Phospho-Proteomic Analysis of Neuroblastoma Tumor Initiating Cell Signaling Pathways: Identification of Src Family and B Cell Receptor Signaling as Novel Drug Targets
by
Milijana Vojvodic
A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Molecular Genetics University of Toronto
© Copyright by Milijana Vojvodic 2010 ! !
Phospho-Proteomic Analysis of Neuroblastoma Tumor Initiating Cell Signaling Pathways: Identification of Src Family and B Cell Receptor Signaling as Novel Drug Targets
Milijana Vojvodic
Master of Science
Department of Molecular Genetics University of Toronto
2010
Abstract
Neuroblastoma (NB) is the most common extra-cranial solid tumor in children. Recently discovered neuroblastoma tumor-initiating cells (NB-TICs) have many properties of cancer stem cells and form tumors with as few as 10 cells. To elucidate the signaling pathways driving NB-
TIC survival and proliferation, we surveyed the phospho-tyrosine containing subset of the NB-
TIC proteome. Over 300 phosphorylated proteins were identified, including 21 tyrosine kinases of which several belong to the Src kinase family. Using bioinformatics tools, several hematopoietic signaling pathways were identified, including the B cell receptor (BCR) pathway.
Further proteomic approaches substantiated molecular hematopoietic features in NB-TICs.
Inhibitors of BCR proximal kinases SYK and SFKs were cytotoxic to NB-TICs. Clinically utilized inhibitors of SFKs induce apoptosis in NB-TICs. Targeting hematopoietic survival pathways in NB-TICs from the bone marrow, which have thus far not been predicted to play a role in this neural malignancy, may provide new drug therapies for NB.
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Acknowledgements
I would like to thank my supervisors, Dr. David Kaplan and Dr. Mike Moran for entrusting me with this project and their ongoing support throughout my studies. I would also like to thank my committee members Dr. Anne-Claude Gingras and Dr. Freda Miller for their guidance.
I wish to acknowledge members of the Kaplan and Moran labs for their support: Dr. Natalie Grinshtein, Dr. Jiefei Tong, Mr. Paul Taylor, Dr. Kristen Smith, Dr. Loen Hansford, and Ms. Tatiana Lipman. I would also like to thank our collaborators: Ms. Olena Morozova, Dr. Marco Marra, Dr. Thomas Kislinger, Ms. Kim Blakely, and Dr. Jason Moffat.
Many thanks to my parents for their immeasurable kindness and support.
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Table of Contents
! Abstract...... ii Acknowledgements ...... iii Table of Contents ...... iv Abbreviations ...... vi List of Tables ...... vii List of Figures...... viii List of Appendices...... x Chapter 1: Introduction ...... 1 1.0 Neuroblastoma – An overview...... 1 1.0.0 Introduction, clinical presentation and disease etiology...... 1 1.0.1 Current clinical therapies and challenges in neuroblastoma treatment...... 4 1.1 Cancer stem cells and neuroblastoma tumor initiating cells ...... 5 1.1.0 The cancer stem cell theory ...... 5 1.1.1 Isolation and characterization of neuroblastoma tumor initiating cells...... 8 1.2 Qualitative phospho-proteomics – An overview ...... 10 1.2.0 An introduction to phospho-proteomics ...... 10 1.2.1 Phospho-peptide and protein enrichment methods...... 11 1.3 The Src kinase family: Signaling effectors and oncogenes...... 14 1.3.0 Src family kinases: Tyrosine kinase structure and function ...... 14 1.3.1 Src family kinases in immune receptor signaling...... 17 1.3.2 Src family signaling in cancer initiation and progression...... 20 1.4 Rationale and aims of thesis...... 21 Chapter 2: Materials and Methods ...... 23 2.0 Patient samples, cell lines and culture conditions...... 23 2.1 Mass spectrometry...... 24 2.1.0 Phospho-tyrosine enrichment and mass spectrometry...... 24 2.1.1 Membrane fraction enrichment and 2D mass spectrometry ...... 27 2.1.2 Data collection and interpretation: GO Annotations, DAVID and Ingenuity analyses29 2.2 Cellular proliferation assays: quantifying growth response in differential growth factor media...... 30 2.3 Testing small molecule kinase inhibitor efficacy ...... 31 2.3.0 Resazurin reduction cytotoxicity assays ...... 31 2.3.1 Confirming SFK and Erk1/2 kinase inhibition ...... 32 2.4 Assessment of concurrent SFK phosphorylation by immunoprecipitation...... 33 2.5 Apoptosis assays: Caspase 3/7 activation, PARP cleavage and phosphatidylserine detection...... 34 2.5.0 Caspase-3/7 activity assay ...... 34 2.5.1 Poly (ADP-ribose) polymerase (PARP) cleavage assays ...... 34
