 3.65 2.62 0.73 1.89 0.72 P4HA1 P13674 2 0.093 ± 0.002 0.149 ± 0.002 0.046 ± 0.007 0.041 ± 0.002 3.19 ± 0.25 5.08 ± 0.05 1.6 0.89 1.59 P4HB P07237 12 0.031 ± 0.005 0.045 ± 0.004 0.021 ± 0.004 0.023 ± 0.003 2.1 ± 0.14 2.92 ± 0.2 1.45 1.1 1.39 PA2G4 Q9UQ80 6 0.071 ± 0.012 0.053 ± 0.003 0.025 ± 0.009 0.031 ± 0.005 3.63 ± 0.52 2.71 ± 0.17 0.75 1.24 0.75 PABPC1 P11940 7 0.085 ± 0.013 0.075 ± 0.011 0.036 ± 0.02 0.038 ± 0.007 3.56 ± 0.68 3.21 ± 0.4 0.88 1.06 0.9 PABPC4L P0CB38 1 0.078 0.07 0.025 0.044 3.73 2.87 0.9 1.76 0.77 PARP1 P09874 3 0.045 ± 0.011 0.057 ± 0.014 0.037 ± 0.007 0.029 ± 0.002 2.55 ± 0.37 3.13 ± 0.39 1.27 0.78 1.23 PCBP1 Q15365 4 0.078 ± 0.013 0.061 ± 0.003 0.027 ± 0.004 0.042 ± 0.001 3.5 ± 0.42 2.75 ± 0.13 0.78 1.56 0.79 PCBP2 Q15366 3 0.077 ± 0.006 0.06 ± 0 0.028 ± 0.01 0.04 ± 0.005 3.26 ± 0.41 2.69 ± 0.12 0.78 1.43 0.82 PDCD5 O14737 1 0.062 0.072 0.025 0.051 2.55 2.49 1.16 2.04 0.98 PDCD6 O75340 3 0.076 ± 0.008 0.049 ± 0.001 0.025 ± 0.011 0.036 ± 0.002 4.21 ± 0.51 2.75 ± 0.11 0.64 1.44 0.65 PDCD6IP Q8WUM4 3 0.08 ± 0.01 0.063 ± 0.004 0.03 ± 0.01 0.046 ± 0.005 3.4 ± 0.65 2.57 ± 0.26 0.79 1.53 0.76 PDHX O00330 1 0.041 0.036 0.025 0.024 3.14 3.48 0.88 0.96 1.11 PDIA3 P30101 9 0.053 ± 0.023 0.054 ± 0.011 0.028 ± 0.005 0.024 ± 0.004 2.82 ± 0.59 3.31 ± 0.45 1.02 0.86 1.18 PDIA4 P13667 2 0.037 ± 0.004 0.045 ± 0.005 0.03 ± 0.007 0.02 ± 0.001 2.22 ± 0.27 2.94 ± 0.23 1.22 0.67 1.32 PDIA6 Q15084 7 0.042 ± 0.004 0.054 ± 0.002 0.024 ± 0.003 0.022 ± 0.004 2.57 ± 0.22 3.41 ± 0.33 1.29 0.92 1.32 191 PDLIM5 Q96HC4 1 0.086 0.093 0.028 0.045 3.25 2.88 1.08 1.61 0.89 PES1 O00541 1 0.065 0.099 0.042 0.042 2.61 3.68 1.52 1 1.41 PFDN5 Q99471 1 0.075 0.042 0.017 0.03 3.59 2.86 0.56 1.76 0.8 PFN1 P07737 4 0.068 ± 0.011 0.052 ± 0.003 0.015 ± 0.003 0.034 ± 0.005 3.71 ± 0.4 2.5 ± 0.17 0.76 2.27 0.67 PGAM1 P18669 1 0.061 0.051 0.018 0.025 4.36 3.59 0.84 1.39 0.82 PGD P52209 1 0.046 0.034 0.014 0.032 3.12 2.33 0.74 2.29 0.75 PGK1 P00558 6 0.054 ± 0.001 0.042 ± 0.01 0.014 ± 0.004 0.035 ± 0.002 3.48 ± 0.3 2.42 ± 0.15 0.78 2.5 0.7 PGRMC1 O00264 2 0.03 ± 0.006 0.036 ± 0.002 0.02 ± 0.004 0.024 ± 0.002 2.46 ± 0.33 2.86 ± 0.34 1.2 1.2 1.16 PHB P35232 1 0.037 0.035 0.028 0.017 3.05 3.71 0.95 0.61 1.22 PHB2 Q99623 2 0.041 ± 0.003 0.039 ± 0.002 0.024 ± 0.002 0.023 ± 0.003 3.35 ± 0.16 3.71 ± 0.04 0.95 0.96 1.11 PHGDH O43175 2 0.039 ± 0.002 0.018 ± 0 0.02 ± 0.001 0.028 ± 0.002 2.24 ± 0.04 1.34 ± 0.01 0.46 1.4 0.6 PKM2 P14618 12 0.071 ± 0.01 0.057 ± 0.006 0.015 ± 0.005 0.032 ± 0.005 3.9 ± 0.45 2.77 ± 0.26 0.8 2.13 0.71 PLEC Q15149 4 0.071 ± 0.011 0.113 ± 0.012 0.036 ± 0.008 0.04 ± 0.011 3.27 ± 0.65 4.27 ± 0.84 1.59 1.11 1.3 POR P16435 2 0.061 ± 0 0.057 ± 0.001 0.03 ± 0.004 0.026 ± 0.005 2.98 ± 0.2 3.29 ± 0.06 0.93 0.87 1.1 POTEKP Q9BYX7 2 0.068 ± 0 0.096 ± 0.002 0.026 ± 0.003 0.025 ± 0.001 3 ± 0.05 3.81 ± 0.1 1.41 0.96 1.27 PPA1 Q15181 4 0.07 ± 0.022 0.046 ± 0.003 0.023 ± 0.004 0.039 ± 0.004 3.77 ± 0.6 2.5 ± 0.17 0.66 1.7 0.66 PPIA P62937 4 0.048 ± 0.004 0.031 ± 0.001 0.021 ± 0.003 0.034 ± 0.001 3.25 ± 0.22 2.2 ± 0.12 0.65 1.62 0.68 PPIAL4A Q9Y536 1 0.052 0.032 0.021 0.035 3.66 2.33 0.62 1.67 0.64 PPIB P23284 6 0.054 ± 0.005 0.064 ± 0.005 0.033 ± 0.008 0.023 ± 0.003 2.66 ± 0.3 3.45 ± 0.16 1.19 0.7 1.3 PPP1CA P62136 6 0.083 ± 0.013 0.079 ± 0.014 0.043 ± 0.005 0.048 ± 0.005 3.09 ± 0.44 3.15 ± 0.46 0.95 1.12 1.02 PPP2R1A P30153 2 0.068 ± 0.025 0.078 ± 0.01 0.03 ± 0.002 0.04 ± 0.003 2.99 ± 0.65 3.07 ± 0.15 1.15 1.33 1.03 PPP4R2 Q9NY27 1 0.105 0.095 0.041 0.044 3.39 3.5 0.9 1.07 1.03

191

Appendix 3. Protein synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

PPT1 P50897 1 0.049 0.027 0.044 0.038 3.12 3.08 0.55 0.86 0.99 PRCP P42785 1 0.03 0.021 0.03 0.031 2.26 2.8 0.7 1.03 1.24 PRDX1 Q06830 7 0.053 ± 0.011 0.031 ± 0.003 0.019 ± 0.007 0.03 ± 0.003 3.39 ± 0.41 2.45 ± 0.11 0.58 1.58 0.72 PRDX2 P32119 1 0.058 0.046 0.018 0.033 4.33 2.94 0.79 1.83 0.68 PRDX3 P30048 2 0.027 ± 0.005 0.027 ± 0.001 0.022 ± 0.002 0.02 ± 0.003 2.68 ± 0.1 3.4 ± 0.14 1 0.91 1.27 PRDX4 Q13162 2 0.052 ± 0.022 0.061 ± 0.001 0.018 ± 0.005 0.018 ± 0.004 3.01 ± 0.28 3.44 ± 0.18 1.17 1 1.14 PRDX5 P30044 4 0.038 ± 0.006 0.032 ± 0.003 0.018 ± 0.003 0.023 ± 0.005 2.96 ± 0.13 2.86 ± 0.18 0.84 1.28 0.97 PRDX6 P30041 5 0.046 ± 0.003 0.027 ± 0.004 0.02 ± 0.006 0.034 ± 0.004 3.19 ± 0.37 2.28 ± 0.24 0.59 1.7 0.71 PRKCSH P14314 3 0.039 ± 0.004 0.04 ± 0.001 0.022 ± 0.006 0.024 ± 0.001 2.57 ± 0.06 2.93 ± 0.06 1.03 1.09 1.14 PRKDC P78527 12 0.032 ± 0.004 0.031 ± 0.004 0.023 ± 0.004 0.023 ± 0.003 2.81 ± 0.16 2.81 ± 0.1 0.97 1 1 PRMT1 Q99873 1 0.084 0.076 0.035 0.025 3.73 3.35 0.9 0.71 0.9 PRPF40A O75400 1 0.058 0.055 0.037 0.038 2.64 2.78 0.95 1.03 1.05 PSAP P07602 4 0.023 ± 0.004 0.049 ± 0.011 0.032 ± 0.003 0.042 ± 0.011 1.37 ± 0.1 2.54 ± 0.19 2.13 1.31 1.85 PSMA4 P25789 1 0.054 0.051 0.024 0.025 3.02 2.77 0.94 1.04 0.92 PSMB1 P20618 1 0.062 0.049 0.017 0.022 3.9 3.22 0.79 1.29 0.83 PSMB7 Q99436 1 0.082 0.059 0.016 0.025 4.15 3.39 0.72 1.56 0.82 192 PSMC2 P35998 2 0.065 ± 0.013 0.062 ± 0.003 0.041 ± 0.001 0.037 ± 0.001 2.87 ± 0.57 3.01 ± 0.42 0.95 0.9 1.05 PSMC3 P17980 1 0.071 0.054 0.044 0.035 2.99 2.95 0.76 0.8 0.99 PSMC4 P43686 1 0.079 0.054 0.027 0.03 3.39 2.7 0.68 1.11 0.8 PSMD11 O00231 2 0.056 ± 0.01 0.051 ± 0 0.029 ± 0.01 0.021 ± 0.004 3.01 ± 0.52 3.03 ± 0.17 0.91 0.72 1.01 PSMD12 O00232 2 0.057 ± 0.003 0.048 ± 0.002 0.029 ± 0.004 0.027 ± 0.004 2.9 ± 0.21 2.81 ± 0.04 0.84 0.93 0.97 PSMD3 O43242 2 0.08 ± 0.004 0.208 ± 0.146 0.023 ± 0.004 0.033 ± 0.002 3.69 ± 0.11 3 ± 0.08 2.6 1.43 0.81 PSMD4 P55036 1 0.081 0.065 0.038 0.064 2.52 2.27 0.8 1.68 0.9 PSMD6 Q15008 1 0.05 0.045 0.033 0.024 2.34 2.79 0.9 0.73 1.19 PSME1 Q06323 2 0.025 ± 0.001 0.036 ± 0.014 0.018 ± 0.001 0.03 ± 0.001 2.02 ± 0.07 1.99 ± 0.05 1.44 1.67 0.99 PTBP1 P26599 2 0.053 ± 0.001 0.056 ± 0.002 0.024 ± 0 0.026 ± 0.004 3.17 ± 0.13 3.55 ± 0.17 1.06 1.08 1.12 PTGES3 Q15185 2 0.063 ± 0.006 0.041 ± 0.001 0.017 ± 0 0.033 ± 0.003 3.76 ± 0.25 2.66 ± 0.11 0.65 1.94 0.71 PTMA P06454 1 0.049 0.031 0.035 0.044 2.49 2.06 0.63 1.26 0.83 PTPN1 P18031 1 0.048 0.066 0.027 0.05 2.49 2.48 1.38 1.85 1 PUF60 Q9UHX1 2 0.073 ± 0.011 0.067 ± 0.009 0.038 ± 0.009 0.052 ± 0.012 3.1 ± 0.59 2.64 ± 0.46 0.92 1.37 0.85 QARS P47897 1 0.05 0.047 0.025 0.024 2.74 2.64 0.94 0.96 0.96 RAB10 P61026 2 0.052 ± 0.008 0.069 ± 0.003 0.032 ± 0.003 0.031 ± 0.001 2.67 ± 0.34 3.43 ± 0.27 1.33 0.97 1.28 RAB11A P62491 1 0.081 0.076 0.035 0.04 3.04 3.31 0.94 1.14 1.09 RAB1A P62820 1 0.048 0.054 0.025 0.028 2.83 3.01 1.13 1.12 1.06 RAB2A P61019 1 0.068 0.063 0.028 0.032 3.26 3.22 0.93 1.14 0.99 RAB31 Q13636 1 0.065 0.084 0.037 0.039 2.97 3.42 1.29 1.05 1.15 RAB3D O95716 1 0.053 0.061 0.038 0.042 2.45 2.96 1.15 1.11 1.21 RAB5A P20339 2 0.059 ± 0.002 0.052 ± 0.003 0.03 ± 0.003 0.039 ± 0 3.01 ± 0.17 2.83 ± 0.22 0.88 1.3 0.94

192

Appendix 3. Protein synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

RAB5C P51148 1 0.059 0.054 0.031 0.034 2.86 2.96 0.92 1.1 1.03 RAB7A P51149 2 0.103 ± 0.003 0.15 ± 0.003 0.036 ± 0.003 0.047 ± 0.004 4.02 ± 0.02 4.85 ± 0.16 1.46 1.31 1.21 RAC1 P63000 1 0.05 0.052 0.04 0.051 2.3 2.27 1.04 1.28 0.99 RAD23B P54727 1 0.068 0.049 0.023 0.033 3.17 2.69 0.72 1.43 0.85 RALA P11233 1 0.05 0.085 0.037 0.03 2.57 3.88 1.7 0.81 1.51 RAN P62826 4 0.06 ± 0.005 0.048 ± 0.002 0.024 ± 0.005 0.03 ± 0.002 3.48 ± 0.38 2.84 ± 0.08 0.8 1.25 0.82 RAP1B P61224 1 0.054 0.06 0.041 0.032 2.27 3.23 1.11 0.78 1.42 RARS P54136 3 0.049 ± 0.007 0.042 ± 0.004 0.022 ± 0.005 0.027 ± 0.008 2.76 ± 0.26 2.6 ± 0.35 0.86 1.23 0.94 RBBP4 Q09028 2 0.037 ± 0.001 0.041 ± 0.001 0.034 ± 0.005 0.03 ± 0.002 2.38 ± 0.17 2.9 ± 0.25 1.11 0.88 1.22 RBM39 Q14498 1 0.064 0.053 0.038 0.056 2.72 2.4 0.83 1.47 0.88 RBM8A Q9Y5S9 2 0.063 ± 0.003 0.059 ± 0.005 0.027 ± 0.006 0.042 ± 0.009 3.13 ± 0.16 2.77 ± 0.31 0.94 1.56 0.88 RBMX P38159 3 0.036 ± 0.002 0.046 ± 0.004 0.026 ± 0.003 0.027 ± 0.005 2.59 ± 0.11 3.28 ± 0.11 1.28 1.04 1.27 RBMXL2 O75526 1 0.04 0.04 0.03 0.022 2.52 3.21 1 0.73 1.27 RCC1 P18754 1 0.023 0.03 0.027 0.024 2.18 3.32 1.3 0.89 1.52 RDX P35241 1 0.073 0.052 0.026 0.05 3.35 2.28 0.71 1.92 0.68 REEP5 Q00765 1 0.029 0.039 0.019 0.025 2.46 2.92 1.34 1.32 1.19 193 RG9MTD1 Q7L0Y3 1 0.062 0.067 0.041 0.03 3.01 3.71 1.08 0.73 1.23 RHOA P61586 1 0.068 0.07 0.058 0.051 2.12 2.47 1.03 0.88 1.17 RHOC P08134 1 0.108 0.126 0.046 0.059 3.52 3.66 1.17 1.28 1.04 RNH1 P13489 1 0.051 0.032 0.022 0.04 3.45 2.33 0.63 1.82 0.68 RNPEP Q9H4A4 2 0.048 ± 0.006 0.029 ± 0.001 0.02 ± 0.006 0.03 ± 0.004 2.86 ± 0.24 2.49 ± 0.19 0.6 1.5 0.87 RPL10 P27635 1 0.085 0.088 0.034 0.054 3.34 3.13 1.04 1.59 0.94 RPL10A P62906 2 0.051 ± 0.001 0.056 ± 0.003 0.018 ± 0.003 0.024 ± 0 3.13 ± 0.24 3.08 ± 0.17 1.1 1.33 0.98 RPL11 P62913 2 0.051 ± 0.003 0.059 ± 0.005 0.022 ± 0 0.027 ± 0.006 3.07 ± 0.09 3.15 ± 0.15 1.16 1.23 1.03 RPL12 P30050 2 0.056 ± 0 0.06 ± 0.002 0.022 ± 0.004 0.024 ± 0 3.28 ± 0.1 3.3 ± 0.02 1.07 1.09 1.01 RPL13 P26373 1 0.044 0.049 0.026 0.015 2.68 2.77 1.11 0.58 1.03 RPL14 P50914 2 0.048 ± 0.006 0.054 ± 0.003 0.02 ± 0.001 0.028 ± 0.002 3.07 ± 0.15 3.13 ± 0.03 1.13 1.4 1.02 RPL15 P61313 2 0.055 ± 0.002 0.051 ± 0.006 0.019 ± 0.001 0.036 ± 0.002 3.41 ± 0.14 2.78 ± 0.28 0.93 1.89 0.81 RPL17 P18621 1 0.059 0.064 0.013 0.024 3.89 3.33 1.08 1.85 0.86 RPL18 Q07020 2 0.049 ± 0.006 0.062 ± 0 0.019 ± 0.002 0.029 ± 0.003 3.16 ± 0.3 3.06 ± 0.16 1.27 1.53 0.97 RPL18A Q02543 2 0.054 ± 0.001 0.056 ± 0.001 0.019 ± 0.001 0.026 ± 0.002 3.13 ± 0.16 2.83 ± 0.34 1.04 1.37 0.9 RPL22 P35268 1 0.033 0.055 0.013 0.033 2.97 2.86 1.67 2.54 0.96 RPL23 P62829 1 0.064 0.067 0.024 0.029 3.59 3.44 1.05 1.21 0.96 RPL23A P62750 1 0.063 0.068 0.021 0.025 3.57 3.46 1.08 1.19 0.97 RPL24 P83731 3 0.056 ± 0.003 0.068 ± 0.012 0.018 ± 0.004 0.031 ± 0.003 3.17 ± 0.11 2.9 ± 0.11 1.21 1.72 0.91 RPL26 P61254 1 0.058 0.056 0.02 0.032 2.89 2.86 0.97 1.6 0.99 RPL27 P61353 1 0.065 0.056 0.022 0.023 3.49 3.15 0.86 1.05 0.9 RPL27A P46776 1 0.058 0.057 0.019 0.028 3.53 3.12 0.98 1.47 0.88

193

Appendix 3. Protein synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

RPL3 P39023 2 0.054 ± 0.003 0.059 ± 0 0.02 ± 0.003 0.029 ± 0.001 3.31 ± 0.12 3.21 ± 0.04 1.09 1.45 0.97 RPL30 P62888 2 0.059 ± 0.004 0.064 ± 0 0.027 ± 0.007 0.026 ± 0.003 3.58 ± 0.44 3.62 ± 0.07 1.08 0.96 1.01 RPL31 P62899 1 0.056 0.061 0.015 0.033 3.4 3.23 1.09 2.2 0.95 RPL32 P62910 1 0.039 0.047 0.021 0.028 2.57 2.41 1.21 1.33 0.94 RPL36 Q9Y3U8 1 0.045 0.055 0.031 0.033 2.72 3.17 1.22 1.06 1.17 RPL36A P83881 1 0.055 0.055 0.023 0.034 3.09 2.79 1 1.48 0.9 RPL4 P36578 6 0.049 ± 0.012 0.063 ± 0.01 0.024 ± 0.005 0.026 ± 0.005 3.01 ± 0.59 3.19 ± 0.24 1.29 1.08 1.06 RPL5 P46777 2 0.048 ± 0.006 0.059 ± 0.008 0.02 ± 0.005 0.021 ± 0.005 3.02 ± 0.12 3.19 ± 0.08 1.23 1.05 1.06 RPL6 Q02878 2 0.062 ± 0.001 0.058 ± 0 0.022 ± 0.001 0.025 ± 0.006 3.33 ± 0.1 2.95 ± 0.21 0.94 1.14 0.88 RPL7 P18124 2 0.055 ± 0.011 0.053 ± 0.001 0.024 ± 0.001 0.034 ± 0.001 3.43 ± 0.31 3.4 ± 0.16 0.96 1.42 0.99 RPL9 P32969 1 0.052 0.058 0.022 0.024 3.11 3.08 1.12 1.09 0.99 RPLP1 P05386 1 0.078 0.074 0.019 0.023 4.08 3.67 0.95 1.21 0.9 RPLP2 P05387 2 0.07 ± 0.007 0.071 ± 0.004 0.02 ± 0.003 0.026 ± 0 3.63 ± 0.1 3.48 ± 0.17 1.01 1.3 0.96 RPN1 P04843 4 0.039 ± 0.007 0.057 ± 0.013 0.029 ± 0.006 0.025 ± 0.006 2.61 ± 0.37 3.28 ± 0.27 1.46 0.86 1.25 RPN2 P04844 1 0.041 0.068 0.027 0.025 2.39 3.37 1.66 0.93 1.41 RPS10 P46783 1 0.086 0.076 0.023 0.025 4.57 3.85 0.88 1.09 0.84

194 RPS11 P62280 1 0.062 0.068 0.02 0.025 3.44 3.22 1.1 1.25 0.94 RPS12 P25398 1 0.048 0.051 0.012 0.03 3.64 2.84 1.06 2.5 0.78 RPS14 P62263 1 0.085 0.082 0.032 0.034 3.73 3.81 0.96 1.06 1.02 RPS15A P62244 1 0.069 0.063 0.017 0.031 3.85 3.37 0.91 1.82 0.88 RPS16 P62249 3 0.062 ± 0.007 0.068 ± 0.004 0.027 ± 0.008 0.031 ± 0.002 3.29 ± 0.44 3.31 ± 0.11 1.1 1.15 1.01 RPS18 P62269 4 0.075 ± 0.015 0.074 ± 0.007 0.035 ± 0.008 0.033 ± 0.004 3.37 ± 0.8 3.32 ± 0.16 0.99 0.94 0.98 RPS19 P39019 2 0.062 ± 0.014 0.067 ± 0 0.029 ± 0 0.029 ± 0.001 3.26 ± 0.4 3.24 ± 0.03 1.08 1 1 RPS2 P15880 2 0.059 ± 0.009 0.06 ± 0.001 0.025 ± 0.006 0.031 ± 0.004 3.09 ± 0.52 2.95 ± 0.25 1.02 1.24 0.95 RPS21 P63220 1 0.079 0.071 0.022 0.029 3.91 3.4 0.9 1.32 0.87 RPS25 P62851 2 0.053 ± 0.002 0.053 ± 0.002 0.025 ± 0.001 0.032 ± 0.003 2.92 ± 0.04 2.69 ± 0.05 1 1.28 0.92 RPS27 P42677 1 0.088 0.063 0.027 0.03 4.03 3.36 0.72 1.11 0.83 RPS27L Q71UM5 1 0.083 0.079 0.026 0.035 3.72 3.41 0.95 1.35 0.92 RPS28 P62857 1 0.065 0.062 0.028 0.028 3.54 3.33 0.95 1 0.94 RPS3 P23396 4 0.064 ± 0.008 0.068 ± 0.004 0.026 ± 0.007 0.028 ± 0.002 3.4 ± 0.39 3.48 ± 0.12 1.06 1.08 1.02 RPS3A P61247 1 0.078 0.072 0.024 0.027 4.1 3.66 0.92 1.13 0.89 RPS4X P62701 4 0.063 ± 0.014 0.07 ± 0.006 0.03 ± 0.007 0.032 ± 0.001 3.18 ± 0.58 3.34 ± 0.23 1.11 1.07 1.05 RPS5 P46782 2 0.064 ± 0.004 0.065 ± 0.002 0.023 ± 0.002 0.028 ± 0.002 3.46 ± 0.19 3.35 ± 0.04 1.02 1.22 0.97 RPS6 P62753 3 0.07 ± 0.004 0.062 ± 0.01 0.027 ± 0.001 0.032 ± 0.006 3.41 ± 0.36 3.1 ± 0.5 0.89 1.19 0.91 RPS8 P62241 6 0.067 ± 0.01 0.066 ± 0.007 0.025 ± 0.005 0.03 ± 0.004 3.52 ± 0.51 3.2 ± 0.32 0.99 1.2 0.91 RPS9 P46781 5 0.067 ± 0.005 0.061 ± 0.004 0.022 ± 0.003 0.031 ± 0.004 3.74 ± 0.23 3.23 ± 0.22 0.91 1.41 0.86 RPSA P08865 4 0.062 ± 0.01 0.064 ± 0.007 0.029 ± 0.008 0.03 ± 0.002 3.28 ± 0.54 3.22 ± 0.31 1.03 1.03 0.98 RRBP1 Q9P2E9 2 0.056 ± 0.015 0.069 ± 0.013 0.051 ± 0.005 0.06 ± 0.004 2.19 ± 0.19 2.59 ± 0.11 1.23 1.18 1.18

194

Appendix 3. Protein synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

RTN4 Q9NQC3 1 0.049 0.093 0.022 0.02 2.31 3.65 1.9 0.91 1.58 RUVBL1 Q9Y265 1 0.044 0.046 0.019 0.022 3.72 3.22 1.05 1.16 0.87 S100A11 P31949 1 0.045 0.042 0.022 0.031 2.48 2.09 0.93 1.41 0.84 S100A13 Q99584 1 0.07 0.042 0.034 0.035 3.4 2.14 0.6 1.03 0.63 S100A14 Q9HCY8 3 0.091 ± 0.03 0.116 ± 0.037 0.034 ± 0.003 0.043 ± 0.014 3.76 ± 1.06 4.31 ± 1.34 1.27 1.26 1.15 S100A16 Q96FQ6 1 0.09 0.122 0.035 0.038 3.63 4.32 1.36 1.09 1.19 S100A6 P06703 2 0.078 ± 0.001 0.051 ± 0.001 0.018 ± 0 0.042 ± 0 4.08 ± 0.02 2.6 ± 0.09 0.65 2.33 0.64 S100A8 P05109 1 0.072 0.031 0.02 0.031 3.77 1.59 0.43 1.55 0.42 S100A9 P06702 3 0.104 ± 0.005 0.047 ± 0.002 0.027 ± 0.007 0.057 ± 0.003 3.84 ± 0.19 1.56 ± 0.05 0.45 2.11 0.41 S100P P25815 1 0.032 0.02 0.014 0.037 1.88 1.59 0.63 2.64 0.85 SAR1A Q9NR31 2 0.064 ± 0.004 0.054 ± 0.001 0.021 ± 0.005 0.032 ± 0.002 3.14 ± 0.25 2.66 ± 0.03 0.84 1.52 0.85 SCFD1 Q8WVM8 1 0.057 0.042 0.029 0.034 2.75 2.72 0.74 1.17 0.99 SCP2 P22307 1 0.022 0.032 0.015 0.03 1.87 2.46 1.45 2 1.32 SCYL1 Q96KG9 1 0.062 0.051 0.028 0.03 3.03 2.52 0.82 1.07 0.83 SDF4 Q9BRK5 1 0.085 0.079 0.054 0.09 2.51 2.62 0.93 1.67 1.04 SDHA P31040 1 0.029 0.033 0.02 0.028 2.73 2.75 1.14 1.4 1.01