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2.5.2 Detection of phosphatidylserine surface expression by annexin-V...... 35 Chapter 3: Results...... 36 3.0 Phosphoproteomics screen of the NB-TIC phospho-tyrosine proteome...... 36 3.0.1 Results of phospho-tyrosine screening of NB-TICs ...... 36 3.0.2 Identification of pY signaling pathways in NB-TIC pY dataset...... 41 3.0.3 Identification of NB-TIC cell surface markers by mass spectrometry ...... 48 3.1 Identification of phosphorylated protein kinases in NB-TICs...... 51 3.1.0 Expression of tyrosine phosphorylated protein kinases in the NB-TIC pY dataset .... 51 3.0.1 Identification of activated Src family kinases in NB-TICs...... 55 3.2: Inhibition of NB-TIC cell growth by Src and SYK kinase inhibitors...... 61 3.2.0 Small molecule SFK inhibitors reduce NB-TIC cell growth...... 61 3.2.1 Validation of SFK inhibition and assessing MAPK activity downstream of SFKs .... 65 3.2.2 Inhibitors of BCR effector kinase SYK reduce NB-TIC growth...... 69 3.3: SFK inhibitors bosutinib and dasatinib induce apoptosis in NB-TICs ...... 71 3.3.0 SFK inhibitor bosutinib potently induces apoptosis in NB-TICs ...... 71 3.3.1 Clinically utilized SFK inhibitor dasatinib is a moderate inducer of apoptotic responses in NB-TICs...... 77 Chapter 4: Discussion...... 81 4.0 Hematopoietic signaling and NB-TICs from bone marrow metastases...... 81 4.0.0 Molecular features of immune system cells expressed in NB-TICs...... 81 4.0.1 Targeting the BCR signaling pathway through SYK tyrosine kinase inhibition...... 82 4.0.2 Hematopoietic signaling pathways in NB-TICs: NB-TIC pY dataset identifies CD19/Lyn signaling pathway ...... 83 4.0.3 How did NB-TICs acquire hematopoietic features? The hypothesis of cell fusion.. 86 4.1 Src family effector kinases: Novel therapeutic targets in neuroblastoma...... 88 4.1.0 The role of Src family kinases in neuroblastoma and NB-TICs...... 88 4.1.1 Treatment of neuroblastoma bone marrow metastatic disease: Utilization of SFK inhibitors dasatinib and bosutinib ...... 90 4.2 Future Directions: Characterization and exploitation of the hematopoietic phenotype of NB-TICs...... 93 4.2.0 Validating BCR signaling: The link between SFK, SYK and B cell membrane receptors...... 94 4.2.1 Testing hematopoietic signatures in non-bone marrow derived NB-TIC lines ...... 95 4.2.2 Testing the efficacy of SFK inhibitors dasatinib and bosutinib in vivo...... 96 4.2.3 Assessing the role of Insulin receptor and Fibroblast growth factor receptor pathways in NB-TICs ...... 97 Bibliography ...... 98 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset...... 113
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Abbreviations
ALCL Anaplastic large cell lymphoma ALK Anaplastic lymphoma kinase AML Acute myeloid leukemia BCR B-cell receptor BEMA ß-elimination/Michael addition bFGF basic fibroblast growth factor CML Chronic myelogenous leukemia CSC Cancer stem cell DAVID Database for Annotation, Visualization and Integrated Discovery EGF Epidermal growth factor EGFR Epidermal growth factor receptor FAK Focal adhesion kinase FGFR Fibroblast growth factor receptor IMAC Immobilized metal Ion affinity chromatography INSS International neuroblastoma staging system IP3 Inositol trisphosphate IRR Immune recognition receptors ITAM immunoreceptor tyrosine-based activation motif KEGG Kyoto Encyclopedia of Genes and Genome LC-MS/MS Liquid Chromatography- Tandem Mass Spectrometry MeCN Acetonitrile MOAC Metal oxide affinity chromatography MRP1 Multidrug resistance protein 1 MuDPIT Multidimensional Protein Identification Technology NB Neuroblastoma NB-TIC Neuroblastoma tumor initiating cell NGF Nerve growth factor NOD/SCID Non-obese diabetic/severe combined immunodeficient NT3 Neurotrophin-3 PI Propidium Iodide PI3K Phosphoinositide 3-kinase PTP Protein Tyrosine Phosphatase p-S/T Phospho-serine/threonine p-Y Phospho-tyrosine SFK Src family kinase SH2 Src homology domain 2 SH3 Src homology domain 3 SH4 Src homology domain 4 SYK Spleen tyrosine kinase TCR T-cell receptor TFA Trifluoroacetic acid TKI Tyrosine kinase inhibitor
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List of Tables