195 SEC11A P67812 1 0.034 0.058 0.034 0.024 2.19 3.12 1.71 0.71 1.42 SEC24C P53992 2 0.07 ± 0.002 0.058 ± 0.001 0.044 ± 0.001 0.046 ± 0.004 2.76 ± 0.09 2.53 ± 0.07 0.83 1.05 0.91 SELENBP1 Q13228 1 0.036 0.016 0.014 0.031 4.27 2.59 0.44 2.21 0.61 SEPt7 Q16181 1 0.068 0.058 0.033 0.037 2.93 3.02 0.85 1.12 1.03 SET Q01105 3 0.038 ± 0.003 0.034 ± 0.003 0.024 ± 0 0.025 ± 0.002 2.66 ± 0.18 2.61 ± 0.11 0.89 1.04 0.98 SETP9 Q9UHD8 1 0.038 0.051 0.042 0.037 1.87 2.52 1.34 0.88 1.35 SF3A3 Q12874 1 0.038 0.042 0.036 0.03 2.06 2.78 1.11 0.83 1.35 SF3B1 O75533 1 0.078 0.084 0.041 0.047 3.05 3.07 1.08 1.15 1.01 SF3B3 Q15393 1 0.041 0.057 0.023 0.025 2.62 3.38 1.39 1.09 1.29 SF3B4 Q15427 1 0.067 0.056 0.054 0.062 2.41 2.41 0.84 1.15 1 SFN P31947 3 0.121 ± 0.032 0.12 ± 0.028 0.029 ± 0.018 0.038 ± 0.011 4.21 ± 1.12 3.94 ± 0.86 0.99 1.31 0.93 SFPQ P23246 4 0.049 ± 0.006 0.062 ± 0.008 0.025 ± 0.007 0.032 ± 0.001 2.89 ± 0.48 3.51 ± 0.31 1.27 1.28 1.21 SHMT1 P34896 1 0.063 0.042 0.029 0.023 3.54 2.95 0.67 0.79 0.83 SLC1A5 Q15758 2 0.124 ± 0.001 0.086 ± 0.024 0.056 ± 0 0.063 ± 0.011 4.17 ± 0.15 3.26 ± 0.77 0.69 1.13 0.78 SLC25A1 P53007 2 0.029 ± 0.001 0.035 ± 0.001 0.017 ± 0.001 0.018 ± 0 2.91 ± 0.17 3.54 ± 0.02 1.21 1.06 1.22 SLC25A12 O75746 1 0.033 0.036 0.024 0.017 2.5 3.4 1.09 0.71 1.36 SLC25A13 Q9UJS0 4 0.032 ± 0.003 0.042 ± 0.004 0.024 ± 0.004 0.02 ± 0.003 2.35 ± 0.18 3.36 ± 0.36 1.31 0.83 1.43 SLC25A24 Q6NUK1 1 0.023 0.049 0.019 0.022 2.11 3.2 2.13 1.16 1.52 SLC25A3 Q00325 2 0.04 ± 0.005 0.052 ± 0.006 0.019 ± 0.001 0.025 ± 0.002 3.03 ± 0.4 3.74 ± 0.41 1.3 1.32 1.24 SLC25A4 P12235 2 0.039 ± 0.002 0.047 ± 0.001 0.022 ± 0.002 0.021 ± 0.001 2.92 ± 0.09 3.65 ± 0.2 1.21 0.95 1.25 SLC25A5 P05141 4 0.032 ± 0.002 0.041 ± 0.003 0.023 ± 0.003 0.025 ± 0.002 2.74 ± 0.14 3.56 ± 0.07 1.28 1.09 1.3 SLC25A6 P12236 1 0.03 0.046 0.032 0.029 2.47 3.03 1.53 0.91 1.23

195

Appendix 3. Protein synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

SLC2A1 P11166 1 0.091 0.164 0.038 0.056 3.43 5.06 1.8 1.47 1.48 SLC3A2 P08195 4 0.058 ± 0.004 0.058 ± 0.005 0.036 ± 0.008 0.038 ± 0.003 2.55 ± 0.16 2.58 ± 0.1 1 1.06 1.01 SLC44A2 Q8IWA5 1 0.031 0.054 0.037 0.039 1.83 2.4 1.74 1.05 1.31 SLC9A3R1 O14745 1 0.094 0.038 0.04 0.055 3.65 1.91 0.4 1.38 0.52 SMC2 O95347 1 0.056 0.05 0.036 0.035 3.17 2.74 0.89 0.97 0.86 SND1 Q7KZF4 6 0.085 ± 0.011 0.079 ± 0.009 0.033 ± 0.003 0.04 ± 0.004 3.5 ± 0.37 3.02 ± 0.32 0.93 1.21 0.86 SNRNP200 O75643 2 0.056 ± 0.001 0.055 ± 0.013 0.052 ± 0.001 0.047 ± 0.005 2.01 ± 0.03 2.64 ± 0.2 0.98 0.9 1.31 SNRNP70 P08621 2 0.079 ± 0.008 0.075 ± 0.005 0.032 ± 0.001 0.025 ± 0.002 3.58 ± 0.22 4.03 ± 0.33 0.95 0.78 1.12 SNRPD3 P62318 1 0.037 0.04 0.036 0.022 2.51 3.31 1.08 0.61 1.32 SNRPEL1 Q5VYJ4 2 0.041 ± 0.002 0.047 ± 0.001 0.038 ± 0.001 0.032 ± 0.001 2.59 ± 0.08 3.23 ± 0.12 1.15 0.84 1.25 SNRPF P62306 1 0.055 0.044 0.026 0.025 3.61 3.34 0.8 0.96 0.93 SOD1 P00441 2 0.047 ± 0.003 0.034 ± 0.009 0.014 ± 0 0.032 ± 0.005 3.38 ± 0.08 2.49 ± 0.09 0.72 2.29 0.74 SOD2 P04179 1 0.063 0.056 0.024 0.015 3.32 3.16 0.89 0.63 0.95 SPTAN1 Q13813 5 0.052 ± 0.006 0.073 ± 0.011 0.038 ± 0.005 0.036 ± 0.006 2.66 ± 0.32 3.14 ± 0.37 1.4 0.95 1.18 SRI P30626 1 0.05 0.042 0.03 0.041 2.45 1.95 0.84 1.37 0.8 SRP14 P37108 1 0.043 0.066 0.04 0.068 2.11 2.64 1.53 1.7 1.25 196 SRP9 P49458 1 0.052 0.059 0.055 0.06 2.09 2.26 1.13 1.09 1.08 SRPR P08240 1 0.084 0.063 0.033 0.048 3.4 2.66 0.75 1.45 0.78 SRRT Q9BXP5 2 0.057 ± 0.006 0.063 ± 0.001 0.028 ± 0.002 0.027 ± 0.002 3 ± 0.28 3.47 ± 0.08 1.11 0.96 1.16 SRSF1 Q07955 4 0.04 ± 0.004 0.051 ± 0.005 0.027 ± 0.007 0.026 ± 0.004 2.67 ± 0.16 3.38 ± 0.15 1.28 0.96 1.27 SRSF10 O75494 1 0.044 0.035 0.04 0.038 2.37 2.48 0.8 0.95 1.05 SRSF3 P84103 1 0.043 0.049 0.025 0.027 2.99 3.37 1.14 1.08 1.13 SSBP1 Q04837 2 0.035 ± 0 0.04 ± 0.001 0.024 ± 0.004 0.025 ± 0 2.66 ± 0.17 3.25 ± 0.04 1.14 1.04 1.22 ST13 P50502 1 0.066 0.054 0.039 0.05 2.83 2.37 0.82 1.28 0.84 STIP1 P31948 2 0.084 ± 0.021 0.057 ± 0.008 0.027 ± 0.011 0.032 ± 0 4.23 ± 1.12 3.11 ± 0.28 0.68 1.19 0.73 STT3A P46977 3 0.042 ± 0.012 0.055 ± 0.006 0.028 ± 0.006 0.031 ± 0.002 2.75 ± 0.59 3.23 ± 0.42 1.31 1.11 1.17 STX3 Q13277 1 0.077 0.11 0.036 0.034 3.06 4.35 1.43 0.94 1.42 SUB1 P53999 2 0.068 ± 0.012 0.043 ± 0.003 0.026 ± 0.004 0.036 ± 0.003 3.44 ± 0.76 2.37 ± 0.1 0.63 1.38 0.69 SUCLA2 Q9P2R7 1 0.036 0.036 0.038 0.026 2.42 3.26 1 0.68 1.35 SUCLG2 Q96I99 1 0.021 0.029 0.019 0.024 2.49 3.4 1.38 1.26 1.37 SULT1A1 P50225 1 0.067 0.033 0.019 0.037 3.2 2.08 0.49 1.95 0.65 SUMF2 Q8NBJ7 1 0.036 0.051 0.038 0.026 2.03 3.01 1.42 0.68 1.48 SUPT16H Q9Y5B9 2 0.048 ± 0.003 0.032 ± 0.001 0.029 ± 0.004 0.027 ± 0.002 2.99 ± 0.2 3.22 ± 0.06 0.67 0.93 1.08 SWAP70 Q9UH65 1 0.068 0.059 0.029 0.036 3.2 2.96 0.87 1.24 0.93 SYNCRIP O60506 3 0.059 ± 0.005 0.057 ± 0.011 0.028 ± 0.004 0.032 ± 0.002 2.89 ± 0.11 2.97 ± 0.31 0.97 1.14 1.03 SYPL1 Q16563 1 0.066 0.042 0.032 0.025 3.23 3.14 0.64 0.78 0.97 TAGLN2 P37802 4 0.056 ± 0.003 0.058 ± 0.004 0.024 ± 0.006 0.038 ± 0.004 2.96 ± 0.17 2.71 ± 0.16 1.04 1.58 0.92 TALDO1 P37837 6 0.042 ± 0.007 0.029 ± 0.001 0.021 ± 0.006 0.03 ± 0.004 2.52 ± 0.12 2.13 ± 0.12 0.69 1.43 0.84

196

Appendix 3. Protein synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

TARDBP Q13148 1 0.048 0.054 0.038 0.041 2.36 2.59 1.13 1.08 1.1 TBCB Q99426 1 0.068 0.05 0.031 0.04 3.28 2.65 0.74 1.29 0.81 TBRG4 Q969Z0 1 0.062 0.095 0.061 0.045 2.45 3.57 1.53 0.74 1.46 TCEB2 Q15370 1 0.079 0.068 0.044 0.058 2.92 2.44 0.86 1.32 0.84 TCOF1 Q13428 1 0.047 0.073 0.032 0.026 2.93 3.53 1.55 0.81 1.2 TCP1 P17987 2 0.071 ± 0.005 0.053 ± 0 0.028 ± 0.007 0.035 ± 0.001 3.7 ± 0.05 2.97 ± 0.08 0.75 1.25 0.8 TECR Q9NZ01 1 0.036 0.039 0.033 0.031 2.13 2.58 1.08 0.94 1.21 TES Q9UGI8 1 0.052 0.047 0.033 0.038 2.61 3.04 0.9 1.15 1.16 TFRC P02786 2 0.114 ± 0.011 0.143 ± 0.015 0.066 ± 0.008 0.077 ± 0.004 3.54 ± 0.14 4.06 ± 0.27 1.25 1.17 1.15 TIMM44 O43615 1 0.042 0.044 0.03 0.018 2.5 3.24 1.05 0.6 1.3 TKT P29401 4 0.044 ± 0.006 0.023 ± 0.003 0.016 ± 0.002 0.024 ± 0.001 3.55 ± 0.43 2.88 ± 0.26 0.52 1.5 0.81 TLN1 Q9Y490 1 0.062 0.064 0.025 0.039 2.77 2.47 1.03 1.56 0.89 TM9SF2 Q99805 1 0.079 0.092 0.04 0.045 2.95 3.42 1.16 1.13 1.16 TMCO1 Q9UM00 1 0.086 0.103 0.032 0.026 3.78 4.46 1.2 0.81 1.18 TMED10 P49755 2 0.031 ± 0 0.042 ± 0.004 0.019 ± 0.002 0.022 ± 0.001 2.48 ± 0.14 2.84 ± 0.17 1.35 1.16 1.15 TMED2 Q15363 2 0.034 ± 0.003 0.038 ± 0.003 0.028 ± 0.005 0.026 ± 0 2.62 ± 0.15 3.04 ± 0.05 1.12 0.93 1.16 197 TMED5 Q9Y3A6 1 0.036 0.052 0.039 0.028 1.77 2.56 1.44 0.72 1.45 TMEM65 Q6PI78 1 0.04 0.04 0.02 0.023 2.5 3.1 1 1.15 1.24 TMEM70 Q9BUB7 1 0.07 0.065 0.02 0.035 4 3.46 0.93 1.75 0.87 TNKS1BP1 Q9C0C2 1 0.053 0.084 0.045 0.037 2.39 2.88 1.58 0.82 1.21 TOR1AIP1 Q5JTV8 1 0.051 0.074 0.023 0.018 2.46 3.6 1.45 0.78 1.46 TPD52 P55327 2 0.063 ± 0 0.043 ± 0.003 0.018 ± 0 0.03 ± 0.002 3.41 ± 0.12 2.34 ± 0.02 0.68 1.67 0.69 TPI1 P60174 5 0.042 ± 0.007 0.03 ± 0.002 0.014 ± 0.002 0.032 ± 0.005 3.03 ± 0.32 2.16 ± 0.1 0.71 2.29 0.71 TPM1 B7Z722 1 0.043 0.08 0.029 0.039 1.61 2.65 1.86 1.34 1.65 TPM1 P09493 3 0.044 ± 0.004 0.068 ± 0.002 0.031 ± 0.005 0.029 ± 0.004 1.82 ± 0.38 2.65 ± 0.33 1.55 0.94 1.46 TPM2 P07951 1 0.049 0.07 0.025 0.024 2.33 2.96 1.43 0.96 1.27 TPM3 P06753 1 0.053 0.063 0.024 0.026 2.79 3.24 1.19 1.08 1.16 TPM4 P67936 1 0.046 0.061 0.018 0.022 2.41 3.09 1.33 1.22 1.28 TPR P12270 1 0.045 0.055 0.033 0.028 2.43 3.42 1.22 0.85 1.41 TRAP1 Q12931 2 0.052 ± 0.006 0.056 ± 0.004 0.024 ± 0.005 0.026 ± 0.003 3.65 ± 0.32 3.79 ± 0.03 1.08 1.08 1.04 TRAPPC3 O43617 1 0.06 0.046 0.039 0.03 2.89 2.57 0.77 0.77 0.89 TRIM25 Q14258 2 0.045 ± 0.008 0.044 ± 0.001 0.033 ± 0.002 0.031 ± 0 2.24 ± 0.18 2.33 ± 0.07 0.98 0.94 1.04 TRIM28 Q13263 4 0.038 ± 0.01 0.046 ± 0.004 0.033 ± 0.008 0.031 ± 0.006 2.95 ± 0.25 3.11 ± 0.19 1.21 0.94 1.06 TUBA1A Q71U36 10 0.055 ± 0.013 0.046 ± 0.013 0.02 ± 0.005 0.034 ± 0.003 3.24 ± 0.46 2.46 ± 0.18 0.84 1.7 0.76 TUBA1B P68363 1 0.059 0.037 0.012 0.032 3.51 2.31 0.63 2.67 0.66 TUBB P07437 2 0.052 ± 0.003 0.033 ± 0.001 0.013 ± 0.001 0.033 ± 0.001 3.17 ± 0.06 2.17 ± 0.02 0.63 2.54 0.68 TUBB1 Q9H4B7 3 0.052 ± 0.006 0.043 ± 0.003 0.023 ± 0.002 0.037 ± 0.003 3.27 ± 0.4 2.6 ± 0.13 0.83 1.61 0.8 TUBB2A Q13885 6 0.055 ± 0.011 0.039 ± 0.005 0.015 ± 0.004 0.03 ± 0.004 3.54 ± 0.52 2.5 ± 0.18 0.71 2 0.71

197

Appendix 3. Protein synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

TUBB2C P68371 2 0.062 ± 0.006 0.045 ± 0.004 0.015 ± 0.004 0.031 ± 0.003 3.53 ± 0.12 2.54 ± 0.05 0.73 2.07 0.72 TUFM P49411 7 0.039 ± 0.007 0.038 ± 0.005 0.028 ± 0.012 0.022 ± 0.002 3.2 ± 0.76 3.48 ± 0.39 0.97 0.79 1.09 TWF2 Q6IBS0 1 0.039 0.038 0.028 0.042 2.8 2.26 0.97 1.5 0.81 TXN P10599 2 0.066 ± 0.007 0.039 ± 0.002 0.016 ± 0 0.039 ± 0 3.96 ± 0.23 2.29 ± 0 0.59 2.44 0.58 TXNDC5 Q8NBS9 4 0.039 ± 0.011 0.049 ± 0.004 0.031 ± 0.013 0.027 ± 0.007 2.45 ± 0.57 2.93 ± 0.11 1.26 0.87 1.2 TXNRD1 Q16881 1 0.039 0.023 0.035 0.024 2.41 1.9 0.59 0.69 0.79 U2AF2 P26368 2 0.06 ± 0.001 0.057 ± 0.002 0.034 ± 0.005 0.034 ± 0.001 3 ± 0.15 3.21 ± 0.03 0.95 1 1.07 UBA1 P22314 4 0.067 ± 0.007 0.043 ± 0.006 0.02 ± 0.005 0.031 ± 0.008 4.11 ± 0.34 2.72 ± 0.32 0.64 1.55 0.66 UBA52 P62987 3 0.108 ± 0.016 0.108 ± 0.018 0.054 ± 0.009 0.069 ± 0.001 3.44 ± 0.51 3.36 ± 0.31 1 1.28 0.98 UBE2O Q9C0C9 1 0.089 0.072 0.033 0.062 3.85 2.39 0.81 1.88 0.62 UBE2V1 Q13404 1 0.081 0.053 0.026 0.051 3.49 2.23 0.65 1.96 0.64 UGDH O60701 1 0.086 0.099 0.026 0.027 3.09 3.41 1.15 1.04 1.1 UPF1 Q92900 1 0.1 0.091 0.034 0.054 3.44 3.18 0.91 1.59 0.92 UQCRB P14927 1 0.033 0.027 0.03 0.014 2.66 3.39 0.82 0.47 1.27 UQCRC2 P22695 1 0.036 0.035 0.02 0.02 3.54 4.02 0.97 1 1.14 USMG5 Q96IX5 1 0.04 0.044 0.048 0.06 1.77 2.22 1.1 1.25 1.25 198 USO1 O60763 1 0.032 0.041 0.023 0.023 2.87 2.89 1.28 1 1.01 USP14 P54578 1 0.063 0.048 0.016 0.032 3.52 2.67 0.76 2 0.76 VAMP8 Q9BV40 2 0.075 ± 0 0.101 ± 0.003 0.041 ± 0.003 0.062 ± 0.003 2.61 ± 0.04 3.24 ± 0.12 1.35 1.51 1.24 VAPA Q9P0L0 2 0.048 ± 0.006 0.056 ± 0.005 0.028 ± 0.013 0.022 ± 0.002 2.65 ± 0.15 3.16 ± 0.1 1.17 0.79 1.19 VAPB O95292 2 0.039 ± 0.001 0.055 ± 0.004 0.02 ± 0.003 0.022 ± 0.001 2.44 ± 0.11 3.12 ± 0.07 1.41 1.1 1.28 VARS P26640 1 0.042 0.037 0.027 0.026 2.72 2.3 0.88 0.96 0.85 VCP P55072 5 0.059 ± 0.006 0.059 ± 0.005 0.028 ± 0.003 0.029 ± 0.004 3.16 ± 0.23 3.38 ± 0.22 1 1.04 1.07 VDAC1 P21796 3 0.024 ± 0.003 0.027 ± 0.006 0.028 ± 0.005 0.02 ± 0.001 2.07 ± 0.34 2.85 ± 0.24 1.13 0.71 1.37 VDAC2 P45880 6 0.05 ± 0.022 0.132 ± 0.18 0.022 ± 0.004 0.019 ± 0.003 2.79 ± 0.19 3.7 ± 0.24 2.64 0.86 1.32 VKORC1L1 Q8N0U8 1 0.038 0.05 0.029 0.024 2.07 3.06 1.32 0.83 1.48 VPS26A O75436 1 0.068 0.055 0.024 0.039 3.19 2.68 0.81 1.63 0.84 VPS35 Q96QK1 1 0.046 0.04 0.02 0.028 3.13 2.56 0.87 1.4 0.82 WDR1 O75083 1 0.067 0.067 0.021 0.034 3 3 1 1.62 1 WDR36 Q8NI36 1 0.069 0.066 0.026 0.023 4.03 3.7 0.96 0.88 0.92 XAB2 Q9HCS7 1 0.054 0.065 0.051 0.037 2.39 3.44 1.2 0.73 1.44 XPO1 O14980 2 0.049 ± 0.009 0.037 ± 0.006 0.019 ± 0.004 0.037 ± 0.004 3.4 ± 0.3 2.72 ± 0.28 0.76 1.95 0.8 XPO5 Q9HAV4 1 0.08 0.065 0.031 0.028 3.83 3.08 0.81 0.9 0.8 XRCC5 P13010 2 0.05 ± 0.008 0.043 ± 0.003 0.021 ± 0.001 0.023 ± 0.004 3.51 ± 0.3 3.02 ± 0.1 0.86 1.1 0.86 XRCC6 P12956 4 0.05 ± 0.008 0.038 ± 0.003 0.025 ± 0.003 0.024 ± 0.001 3.12 ± 0.33 2.9 ± 0.18 0.76 0.96 0.93 XRN2 Q9H0D6 1 0.062 0.069 0.043 0.039 2.38 2.98 1.11 0.91 1.25 YBX1 P67809 1 0.119 0.094 0.071 0.038 3.41 3.7 0.79 0.54 1.09 YWHAB P31946 5 0.052 ± 0.01 0.048 ± 0.004 0.02 ± 0.009 0.03 ± 0.006 3.01 ± 0.44 2.57 ± 0.18 0.92 1.5 0.85

198

Appendix 3. Protein synthesis rates, degradation rate constants and relative abundances at 24 h in SKBR3 cells and SKBR3_shVPS4B cells (continued).

YWHAE P62258 3 0.046 ± 0.006 0.048 ± 0.01 0.02 ± 0.007 0.029 ± 0.001 3.07 ± 0.36 2.55 ± 0.17 1.04 1.45 0.83 YWHAG P61981 2 0.069 ± 0.01 0.051 ± 0.004 0.017 ± 0.003 0.03 ± 0.002 3.62 ± 0.37 2.81 ± 0.27 0.74 1.76 0.78 YWHAH Q04917 1 0.082 0.049 0.014 0.042 4.04 2.73 0.6 3 0.68 YWHAQ P27348 5 0.053 ± 0.007 0.053 ± 0.012 0.024 ± 0.006 0.032 ± 0.004 2.85 ± 0.37 2.68 ± 0.23 1 1.33 0.94 YWHAZ P63104 5 0.059 ± 0.011 0.054 ± 0.004 0.021 ± 0.002 0.031 ± 0.007 3.17 ± 0.47 2.76 ± 0.21 0.92 1.48 0.87 ZC3H18 Q86VM9 1 0.09 0.07 0.029 0.043 3.52 3.13 0.78 1.48 0.89 ZMPSTE24 O75844 1 0.049 0.07 0.017 0.024 3.11 3.72 1.43 1.41 1.2 B2RCX0 1 0.046 0.035 0.021 0.035 2.76 2.4 0.76 1.67 0.87 B4E3B6 1 0.085 0.051 0.03 0.031 3.5 2.8 0.6 1.03 0.8 Q6NVV1 1 0.057 0.054 0.018 0.023 3.23 3.1 0.95 1.28 0.96 Q8NHW5 3 0.043 ± 0.007 0.052 ± 0.004 0.023 ± 0.003 0.027 ± 0.003 2.95 ± 0.06 3.03 ± 0.07 1.21 1.17 1.03 A8MWD9 1 0.039 0.047 0.033 0.021 2.71 3.29 1.21 0.64 1.21 A6NL28 1 0.064 0.071 0.029 0.025 2.87 3.48 1.11 0.86 1.21 199

199

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant.

Note: The main modules for peptide and protein quantitation under the namespace of “NewIsoQuant” are listed: ProcessPeptideQuantitation, ProcessProteinQuantitation and class MS1quant

namespace NewIsoQuant {

// ***************************************************************************************** // Peptide quantitation;

public void ProcessPeptideQuantitation(string filestring, ref int NumberOfFiles) { starttime = DateTime.Now;

MS1quant test = new MS1quant(); string outputMS1Quantitation = textBox4.Text + Path.GetFileNameWithoutExtension(filestring) + "_new.txt";

string inputMS1file = textBox1.Text; string inputSequestResults = filestring; string outputSequestResults;

outputSequestResults = textBox4.Text + Path.GetFileNameWithoutExtension(filestring); outputSequestResults += ".id";

int totalpeaks; int numberofpairs;

//readSequestXMLfile DialogResult formresult = new DialogResult();

ProcessSequestXML inputSequestXML = new ProcessSequestXML(); ProcessMascotXML inputMascotXML = new ProcessMascotXML();

if (comboBox1.SelectedIndex == 0 && comboBox2.SelectedIndex == 0) //Sequest { inputSequestXML.SequestScoreFilter = new double[4];

inputSequestXML.SequestScoreFilter[0] = double.Parse(textBox5.Text); inputSequestXML.SequestScoreFilter[1] = double.Parse(textBox6.Text); inputSequestXML.SequestScoreFilter[2] = double.Parse(textBox7.Text); inputSequestXML.SequestScoreFilter[3] = double.Parse(textBox8.Text);

inputSequestXML.Process(inputSequestResults, outputSequestResults);

if (Math.Abs(inputSequestXML.massK - 8) < 0.1) checkBox5.Checked = true; else if (Math.Abs(inputSequestXML.massK - 4) < 0.001) checkBox3.Checked = true; else if (Math.Abs(inputSequestXML.massK - 6) < 0.1) checkBox4.Checked = true; else if (Math.Abs(inputSequestXML.massK - 3.99) < 0.001) checkBox12.Checked = true;

200

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

if (Math.Abs(inputSequestXML.massR - 6) < 0.1) checkBox1.Checked = true; else if (Math.Abs(inputSequestXML.massR-10) < 0.1) checkBox2.Checked = true; else if (Math.Abs(inputSequestXML.massR-3.99) < 0.1) checkBox11.Checked = true;

if (NumberOfFiles == 0) { formresult = MessageBox.Show("SILAC labels:\n" + "K " + inputSequestXML.tagK + " " + inputSequestXML.massK + " R " + inputSequestXML.tagR + " " + inputSequestXML.massR + "\n\nDatabase:\n" + Path.GetFileName(inputSequestXML.Databasefile), "Confirm", MessageBoxButtons.YesNo, MessageBoxIcon.Question);

if (formresult == System.Windows.Forms.DialogResult.No) { Close(); return; } NumberOfFiles++; } } else if (comboBox1.SelectedIndex == 0 && comboBox2.SelectedIndex == 1) //Mascot { inputMascotXML.MascotScoreFilter = double.Parse(textBox9.Text); inputMascotXML.Process(inputSequestResults, outputSequestResults);

if (Math.Abs(inputMascotXML.massK - 8) < 0.1) checkBox5.Checked = true; else if (Math.Abs(inputMascotXML.massK - 4) < 0.1) checkBox3.Checked = true; else if (Math.Abs(inputMascotXML.massK - 6) < 0.1) checkBox4.Checked = true;

if (Math.Abs(inputMascotXML.massR - 6) < 0.1) checkBox1.Checked = true; else if (Math.Abs(inputMascotXML.massR) < 0.1) checkBox2.Checked = true;

if (NumberOfFiles == 0) { formresult = MessageBox.Show("SILAC labels:\n" + "K " + inputMascotXML.tagK + " " + inputMascotXML.massK + " R " + inputMascotXML.tagR + " " + inputMascotXML.massR + "\n\nDatabase:\n" + Path.GetFileName(inputMascotXML.Databasefile), "Confirm", MessageBoxButtons.YesNo, MessageBoxIcon.Question);

if (formresult == System.Windows.Forms.DialogResult.No) { Close();

return; } NumberOfFiles++;