Table 1 Clinical features of NB-TICs used in this study………………………... 24 Table 2 Summary of phospho-proteomic screening of 6 NB-TIC lines……...... 39 Table 3 KEGG pathway assignments across the NB-TIC pY protein dataset….. 45 Table 4 Membrane and whole cell fraction proteomics identifies surface markers characteristic of hematopoietic lineage cells in NB-TICs…….. 50 Table 5 SFK peptides identified by phosphoproteomics across NB-TIC lines NB12, NB88, NB122R, NB61 and NB25……………………………… 57
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List of Figures
Figure 1 Representation of phospho-tyrosine peptide enrichment and identification procedure developed by Rush et al ……………………..... 14 Figure 2 Structural domains of Src family kinases and their putative function in kinase interaction and activity…………………………………………… 17 Figure 3 Cellular distribution and function of tyrosine phosphorylated proteins identified by phospho-proteomic screening of NB-TICs………………... 40 Figure 4 Ten high-probability pathways identified across NB-TIC pY protein dataset by Ingenuity Pathway Analysis………………………………….. 44 Figure 5 NB-TICs respond to growth factors bFGF and insulin with an increase in proliferation…………………………………………………………… 46 Figure 6 NB-TICs express numerous phosphorylated effectors of BCR signaling as annotated by the KEGG database……………………………………... 47 Figure 7 Protein kinases identified by phosphoproteomics across NB-TIC lines…. 53 Figure 8 NB-TICs express effector kinases Lyn, LCK, Fgr, SYK, and BTK downstream of the B-Cell, T-Cell and Fc-! receptors…………………… 53 Figure 9 Sequence alignment of activation loops in mammalian SFKs Lyn, HCK, LCK, BLK, Src, Yes and Fgr depicts non-identical sequence homology in tryptic fragment containing pY416 residue for all SFKs except Src/Yes and HCK/Lyn…………………………………………………… 58 Figure 10 Representative MS/MS spectra of SFK activation loop tryptic fragments suggest that individual SFKs are in an activated state in NB-TICs……… 59 Figure 11 Reciprocal IP approach confirms expression and phosphorylation of SFKs Lyn, HCK, Fgr, Src and LCK in NB-TICs………………………... 60 Figure 12 Small molecule SFK inhibitors SU6656, PD18970, bosutinib (SKI-606) and dasatinib (BMS-354825) potently inhibit NB-TIC growth………….. 61 Figure 13 SFK inhibitor SU6656 induces enlargement of cell volume in individual NB-TICs.………………………...………………………...……………... 64 Figure 14 Src family kinase inhibitors reduces phosphorylation in the activating kinase domain of the SFKs………………………………………………. 67
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Figure 15 SFKs inhibitors PD180970 and dasatinib decrease levels of active ERK1/2………………………………………………………………...... 68 Figure 16 SYK Kinase inhibitors Rigel 406, ER 25319 and BAY 61-3606 inhibit NB-TIC growth in vitro………………………………………………...... 70 Figure 17 Bosutinib increases Caspase 3/7 activity in NB88 cells…………………. 73 Figure 18 Bosutinib induces Poly ADP-ribose polymerase (PARP) cleavage in NB-TICs………………………………………………………………….. 74 Figure 19 Bosutinib potently induces early apoptosis in NB-TICs as indicated by the exposure of phosphatidylserine lipids on the cell surface…………… 76 Figure 20 Dasatinib increases caspase 3/7 activity in NB88 cells………………….. 78 Figure 21 Dasatinib induces Poly ADP-ribose polymerase (PARP) cleavage in NB-TICs………………………………………………………………….. 78 Figure 22 Dasatinib induces early apoptosis in NB-TICs as indicated by surface exposure of phosphatidylserine………………………………………….. 79 Figure 23 Proposed tyrosine phosphorylation relay model downstream of BCR accessory receptor CD19 and Lyn in NB-TICs………………………….. 85
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List of Appendices
Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset… 113
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Chapter 1: Introduction
1.0 Neuroblastoma – An overview
1.0.0 Introduction, clinical presentation and disease etiology
Epidemiology and clinical presentation
Neuroblastoma (NB) is an extra-cranial heterogeneous tumor of the sympathetic nervous system (1). The first description of pediatric tumors under the term ‘neuroblastoma’ was made by Dr. James Homer Wright of the Massachusetts General Hospital as early as 1910 (2). Today approximately 600 new cases are diagnosed in the US and 65 in Canada each year, rendering this tumor the most common extra-cranial pediatric cancer (1; 3). In 95% of cases, neuroblastoma is diagnosed before the age of 5 years where it leads to 15% of all cancer-related fatalities in infancy and childhood (4). Despite a century of extensive clinical and basic research efforts, the biology and clinical progression of the disease continues to present a multi-faceted clinical enigma in pediatric oncology.