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Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

} }

//readSEQUESTTextfile List lstMSMS = new List(); List lstMS = new List(); int numberofunipep = 0; numberofpairs = 0;

if (comboBox1.SelectedIndex == 0 && comboBox2.SelectedIndex == 0) { StreamReader inputSequest = File.OpenText(outputSequestResults); numberofpairs = test.readSequestFile(ref inputSequest, lstMSMS, inputSequestXML.tagK, inputSequestXML.tagR, inputSequestXML.massK, inputSequestXML.massR); numberofunipep = test.GetMSInfo(lstMS, lstMSMS); } else if (comboBox1.SelectedIndex == 0 && comboBox2.SelectedIndex == 1) { StreamReader inputSequest = File.OpenText(outputSequestResults); numberofpairs = test.readSequestFile(ref inputSequest, lstMSMS, inputSequestXML.tagK, inputSequestXML.tagR, inputSequestXML.massK, inputSequestXML.massR); numberofunipep = test.GetMSInfo(lstMS, lstMSMS); }

else if (comboBox1.SelectedIndex == 1) // only support medium R6, K4 and heavy R10 and K8 { inputSequestXML.Process3(inputSequestResults, outputSequestResults); StreamReader inputSequest = File.OpenText(outputSequestResults); triplelabel triple;

triple.massK1 = inputSequestXML.massK; triple.massK2 = inputSequestXML.massK2; triple.massR1 = inputSequestXML.massR; triple.massR2 = inputSequestXML.massR2; triple.tagK1 = inputSequestXML.tagK; triple.tagK2 = inputSequestXML.tagK2; triple.tagR1 = inputSequestXML.tagR; triple.tagR2 = inputSequestXML.tagR2;

if (NumberOfFiles == 0) { formresult = MessageBox.Show("SILAC labels:\n" + "K " + triple.tagK1 + " " + triple.massK1 + " R " + triple.tagR1 + " " + triple.massR1 + "\n" + "K " + triple.tagK2 + " " + triple.massK2 + " R " + triple.tagR2 + " " + triple.massR2 + "\nDatabase:\n" + Path.GetFileName(inputSequestXML.Databasefile), "Confirm", MessageBoxButtons.YesNo, MessageBoxIcon.Question);

if (formresult == System.Windows.Forms.DialogResult.No) { Close(); return;

202

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

} NumberOfFiles++; }

numberofpairs = test.readSequestFile3(ref inputSequest, lstMSMS, triple); numberofunipep = test.GetMSInfo(lstMS, lstMSMS);

} toolStripProgressBar1.Visible = true; toolStripStatusLabel1.Visible = true; this.toolStripStatusLabel1.Text = "Preparing data"; this.Refresh();

//read MS1 peaks List inputpeaks = new List();

toolStripProgressBar1.Visible = true; toolStripStatusLabel1.Visible = true; this.toolStripStatusLabel1.Text = "Quantitation in progress"; this.Refresh();

toolStripProgressBar1.Maximum = lstMS.Count;

StreamReader inputMS1 = File.OpenText(inputMS1file); totalpeaks = test.readMS1File(ref inputMS1, inputpeaks);

StreamWriter ofile = new StreamWriter(outputMS1Quantitation);

List lstresult = new List(); List lstresult3 = new List();

if (comboBox1.SelectedIndex == 0) { statnumber result = new statnumber(); for (int m = 0; m < lstMS.Count; m++) { ofile.WriteLine((m + 1) + ":Comparing " + "{0:F3}" + " : " + "{1:F3}" + " Seq:" + lstMS[m].nonlabelpeptide, lstMS[m].sellmass, lstMS[m].selhmass); test.FindRatios_RT(ref ofile, lstMS[m], inputpeaks, totalpeaks, ref result); lstresult.Add(result); if (result.count == 0) { ofile.WriteLine(lstMS[m].nonlabelpeptide + " Count:" + result.count + " Av:" + result.average + "|" + result.LH_average + " Std:" + result.std + "|" + result.LH_std + " WA:" + result.weightedaverage + "|" + result.LH_weightaverage + " Median:" + result.median + "|" + result.LH_median + " RO_av:" + result.average_removeoutlier + "|" + result.LH_average_removeoutlier + " RO_wa:" + result.weightedaverage_removeoutlier + "|" + result.LH_weightedaverage_removeoutlier + " RO_wa_Std:" + result.std_removeoutlier + "|" + result.LH_std_removeoutlier + " Av_int:" + result.average_intensity + "|" + result.LH_average_intensity); } else {

203

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

ofile.WriteLine(lstMS[m].nonlabelpeptide + " Count:" + result.count + " Av:" + "{0:F3}|{1:F3}" + " Std:" + "{2:F3}|{3:F3}" + " WA:" + "{4:F3}|{5:F3}" + " Median:" + "{6:F3}|{7:F3}" + " RO_av:" + "{8:F3}|{9:F3}" + " RO_wa:" + "{10:F3}|{11:F3}" + " RO_wa_Std:" + "{12:F3}|{13:F3}" + " Av_int:" + "{14:F0}|{15:F0}", result.average, result.LH_average, result.std, result.LH_std, result.weightedaverage, result.LH_weightaverage, result.median, result.LH_median, result.average_removeoutlier, result.LH_average_removeoutlier, result.weightedaverage_removeoutlier, result.LH_weightedaverage_removeoutlier, result.std_removeoutlier, result.LH_std_removeoutlier, result.average_intensity, result.LH_average_intensity);

} ofile.WriteLine();

toolStripProgressBar1.Value = m; if (m % 10 == 0) Application.DoEvents(); }

ofile.Close();

toolStripProgressBar1.Visible = false; toolStripProgressBar1.Value = 0;

this.toolStripStatusLabel1.Text = "Summarize results"; this.Refresh(); string outputPeptideQuantitation = textBox4.Text + Path.GetFileNameWithoutExtension(filestring) + "_peptide.txt"; StreamWriter ofile1 = new StreamWriter(outputPeptideQuantitation); for (int mm = 0; mm < numberofunipep; mm++) { if (lstresult[mm].average_removeoutlier > 0) { ofile1.WriteLine(lstMS[mm].proteinid + '\t' + lstMS[mm].selscan + '\t' + lstMS[mm].netpeptide + '\t' + lstMS[mm].nonlabelpeptide + '\t' + lstresult[mm].average_removeoutlier.ToString("F2") + "|" + lstresult[mm].LH_average_removeoutlier.ToString("F2") + '\t' + lstresult[mm].std_removeoutlier.ToString("F2")

+ "|" + lstresult[mm].LH_std_removeoutlier.ToString("F2") + '\t' + lstresult[mm].count.ToString("F0") + '\t' + lstresult[mm].average_intensity.ToString("F0") + '\t' + lstMS[mm].proteindis + '\t' + lstMS[mm].xcorr.ToString("F2") + '\t' + lstMS[mm].DelCn.ToString("F2") + '\t' + lstMS[mm].IonMatch + '\t' + lstMS[mm].sellmass.ToString("F3") + '\t' + lstMS[mm].selhmass.ToString("F3")); //+ '\t' + lstMS[mm].selcharge + '\t' + lstresult[mm].LH_average_intensity } } ofile1.Close();

this.toolStripStatusLabel1.Text = "Peptide Quantation finished";

this.Refresh(); button2.Enabled = true; }

204

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

else if (comboBox1.SelectedIndex == 1) { statnumber2 result3 = new statnumber2(); for (int m = 0; m < lstMS.Count; m++) { ofile.WriteLine((m + 1) + ":Comparing " + "{0:F3}" + " : " + "{1:F3}" + " : " + "{2:F3}" + " Seq:" + lstMS[m].nonlabelpeptide, lstMS[m].sellmass, lstMS[m].selmmass, lstMS[m].selhmass); test.FindRatios_RT3(ref ofile, lstMS[m], inputpeaks, totalpeaks, ref result3); lstresult3.Add(result3); if (result3.count == 0) ofile.WriteLine(lstMS[m].nonlabelpeptide + " Count:" + result3.count + " Av:" + result3.average1 + "|" + result3.average2 + "|" + result3.average3 + " Std:" + result3.std1 + "|" + result3.std2 + "|" + result3.std3 + " WA:" + result3.weightedaverage1 + "|" + result3.weightedaverage2 + "|" + result3.weightedaverage3 + " Median:" + result3.median1 + "|" + result3.median2 + "|" + result3.median3 + " RO_av:" + result3.average_removeoutlier1 + "|" + result3.average_removeoutlier2 + "|" + result3.average_removeoutlier3 + " RO_wa:" + result3.weightedaverage_removeoutlier1 + "|" + result3.weightedaverage_removeoutlier2 + "|" + result3.weightedaverage_removeoutlier3 + " RO_wa_Std:" + result3.std_removeoutlier1 + "|" + result3.std_removeoutlier2 + "|" + result3.std_removeoutlier3 + " Av_int:" + result3.average_intensity1 + "|" + result3.average_intensity2 + "|" + result3.average_intensity3); else ofile.WriteLine(lstMS[m].nonlabelpeptide + " Count:" + result3.count + " Av:" + "{0:F3}|{1:F3}|{2:F3}" + " Std:" + "{3:F3}|{4:F3}|{5:F3}" + " WA:" + "{6:F3}|{7:F3}|{8:F3}" + " Median:" + "{9:F3}|{10:F3}|{11:F3}" + " RO_av:" + "{12:F3}|{13:F3}|{14:F3}" + " RO_wa:" + "{15:F3}|{16:F3}|{17:F3}" + " RO_wa_Std:" + "{18:F3}|{19:F3}|{20:F3}" + " Av_int:" + "{21:F0}|{22:F0}|{23:F0}", result3.average1, result3.average2, result3.average3, result3.std1, result3.std2, result3.std3, result3.weightedaverage1, result3.weightedaverage2, result3.weightedaverage3, result3.median1, result3.median2, result3.median3, result3.average_removeoutlier1, result3.average_removeoutlier2, result3.average_removeoutlier3, result3.weightedaverage_removeoutlier1, result3.weightedaverage_removeoutlier2, result3.weightedaverage_removeoutlier3, result3.std_removeoutlier1, result3.std_removeoutlier2, result3.std_removeoutlier3, result3.average_intensity1, result3.average_intensity2, result3.average_intensity3);

ofile.WriteLine(); toolStripProgressBar1.Value = m; if (m % 10 == 0) Application.DoEvents(); } ofile.Close();

toolStripProgressBar1.Visible = false; toolStripProgressBar1.Value = 0;

this.toolStripStatusLabel1.Text = "Summarize results"; this.Refresh();

string outputPeptideQuantitation = textBox4.Text + Path.GetFileNameWithoutExtension(filestring) + "_peptide.txt"; StreamWriter ofile1 = new StreamWriter(outputPeptideQuantitation); for (int mm = 0; mm < lstMS.Count; mm++)

205

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued). { if (lstresult3[mm].average_removeoutlier1 > 0) {

ofile1.WriteLine(lstMS[mm].proteinid + '\t' + lstMS[mm].selscan + '\t' + lstMS[mm].netpeptide + '\t' + lstMS[mm].nonlabelpeptide + '\t' + lstresult3[mm].average_removeoutlier1.ToString("F2") + "|" + lstresult3[mm].average_removeoutlier2.ToString("F2") + "|" + lstresult3[mm].average_removeoutlier3.ToString("F2") + '\t' + lstresult3[mm].std_removeoutlier1.ToString("F2") + "|" + lstresult3[mm].std_removeoutlier2.ToString("F2") + "|" + lstresult3[mm].std_removeoutlier3.ToString("F2") + '\t' + lstresult3[mm].count.ToString("F0") + '\t' + lstresult3[mm].average_intensity1.ToString("F0") + '|' + lstresult3[mm].average_intensity2.ToString("F0") + '|' + lstresult3[mm].average_intensity3.ToString("F0") + '\t' + lstMS[mm].proteindis + '\t' + lstMS[mm].xcorr.ToString("F2") + '\t' + lstMS[mm].DelCn.ToString("F2") + '\t' + lstMS[mm].IonMatch + '\t' + lstMS[mm].sellmass.ToString("F3") + '\t' + lstMS[mm].selmmass.ToString("F3") + '\t' + lstMS[mm].selhmass.ToString("F3")); //+ '\t' + lstMS[mm].selcharge } }

ofile1.Close();

this.toolStripStatusLabel1.Text = "Peptide quantation finished"; this.Refresh(); button2.Enabled = true;

} }

//**************************************************************************************** // Protein quantitation; public void ProcessProteinQuantitation(string outputPeptideQuantitation, string outputProteinGroup, string outputProtein) { ProteinGroup test = new ProteinGroup();

toolStripProgressBar1.Maximum = 0; toolStripProgressBar1.Visible = true; toolStripStatusLabel1.Visible = true; this.toolStripStatusLabel1.Text = "Protein quantitation"; this.Refresh();

StreamWriter ofile = new StreamWriter(outputProteinGroup);

// reading peptideQuantition Files; List PeptideQuantitationFileLines = File.ReadAllLines(outputPeptideQuantitation).ToList();

List ProteinCluster=new List ();

if (comboBox1.SelectedIndex == 0) { List peptideresult = new List(); List lstprotein = new List();

206

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

peptideresult = test.ReadPeptideQuant(outputPeptideQuantitation); int count=1;

toolStripProgressBar1.Maximum = peptideresult.Count;

for (int i = 0; i < peptideresult.Count; i++) { ProteinCluster.Add(peptideresult [i].proteinid ); foreach (string item in ProteinCluster) ofile.Write(item + "\t"); ofile.WriteLine(count + " " + peptideresult[i].unmodpeptide + " " + peptideresult[i].ratio + "|" + peptideresult[i].LH_ratio + " " + peptideresult[i].std + "|" + peptideresult[i].LH_std + " " + peptideresult[i].intensity + " " + peptideresult[i].count);

toolStripProgressBar1.Value = i; if (i % 10 == 0) Application.DoEvents(); } ofile.Close();

List uniqueprotein = new List(); uniqueprotein = ProteinCluster.Distinct().ToList(); ProteinCluster.Clear(); ProteinCluster = uniqueprotein.ToList();

toolStripProgressBar1.Visible = false; this.Refresh(); ProteinQuant proteinresult = new ProteinQuant(); proteinresult.CalculateProteinQuant(outputProtein, ProteinCluster, peptideresult, test); this.toolStripStatusLabel1.Text = "Protein quantitation finished"; toolStripProgressBar1.Visible = false; this.Refresh();

} else if (comboBox1.SelectedIndex == 1) { List peptideresult = new List(); peptideresult = test.ReadPeptideQuant3(outputPeptideQuantitation); int count=1; toolStripProgressBar1.Maximum = peptideresult.Count; for (int i = 0; i < peptideresult.Count; i++) { ProteinCluster.Add(peptideresult[i].proteinid);

foreach (string item in ProteinCluster) ofile.Write(item + "\t"); ofile.WriteLine(count + " " + peptideresult[i].unmodpeptide + " " + peptideresult[i].ratios[0] + "|" + peptideresult[i].ratios[1] + "|" + peptideresult[i].ratios[2] + " " + peptideresult[i].std[0] + "|" + peptideresult[i].std[1] + "|" + peptideresult[i].std[2] + " " + peptideresult[i].ave_int[0] + "|" + peptideresult[i].ave_int[1] + "|" + peptideresult[i].ave_int[2] + " " + peptideresult[i].count);

207

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

toolStripProgressBar1.Value = i; if (i % 10 == 0) Application.DoEvents(); } ofile.Close();

toolStripProgressBar1.Visible = false; this.Refresh(); List uniqueprotein = new List(); uniqueprotein = ProteinCluster.Distinct().ToList(); ProteinCluster.Clear(); ProteinCluster = uniqueprotein.ToList();

ProteinQuant proteinresult = new ProteinQuant(); proteinresult.CalculateProteinQuant3(outputProtein, ProteinCluster, peptideresult, test); this.toolStripStatusLabel1.Text = "Protein quantitation finished"; toolStripProgressBar1.Visible = false; this.Refresh();

}

TimeSpan timespent = new TimeSpan(); timespent = DateTime.Now - starttime; MessageBox.Show("Quantitation Finished in " + timespent.Minutes + "m " + timespent.Seconds + "s"); this.Close();

}

//*************************************************************************************** //Ratio calculation class MS1quant { public void processstring(string tempstring, ref int scannumber, ref string proteinid, ref string proteindis, ref double mw, ref double delta, ref int charge, ref string peptide, ref double xcorr, ref double deltacn, ref string ionmatch) {

int npos1; npos1 = tempstring.IndexOf('\t'); string scanstring = tempstring.Substring(0, npos1); if (scanstring.IndexOf('-') != -1) scanstring = scanstring.Substring(0, scanstring.IndexOf('-'));

scannumber = int.Parse(scanstring); //scannumber

tempstring = tempstring.Substring(npos1 + 1, tempstring.Length - npos1 - 1); npos1 = tempstring.IndexOf(' '); proteinid = tempstring.Substring(0, npos1); //proteinid

tempstring = tempstring.Substring(npos1 + 1, tempstring.Length - npos1 - 1);

208

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

npos1 = tempstring.IndexOf('\t'); proteindis = tempstring.Substring(0, npos1); //proteindis

tempstring = tempstring.Substring(npos1 + 1, tempstring.Length - npos1 - 1); npos1 = tempstring.IndexOf('\t'); mw = double.Parse(tempstring.Substring(0, npos1)); //mw

tempstring = tempstring.Substring(npos1 + 1, tempstring.Length - npos1 - 1); npos1 = tempstring.IndexOf('\t'); delta = double.Parse(tempstring.Substring(0, npos1)); //delta

tempstring = tempstring.Substring(npos1 + 1, tempstring.Length - npos1 - 1); npos1 = tempstring.IndexOf('\t'); charge = int.Parse(tempstring.Substring(0, npos1)); //charge

tempstring = tempstring.Substring(npos1 + 1, tempstring.Length - npos1 - 1); npos1 = tempstring.IndexOf('\t'); peptide = tempstring.Substring(0, npos1); //peptide

tempstring = tempstring.Substring(npos1 + 1, tempstring.Length - npos1 - 1); npos1 = tempstring.IndexOf('\t'); xcorr = double.Parse(tempstring.Substring(0, npos1)); //xcorr

tempstring = tempstring.Substring(npos1 + 1, tempstring.Length - npos1 - 1); npos1 = tempstring.IndexOf('\t'); deltacn = double.Parse(tempstring.Substring(0, npos1)); //delta

ionmatch = tempstring.Substring(npos1 + 1, tempstring.Length - npos1 - 1); //ionmatch }

double percentile(double[] sortedData, double p) { if (p >= 100.0d) return sortedData[sortedData.Length - 1];

double position = (double)(sortedData.Length + 1) * p / 100.0;

double leftNumber = 0.0d, rightNumber = 0.0d;

double n = p / 100.0d * (sortedData.Length - 1) + 1.0d;

if (position >= 1 && sortedData.Length > 1) { leftNumber = sortedData[(int)System.Math.Floor(n) - 1]; rightNumber = sortedData[(int)System.Math.Floor(n)]; } else if (position < 1 && sortedData.Length > 1) {

209

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

leftNumber = sortedData[0]; // first data rightNumber = sortedData[1]; // first data } else { leftNumber = rightNumber = sortedData[0]; }

if (leftNumber == rightNumber) return leftNumber; else { double part = n - System.Math.Floor(n); return leftNumber + part * (rightNumber - leftNumber); }

}

// for triple label int getlabelinfo3(ref StreamReader inputSequest, List lstMSMS, triplelabel triple) { //correct mass shift if (Math.Abs(triple.massK1 - 8) < 0.1) triple.massK1 = 8.01420; else if (Math.Abs(triple.massK1 - 4) < 0.1) triple.massK1 = 4.02511;

if (Math.Abs(triple.massK2 - 8) < 0.1) triple.massK2 = 8.01420; else if (Math.Abs(triple.massK2 - 4) < 0.1) triple.massK2 = 4.02511;

if (Math.Abs(triple.massR1 - 10) < 0.1) triple.massR1 = 10.00827; else if (Math.Abs(triple.massR1 - 6) < 0.1) triple.massR1 = 6.02013;

if (Math.Abs(triple.massR2 - 10) < 0.1) triple.massR2 = 10.00827; else if (Math.Abs(triple.massR2 - 6) < 0.1)

triple.massR2 = 6.02013;

string peptide = ""; double mass = 0; int scannumber = 0; string proteinid = "", proteindis = ""; double mw = 0, delta = 0.0; double xcorrvalue = 0, deltacnvalue = 0; string ionmatchvalue = ""; int ioncharge = 0;

int npos1, npos2; int i;

210

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

string tempstring;

while ((tempstring = inputSequest.ReadLine()) != null) { processstring(tempstring, ref scannumber, ref proteinid, ref proteindis, ref mw, ref delta, ref ioncharge, ref peptide, ref xcorrvalue, ref deltacnvalue, ref ionmatchvalue);

mass = (mw - 1.0078 - delta + ioncharge * 1.0078) / ioncharge;

npos1 = peptide.IndexOf('.'); npos2 = peptide.LastIndexOf('.');

peptide = peptide.Substring(npos1 + 1, npos2 - npos1 - 1);

int Knum = 0; int Rnum = 0; int llabel = 0; int hlabel = 0; string unlabelpep = ""; string netpep = ""; for (i = 0; i < peptide.Length; i++) {

if (peptide[i] == triple.tagK1 || peptide[i] == triple.tagR1) { llabel = llabel + 1; } if (peptide[i] == triple.tagK2 || peptide[i] == triple.tagR2) { hlabel = hlabel + 1; } if (peptide[i] == 'K') { Knum = Knum + 1; } if (peptide[i] == 'R') { Rnum = Rnum + 1;

} if (peptide[i] != triple.tagK1 && peptide[i] != triple.tagR1 && peptide[i] != triple.tagK2 && peptide[i] != triple.tagR2) { unlabelpep = unlabelpep + peptide[i]; if (Char.IsLetter(peptide, i) == true) { netpep = netpep + peptide[i]; } } }

List lmhmass = new List(); MSMSInfo MSMS = new MSMSInfo();

if (llabel == 0 && hlabel == 0)//non label {

211

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

double shift2medium = (triple.massK1 * Knum + triple.massR1 * Rnum) / ioncharge; double shift2heavy = (triple.massK2 * Knum + triple.massR2 * Rnum) / ioncharge; lmhmass.Add(mass); lmhmass.Add(mass + shift2medium); lmhmass.Add(mass + shift2heavy); lmhmass.Sort();

MSMS.lightmass = lmhmass[0]; MSMS.mediummass = lmhmass[1]; MSMS.heavymass = lmhmass[2]; MSMS.charge = ioncharge; MSMS.DeltaCn = deltacnvalue; MSMS.IonMatch = ionmatchvalue; MSMS.peptide = peptide; MSMS.proteinid = proteinid; MSMS.proteindis = proteindis; MSMS.netpeptide = netpep; MSMS.nonlablepeptide = unlabelpep; MSMS.Xcorr = xcorrvalue; MSMS.Scan = scannumber; lstMSMS.Add(MSMS); MSMS = new MSMSInfo(); }

else//medium or heavy label { int KRnum = 0; int labelnum = 0; KRnum = Knum + Rnum;

if (llabel > hlabel) { labelnum = llabel; } else { labelnum = hlabel; }

if (KRnum == labelnum) { if (llabel > 0 && hlabel == 0)//medium label

{ double shift2light = (triple.massK1 * Knum + triple.massR1 * Rnum) / ioncharge; double shift2heavy = ((triple.massK2 - triple.massK1) * Knum + (triple.massR2 - triple.massR1) * Rnum) / ioncharge; lmhmass.Add(mass - shift2light); lmhmass.Add(mass); lmhmass.Add(mass + shift2heavy); } if (llabel == 0 && hlabel > 0)//heavy label { double shift2light = (triple.massK2 * Knum + triple.massR2 * Rnum) / ioncharge; double shift2medium = ((triple.massK2 - triple.massK1) * Knum + (triple.massR2 - triple.massR1) * Rnum) / ioncharge; lmhmass.Add(mass - shift2light); lmhmass.Add(mass - shift2medium); lmhmass.Add(mass); }

212

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

lmhmass.Sort(); MSMS.lightmass = lmhmass[0]; MSMS.mediummass = lmhmass[1]; MSMS.heavymass = lmhmass[2]; MSMS.charge = ioncharge; MSMS.DeltaCn = deltacnvalue; MSMS.IonMatch = ionmatchvalue; MSMS.peptide = peptide; MSMS.proteinid = proteinid; MSMS.proteindis = proteindis; MSMS.netpeptide = netpep; MSMS.nonlablepeptide = unlabelpep; MSMS.Xcorr = xcorrvalue; MSMS.Scan = scannumber; lstMSMS.Add(MSMS); MSMS = new MSMSInfo(); } } } return lstMSMS.Count; }

// for double label int getlabelinfo(ref StreamReader inputSequest, List lstMSMS, char tagK, char tagR, double massK, double massR) { //correct mass shift if (Math.Abs(massK - 8) < 0.1) massK = 8.01420; else if (Math.Abs(massK - 4) < 0.001) massK = 4.02511; else if (Math.Abs(massK - 3.99) < 0.001)//R+3.99 massK = 3.9891;

if (Math.Abs(massR - 10) < 0.1) massR = 10.00827; else if (Math.Abs(massR - 6) < 0.1) massR = 6.02013;

else if (Math.Abs(massR - 3.9) < 0.1)//R+3.9 massR = 3.98814;

string peptide = ""; double mass; int scannumber = 0; string proteinid = "", proteindis = ""; double mw = 0, delta = 0.0; double xcorrvalue = 0, deltacnvalue = 0; string ionmatchvalue = ""; int ioncharge = 0; int npos1, npos2; int k = 0; int i;

213

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

string tempstring;

while ((tempstring = inputSequest.ReadLine()) != null) { processstring(tempstring, ref scannumber, ref proteinid, ref proteindis, ref mw, ref delta, ref ioncharge, ref peptide,

ref xcorrvalue, ref deltacnvalue, ref ionmatchvalue);

mass = (mw - 1.0078 - delta + ioncharge * 1.0078) / ioncharge;

npos1 = peptide.IndexOf('.'); npos2 = peptide.LastIndexOf('.');

peptide = peptide.Substring(npos1 + 1, npos2 - npos1 - 1);

int Knum = 0; int Rnum = 0; int Label = 0; string unlabelpep = ""; string netpep = ""; for (i = 0; i < peptide.Length; i++) { if (peptide[i] == tagK || peptide[i] == tagR) { Label = 1; } if (peptide[i] == 'K') { Knum = Knum + 1; } if (peptide[i] == 'R') {

Rnum = Rnum + 1; } if (peptide[i] != tagK && peptide[i] != tagR) { unlabelpep = unlabelpep + peptide[i]; if (Char.IsLetter(peptide, i) == true) { netpep = netpep + peptide[i]; } }

}

MSMSInfo MSMS = new MSMSInfo();

MSMS.charge = ioncharge; MSMS.DeltaCn = deltacnvalue; MSMS.IonMatch = ionmatchvalue; MSMS.peptide = peptide; MSMS.proteinid = proteinid; MSMS.proteindis = proteindis;

214

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

MSMS.netpeptide = netpep; MSMS.nonlablepeptide = unlabelpep; MSMS.Xcorr = xcorrvalue; MSMS.Scan = scannumber;

double massshift = (massK * Knum + massR * Rnum) / ioncharge;

MSMS.lightmass = mass - Label * massshift; MSMS.heavymass = mass + (1 - Label) * massshift; MSMS.mediummass = 0;

lstMSMS.Add(MSMS); MSMS = new MSMSInfo(); } return k + 1; }

public int readSequestFile(ref StreamReader inputSequest, List lstMSMS, char tagK, char tagR, double massK, double massR) { int numberofpairs; numberofpairs = getlabelinfo(ref inputSequest, lstMSMS, tagK, tagR, massK, massR); return lstMSMS.Count; }

public int readSequestFile3(ref StreamReader inputSequest, List lstMSMS, triplelabel triple) { int numberofpairs; numberofpairs = getlabelinfo3(ref inputSequest, lstMSMS, triple); return numberofpairs; }

//calculate ratio for double label public void FindRatios_RT(ref StreamWriter ofile, MSInfo MS, List inputpeaks, int total, ref statnumber results) { int i; int totalscan = 0; double ratio = 0; double lightInt = 0.0, heavyInt = 0.0;

results = new statnumber(); totalscan = MS.fscan.Count; int pos = 0;

List lstratiohl = new List(); List lstratiolh = new List(); List lstscan = new List(); List lsthint = new List(); List lstlint = new List();

215

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

List testscan = new List(); for (i = 0; i < totalscan; i++)

{ for (int k = pos; k < total; k++) { if (inputpeaks[k].scannumber >= MS.fscan[i] && inputpeaks[k].scannumber <= MS.lscan[i]) { testscan.Add(inputpeaks[k].scannumber);

FindQuanRatios(ref ratio, inputpeaks[k], MS.lightmass[i], MS.heavymass[i], MS.charge[i], ref heavyInt, ref lightInt); if (ratio > 0) { lstscan.Add(inputpeaks[k].scannumber); lstratiohl.Add(ratio); lstratiolh.Add(1 / ratio); lstlint.Add(lightInt); lsthint.Add(heavyInt);

ofile.WriteLine(inputpeaks[k].scannumber + " {0:F3} {1:F0} : {2:F0}", ratio, heavyInt, lightInt); } } if (inputpeaks[k].scannumber > MS.lscan[i]) { pos = k; break; } } }

if (lstratiohl.Count > 0) { results.count = lstratiohl.Count; results.average_intensity = lstlint.Average(); results.LH_average_intensity = lsthint.Average(); double sum = 0; double avg = 0; double sd2 = 0; double sd = 0; int count = 0;

//h to l ratio count = lstratiohl.Count; sum = lstratiohl.Sum(); avg = lstratiohl.Average(); foreach (double a in lstratiohl) { sd2 = sd2 + Math.Pow((a - avg), 2); } sd = Math.Sqrt(sd2 / count); results.average = avg; results.std = sd;

//l to h ratio sum = 0; avg = 0; count = 0; sd2 = 0; sd = 0; sum = lstratiolh.Sum();

216

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

avg = lstratiolh.Average(); count = lstratiolh.Count; foreach (double a in lstratiolh) { sd2 = sd2 + Math.Pow((a - avg), 2); } sd = Math.Sqrt(sd2 / count); results.LH_average = avg; results.LH_std = sd;

//weightedaverage //h to l ratio sum = 0; avg = 0;

for (int x = 0; x < lstratiohl.Count; x++) { sum = sum + lstratiohl[x] * lsthint[x]; } avg = sum / lsthint.Sum(); results.weightedaverage = avg; //l to h ratio sum = 0; avg = 0; for (int x = 0; x < lstratiolh.Count; x++) { sum = sum + lstratiolh[x] * lstlint[x]; } avg = sum / lstlint.Sum(); results.LH_weightaverage = avg;

//outlier remove //calculate quantile

if (lstratiohl.Count > 0) { double[] arraypercentile = lstratiohl.ToArray(); Array.Sort(arraypercentile);

results.median = percentile(arraypercentile, 50); results.first_quantile = percentile(arraypercentile, 25); results.third_quantile = percentile(arraypercentile, 75);

double[] LH_arraypercentile = lstratiolh.ToArray(); Array.Sort(LH_arraypercentile);

results.LH_median = percentile(LH_arraypercentile, 50); results.LH_first_quantile = percentile(LH_arraypercentile, 25); results.LH_third_quantile = percentile(LH_arraypercentile, 75); } else { results.median = 0; results.first_quantile = 0; results.third_quantile = 0; results.LH_median = 0; results.LH_first_quantile = 0; results.LH_third_quantile = 0; }

217

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

//remove outlier

List lstratiohlro = new List(); List lstratiolhro = new List(); List lsthintro = new List(); List lstlintro = new List(); for (int x = 0; x < lstratiohl.Count; x++) { double temp = lstratiohl[x]; if (temp > 0 && temp <= results.median + 1.5 * (results.third_quantile - results.median) && temp >= results.median - 1.5 * (results.median - results.first_quantile)) { lstratiohlro.Add(temp); lsthintro.Add(lsthint[x]); } temp = lstratiolh[x]; if (temp > 0 && temp <= results.LH_median + 1.5 * (results.LH_third_quantile - results.LH_median) && temp >= results.LH_median - 1.5 * (results.LH_median - results.LH_first_quantile)) { lstratiolhro.Add(temp); lstlintro.Add(lstlint[x]); } }

//h to l ratio with outlier remove if (lstratiohlro.Count > 0) { sum = 0; avg = 0; count = 0; sd2 = 0; sd = 0; sum = lstratiohlro.Sum(); avg = lstratiohlro.Average(); count = lstratiohlro.Count; foreach (double a in lstratiohlro) { sd2 = sd2 + Math.Pow((a - avg), 2); } sd = Math.Sqrt(sd2 / count); results.average_removeoutlier = avg; results.std_removeoutlier = sd; results.average_intensity = lsthint.Average();

//l to h ratio sum = 0; avg = 0; count = 0; sd2 = 0; sd = 0; sum = lstratiolhro.Sum(); avg = lstratiolhro.Average(); count = lstratiolhro.Count; foreach (double a in lstratiolhro) { sd2 = sd2 + Math.Pow((a - avg), 2); } sd = Math.Sqrt(sd2 / count); results.LH_average_removeoutlier = avg; results.LH_std_removeoutlier = sd; results.LH_average_intensity = lstlint.Average(); //weightedaverage //h to l ratio sum = 0; avg = 0; for (int x = 0; x < lstratiohlro.Count; x++) { sum = sum + lstratiohlro[x] * lsthintro[x]; }

218

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

avg = sum / lsthintro.Sum(); results.weightedaverage_removeoutlier = avg;

//l to h ratio sum = 0; avg = 0; for (int x = 0; x < lstratiolhro.Count; x++) { sum = sum + lstratiolhro[x] * lstlintro[x]; } avg = sum / lstlintro.Sum(); results.LH_weightedaverage_removeoutlier = avg; } } }

//Calculate triple label ratio public void FindRatios_RT3(ref StreamWriter ofile, MSInfo MS, List inputpeaks, int total, ref statnumber2 results) { int i;

double ratio1 = 0.0, ratio2 = 0.0, ratio3 = 0.0;

double lightInt = 0.0, mediumInt = 0.0, heavyInt = 0.0;

results = new statnumber2(); int totalscan = MS.fscan.Count; int pos = 0;

List lstratiohl = new List(); List lstratioml = new List(); List lstratiohm = new List(); List lstscan = new List(); List lsthint = new List(); List lstmint = new List(); List lstlint = new List();

for (i = 0; i < totalscan; i++) {

for (int k = pos; k < total; k++) { if (inputpeaks[k].scannumber >= MS.fscan[i] && inputpeaks[k].scannumber <= MS.lscan[i]) { FindQuanRatios3(ref ratio1, ref ratio2, ref ratio3, inputpeaks[k], MS.lightmass[i], MS.mediummass[i], MS.heavymass[i], MS.charge[i], ref lightInt, ref mediumInt, ref heavyInt); if (ratio1 > 0 && ratio2 > 0 && ratio3 > 0) { lstscan.Add(inputpeaks[k].scannumber); lstratiohl.Add(ratio1); lstratioml.Add(ratio2); lstratiohm.Add(ratio3);

lsthint.Add(lightInt);

219

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

lstmint.Add(mediumInt); lstlint.Add(heavyInt);

ofile.WriteLine(inputpeaks[k].scannumber + " {0:F3}(H/L)/{1:F3}(M/L)/{2:F3}(H/M) {3:F0} : {4:F0} : {5:F0}", ratio1, ratio2, ratio3, lightInt, mediumInt, heavyInt);

} } if (inputpeaks[k].scannumber > MS.lscan[i]) { pos = k; break; } } }

double sum = 0; double avg = 0; double sd2 = 0; double sd = 0; int count = 0;

if (lstratiohl.Count > 0 && lstratioml.Count > 0 && lstratiohm.Count > 0) {

results.count = lstratiohl.Count; results.average_intensity1 = lstlint.Average(); results.average_intensity2 = lstmint.Average(); results.average_intensity3 = lsthint.Average();

//m to l ratio sum = 0; avg = 0; count = 0; sd2 = 0; sd = 0; sum = lstratioml.Sum(); avg = lstratioml.Average(); count = lstratioml.Count; foreach (double a in lstratioml) { sd2 = sd2 + Math.Pow((a - avg), 2); } sd = Math.Sqrt(sd2 / count); results.average2 = avg; results.std2 = sd;

//h to m ratio sum = 0; avg = 0; count = 0; sd2 = 0; sd = 0; sum = lstratiohm.Sum(); avg = lstratiohm.Average(); count = lstratiohm.Count;

220

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

foreach (double a in lstratiohm) { sd2 = sd2 + Math.Pow((a - avg), 2); } sd = Math.Sqrt(sd2 / count); results.average3 = avg; results.std3 = sd;

//weightedaverage //h to l ratio sum = 0; avg = 0; for (int x = 0; x < lstratiohl.Count; x++) { sum = sum + lstratiohl[x] * lsthint[x]; } avg = sum / lsthint.Sum(); results.weightedaverage1 = avg;

//m to l ratio sum = 0; avg = 0; for (int x = 0; x < lstratioml.Count; x++) { sum = sum + lstratioml[x] * lstmint[x]; } avg = sum / lstmint.Sum(); results.weightedaverage2 = avg;

//h to m ratio sum = 0; avg = 0; for (int x = 0; x < lstratiohm.Count; x++) { sum = sum + lstratiohm[x] * lsthint[x]; } avg = sum / lsthint.Sum(); results.weightedaverage3 = avg;

//outlier remove if (results.count > 0) { double[] arraypercentile1 = lstratiohl.ToArray(); double[] arraypercentile2 = lstratioml.ToArray();

double[] arraypercentile3 = lstratiohm.ToArray();

Array.Sort(arraypercentile1); Array.Sort(arraypercentile2); Array.Sort(arraypercentile3);

results.median1 = percentile(arraypercentile1, 50); results.median2 = percentile(arraypercentile2, 50); results.median3 = percentile(arraypercentile3, 50);

results.first_quantile1 = percentile(arraypercentile1, 25); results.first_quantile2 = percentile(arraypercentile2, 25); results.first_quantile3 = percentile(arraypercentile3, 25);

results.third_quantile1 = percentile(arraypercentile1, 75);

221

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

results.third_quantile2 = percentile(arraypercentile2, 75); results.third_quantile3 = percentile(arraypercentile3, 75); } else { results.median1 = results.median2 = results.median3 = 0; results.first_quantile1 = results.first_quantile2 = results.first_quantile3 = 0; results.third_quantile1 = results.third_quantile2 = results.third_quantile3 = 0; }

//remove outlier List lstratiohlro = new List(); List lstratiomlro = new List(); List lstratiohmro = new List(); List lsthintro = new List(); List lsthmintro = new List(); List lstmintro = new List();

for (int x = 0; x < lstratiohl.Count; x++) { double temp = lstratiohl[x]; if (temp > 0 && temp <= results.median1 + 1.5 * (results.third_quantile1 - results.median1) && temp >= results.median1 - 1.5 * (results.median1 - results.first_quantile1)) { lstratiohlro.Add(temp); lsthintro.Add(lsthint[x]); }

temp = lstratioml[x]; if (temp > 0 && temp <= results.median2 + 1.5 * (results.third_quantile2 - results.median2) && temp >= results.median2 - 1.5 * (results.median2 - results.first_quantile2)) { lstratiomlro.Add(temp); lstmintro.Add(lsthint[x]); }

temp = lstratiohm[x]; if (temp > 0 && temp <= results.median3 + 1.5 * (results.third_quantile3 - results.median3) && temp >= results.median3 - 1.5 * (results.median3 - results.first_quantile3)) { lstratiohmro.Add(temp); lsthmintro.Add(lsthint[x]); } }

//h to l ratio if (lstratiohlro.Count > 1) { sum = 0; count = 0; avg = 0; sd2 = 0; sd = 0; count = lstratiohlro.Count; sum = lstratiohlro.Sum(); avg = lstratiohlro.Average(); foreach (double a in lstratiohlro) { sd2 = sd2 + Math.Pow((a - avg), 2); } sd = Math.Sqrt(sd2 / count); results.average_removeoutlier1 = avg;

222

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

results.std_removeoutlier1 = sd;

//weightedaverage sum = 0; avg = 0; for (int x = 0; x < lstratiohlro.Count; x++) { sum = sum + lstratiohlro[x] * lsthintro[x]; } avg = sum / lsthintro.Sum(); results.weightedaverage_removeoutlier1 = avg; } else { results.average_removeoutlier1 = results.average1; results.weightedaverage_removeoutlier1 = results.weightedaverage1; results.std_removeoutlier1 = results.std1; }

//m to l ratio if (lstratiomlro.Count > 1) { sum = 0; avg = 0; count = 0; sd2 = 0; sd = 0; sum = lstratiomlro.Sum(); avg = lstratiomlro.Average(); count = lstratiomlro.Count; foreach (double a in lstratiomlro) { sd2 = sd2 + Math.Pow((a - avg), 2); } sd = Math.Sqrt(sd2 / count); results.average_removeoutlier2 = avg; results.std_removeoutlier2 = sd;

//weighted average sum = 0; avg = 0; for (int x = 0; x < lstratiomlro.Count; x++) {

sum = sum + lstratiomlro[x] * lstmintro[x]; } avg = sum / lstmintro.Sum(); results.weightedaverage_removeoutlier2 = avg; } else { results.average_removeoutlier2 = results.average2; results.weightedaverage_removeoutlier2 = results.weightedaverage2; results.std_removeoutlier2 = results.std2; }

//h to m ratio if (lstratiomlro.Count > 1) { sum = 0; avg = 0; count = 0; sd2 = 0; sd = 0; sum = lstratiohmro.Sum(); avg = lstratiohmro.Average(); count = lstratiohmro.Count; foreach (double a in lstratiohmro) { sd2 = sd2 + Math.Pow((a - avg), 2); }

223

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

sd = Math.Sqrt(sd2 / count); results.average_removeoutlier3 = avg; results.std_removeoutlier3 = sd;

//weighted average sum = 0; avg = 0; for (int x = 0; x < lstratiohmro.Count; x++) { sum = sum + lstratiohmro[x] * lsthmintro[x]; } avg = sum / lsthintro.Sum(); results.weightedaverage_removeoutlier3 = avg; } else { results.average_removeoutlier3 = results.average3; results.weightedaverage_removeoutlier3 = results.weightedaverage3; results.std_removeoutlier3 = results.std3; } } }

public void FindQuanRatios3(ref double ratio1, ref double ratio2, ref double ratio3, mspeaks peaklist, double unlabeledmass, double mediumlabelmass, double labeledmass, int charge, ref double heavyInt, ref double mediumInt, ref double lightInt) { List lInt = new List(); List mInt = new List(); List hInt = new List();

ratio1 = -1; ratio2 = -1; ratio3 = -1; heavyInt = 0; mediumInt = 0; lightInt = 0;

int pos = 0; FindMZwithProfile(peaklist, unlabeledmass, charge, ref lInt, ref pos); FindMZwithProfile(peaklist, mediumlabelmass, charge, ref mInt, ref pos); FindMZwithProfile(peaklist, labeledmass, charge, ref hInt, ref pos);

List cnt = new List(); cnt.Add(lInt.Count); cnt.Add(mInt.Count); cnt.Add(hInt.Count);

if (cnt.Min() == 3) { lightInt = lInt.Sum(); mediumInt = mInt.Sum(); heavyInt = hInt.Sum(); ratio1 = heavyInt / lightInt; ratio2 = mediumInt / lightInt; ratio3 = heavyInt / mediumInt;

}

224

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

if (cnt.Min() == 2) { lightInt = lInt[0] + lInt[1]; mediumInt = mInt[0] + mInt[1]; heavyInt = hInt[0] + lInt[1]; ratio1 = heavyInt / lightInt; ratio2 = mediumInt / lightInt; ratio3 = heavyInt / mediumInt;

} }

public void FindQuanRatios(ref double ratio, mspeaks peaklist, double unlabeledmass, double labeledmass, int charge, ref double intensity_heavy, ref double intensity_light) { ratio = 0; List ulbint = new List(); List lbint = new List(); int pos = 0;

FindMZwithProfile(peaklist, unlabeledmass, charge, ref ulbint, ref pos); FindMZwithProfile(peaklist, labeledmass, charge, ref lbint, ref pos);

List cnt = new List(); cnt.Add(ulbint.Count); cnt.Add(lbint.Count); if (cnt.Min() == 3) { intensity_light = ulbint.Sum(); intensity_heavy = lbint.Sum(); ratio = intensity_heavy/intensity_light ; }

else if (cnt.Min() == 2) { intensity_light = ulbint[0] + ulbint[1]; intensity_heavy = lbint[0] + lbint[1]; ratio = intensity_heavy/intensity_light ; }

}

public int readMS1File(ref StreamReader inputMS1, List inputpeaks) { int i; mspeaks temp = new mspeaks(); string tempstring; int scannumber = 0; double rt = 0.0;

double tempmass = 0.0; double tempintensity = 0.0;

225

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

while ((tempstring = inputMS1.ReadLine()) != null) { pickheader(tempstring, ref scannumber, ref rt); i = 0;

while ((tempstring = inputMS1.ReadLine()) != "") { pickmspeaks(tempstring, ref tempmass, ref tempintensity); temp.mass.Add(tempmass); temp.intensity.Add(tempintensity); i++; } temp.length = i; temp.scannumber = scannumber;

inputpeaks.Add(temp); temp = new mspeaks(); }

return inputpeaks.Count;

}

public void pickheader(string sequence, ref int scannumber, ref double rt) {

int npos1, npos2; npos1 = sequence.IndexOf(':'); npos2 = sequence.IndexOf(' ');

string tempstring1, tempstring2; tempstring1 = sequence.Substring(npos1 + 1, sequence.Length - npos1 - 1); tempstring2 = sequence.Substring(npos1 + 1, npos2 - npos1 - 1); scannumber = int.Parse(tempstring2);

npos1 = tempstring1.IndexOf(':'); tempstring2 = tempstring1.Substring(npos1 + 1, tempstring1.Length - npos1 - 1); rt = double.Parse(tempstring2);

}

public void pickmspeaks(string tempstring, ref double mass, ref double intensity) { int npos; npos = tempstring.IndexOf(' ');

string tempstring1;

tempstring1 = tempstring.Substring(npos + 1, tempstring.Length - npos - 1); intensity = double.Parse(tempstring1);

mass = double.Parse(tempstring.Substring(0, npos)); }

226

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

public void FindMZwithProfile(mspeaks peaklist, double mass, int charge, ref List inten, ref int pointer) { int i = 0; int j = 0; int end = peaklist.length; double a = 0; a = mass; for (i = pointer; i < end; i++) { double b = 0; b = peaklist.mass[i];

if (Math.Abs(a - b) < 0.009) { inten.Add(peaklist.intensity[i]); a = b + 1.0078 / charge; j = j + 1; pointer = i - 1; end = i + 5 - j; if (end > peaklist.length) { end = peaklist.length; } } if (pointer < 1) { pointer = 0; }

if (j > 2) { break; } } if (j == 0) { pointer = 0; } }

public int GetMSInfo(List lstMS, List lstMSMS) { //extract MS information from MSMS, determine MS1 scan range for MS1 quantification MSInfo temp = new MSInfo();

List lststrMSMS = new List(); List lstnonlabelpep = new List(); List lstuninonlabel = new List();

for (int i = 0; i < lstMSMS.Count; i++) { string a = lstMSMS[i].nonlablepeptide + '\t' + lstMSMS[i].Scan + '\t' + lstMSMS[i].charge + '\t' + lstMSMS[i].lightmass + '\t' + lstMSMS[i].heavymass; a = a + '\t' + lstMSMS[i].Xcorr + '\t' + lstMSMS[i].proteinid + '\t' + lstMSMS[i].proteindis + '\t' + lstMSMS[i].netpeptide; a = a + '\t' + lstMSMS[i].DeltaCn + '\t' + lstMSMS[i].IonMatch + '\t' + lstMSMS[i].mediummass; if (a != "\t0\t0\t0\t0\t\t\t") { lststrMSMS.Add(a); lstnonlabelpep.Add(lstMSMS[i].nonlablepeptide); } } lststrMSMS.Sort(); lstuninonlabel = lstnonlabelpep.Distinct().ToList(); lstuninonlabel.Sort(); int pointer = 0;

227

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

for (int i = 0; i < lstuninonlabel.Count; i++) { string a = lstuninonlabel[i];

//get the sorted block of a give nonlabelpeptide a List sublstuninonlabel = new List(); for (int j = pointer; j < lststrMSMS.Count; j++) { string[] b = lststrMSMS[j].Split(new char[] { '\t' }); if (b[0] == a) { sublstuninonlabel.Add(lststrMSMS[j]); } else { pointer = j; break; } }

//get info the sorted block List lstxcorr = new List(); for (int k = 0; k < sublstuninonlabel.Count; k++) { //prepare scan range for MS1 quantitation int scan1 = 0; int scan2 = 0; int scan3 = 0; string[] col2 = sublstuninonlabel[k].Split(new char[] { '\t' }); scan2 = Convert.ToInt32(col2[1]);

if (sublstuninonlabel.Count > 1) { if (k > 0 && k < sublstuninonlabel.Count - 1) { string[] col1 = sublstuninonlabel[k - 1].Split(new char[] { '\t' }); scan1 = Convert.ToInt32(col1[1]); string[] col3 = sublstuninonlabel[k + 1].Split(new char[] { '\t' }); scan3 = Convert.ToInt32(col3[1]); } if (k == 0) { if (scan2 - 100 > 0) { scan1 = scan2 - 100; } else { scan1 = 0; } if (sublstuninonlabel.Count > 1) { string[] col3 = sublstuninonlabel[k + 1].Split(new char[] { '\t' }); scan3 = Convert.ToInt32(col3[1]); } }

if (k == sublstuninonlabel.Count - 1) { string[] col1 = sublstuninonlabel[k - 1].Split(new char[] { '\t' }); scan1 = Convert.ToInt32(col1[1]);

scan3 = scan2 + 100; } } else {

228

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

if (scan2 - 100 > 0) { scan1 = scan2 - 100; } else { scan1 = 0; } scan3 = scan2 + 100; }

if (scan2 - scan1 > 100) { scan1 = scan2 - 100; } if (scan3 - scan2 > 100) { scan3 = scan2 + 100; }

int scanend = 0; if ((scan3 - scan2) % 2 == 0) { scanend = scan2 + (scan3 - scan2) / 2 - 1; } else { scanend = scan2 + (scan3 - scan2) / 2; }

int scanstart = scan2 - (scan2 - scan1) / 2;

temp.fscan.Add(scanstart); temp.lscan.Add(scanend); temp.scan.Add(scan2); temp.charge.Add(Convert.ToInt32(col2[2])); temp.lightmass.Add(Convert.ToDouble(col2[3])); temp.heavymass.Add(Convert.ToDouble(col2[4])); temp.mediummass.Add(Convert.ToDouble(col2[11]));

double xcorr = 0; xcorr = Convert.ToDouble(col2[5]); lstxcorr.Add(xcorr);

}

//find the scan with maxium xcorr, which will be used in final peptide report double maxcorr = 0; int pos = 0; for (int k = 0; k < lstxcorr.Count; k++) { if (lstxcorr[k] > maxcorr) { maxcorr = lstxcorr[k]; pos = k; } }

string[] col = sublstuninonlabel[pos].Split(new char[] { '\t' }); temp.selscan = Convert.ToInt32(col[1]); temp.nonlabelpeptide = col[0]; temp.selcharge = Convert.ToInt32(col[2]); temp.sellmass = Convert.ToDouble(col[3]); temp.selhmass = Convert.ToDouble(col[4]); temp.selmmass = Convert.ToDouble(col[11]); temp.xcorr = Convert.ToDouble(col[5]); temp.proteinid = col[6]; temp.proteindis = col[7]; temp.netpeptide = col[8]; temp.DelCn = Convert.ToDouble(col[9]); temp.IonMatch = col[10];

if (temp.DelCn < 0.1) { temp = new MSInfo(); } else { lstMS.Add(temp);

229

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

temp = new MSInfo(); }

} return lstMS.Count; } }

public class mspeaks { public List mass; public List intensity; public int length; public int scannumber;

public mspeaks() { mass = new List(); intensity = new List(); }

public void SetLength(int newlength) { length = newlength; }

public void SetScannumber(int newscan) { scannumber = newscan; }

}

public class MSInfo { public List fscan; public List lscan; public List scan; public List charge; public int selscan; public string nonlabelpeptide; public string proteinid; public string proteindis; public string netpeptide; public double xcorr; public double DelCn; public string IonMatch; public int selcharge; public double sellmass; public double selhmass; public List lightmass; public List heavymass; public double selmmass; public List mediummass;

230

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

public MSInfo() { fscan = new List(); lscan = new List(); scan = new List(); lightmass = new List(); mediummass = new List(); heavymass = new List(); charge = new List(); } }

public class MSMSInfo { public string peptide; public string proteinid; public string proteindis; public double Xcorr; public string IonMatch; public int Scan; public int charge; public double lightmass; public double mediummass; public double heavymass; public double DeltaCn; public string netpeptide; public string nonlablepeptide; }

struct statnumber { //heavy to light public double average; public double std; public double weightedaverage; public double median; public double first_quantile; public double third_quantile; public double average_removeoutlier; public double weightedaverage_removeoutlier; public double std_removeoutlier; public double average_intensity; //light to heavy public double LH_average; public double LH_std; public double LH_weightaverage; public double LH_median; public double LH_first_quantile; public double LH_third_quantile; public double LH_weightedaverage_removeoutlier; public double LH_std_removeoutlier; public double LH_average_removeoutlier; public double LH_average_intensity; public int count; }

231

Appendix 4. Source codes for peptide and protein quantitation in IsoQuant (continued).

struct statnumber2 { public double average1;//heavy to light public double average2;//medium to light public double average3;//heavy to medium public double std1; public double std2; public double std3; public double weightedaverage1; public double weightedaverage2; public double weightedaverage3; public double median1; public double median2; public double median3; public double first_quantile1; public double first_quantile2; public double first_quantile3; public double third_quantile1; public double third_quantile2; public double third_quantile3; public double average_removeoutlier1; public double average_removeoutlier2; public double average_removeoutlier3; public double weightedaverage_removeoutlier1; public double weightedaverage_removeoutlier2; public double weightedaverage_removeoutlier3; public double std_removeoutlier1; public double std_removeoutlier2; public double std_removeoutlier3; public double average_intensity1; public double average_intensity2; public double average_intensity3; public int count; }

struct triplelabel { public char tagK1; public char tagK2; public char tagR1; public char tagR2; public double massK1; public double massK2; public double massR1; public double massR2; } }

232

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