Clinical presentation
Neuroblastoma belongs to the family of neuroblastic tumors that also includes ganglioneuroblastoma and ganglioneuroma (5). It is believed to arise from primordial neural crest cells, which form part of the sympathetic nervous system during embryonic development
(1). As part of the neuro-endocrine cellular lineage, neuroblastoma typically presents as a primary tumor in the abdomen, commonly the adrenal medulla (1). High-risk disease is also associated with disseminated tumors in distal regions with sympathetic nervous system innervations such as the neck and chest (6). Approximately 70% of patients present with
! 1 ! ! metastases at diagnosis (1). Neuroblastoma metastases can take lymphatic as well as hematologic routes often leading to widespread infiltrations in the liver, spleen, lymph nodes and bone marrow, amongst other sites, in advanced stage disease (1).
Neuroblastoma presents with vastly heterogeneous clinical behavior across the pediatric patient pool. In recognition of this diversity of manifestations and outcomes of the disease, the
International Neuroblastoma Staging System (INSS) was developed to classify patients into increasing risk stages from 1 to 4 and 4S (as described in (4) and (6)). The highest risk category of neuroblastoma, stage 4, continues to be one of the most challenging problems in current pediatric oncology with a survival rate of less than 30% (2). It is characterized by primary tumors as well as bilaterally disseminated lesions including infiltrations of distal lymph nodes and bone (4). Interestingly, stage 4S infant patients that are under 1 year of age and that have a localized primary tumor and limited dissemination have a high incidence of spontaneous tumor regression without surgical or chemotherapeutic intervention (1; 4). Neuroblastoma is thus a phenotypically diverse disease and requires a stage-specific therapeutic protocol to derive the most effective treatment.
Etiology
Currently, familial neuroblastoma comprises less than 5% of the total patient population
(4). In the case of familial neuroblastoma the disease phenotype has been linked to autosomal dominant transmission in accordance with a two-hit model of tumor occurrence (1). Studies on the PHOX2B gene suggested mutations in this gene to be linked to a small subset of familial neuroblastomas that are also affiliated with other sympathetic nervous system disorders (7).
More recent genetic analyses have associated activating mutations in the Anaplastic Lymphoma
Kinase (ALK) gene on chromosome 2p23 with familial neuroblastoma, identifying this as the
! 2 ! ! first hereditary oncogene in neuroblastoma (8; 9). Interestingly, there are no etiological factors, environmental or innate, that have been conclusively linked to be causative of sporadic, non- familial disease. With sporadic neuroblastoma comprising over 95% of new diagnoses, the need to identify new genes and proteins linked to the formation of this tumor is an imperative line of investigation (4).
Prognostic features and pathobiology
Several prognostic features are currently employed in order to resolve possible disease outcomes. The transcription factor MYCN is the most well studied oncogene in neuroblastoma.
MYCN is amplified across approximately 25% of neuroblastoma cases across all stages and age groups (6). As a prognostic marker, the amplification of the MYCN gene is one of the most important molecular features in neuroblastoma, correlating with rapid disease progression and poor prognosis (10). MYCN amplification is associated with characteristic transcriptome profiles across tumor samples, where it has been shown to be involved in the up-regulation of multidrug- resistance associated protein 1 (MRP) thereby influencing cellular resistance to cytotoxic drugs, and MDM2, an anti-apoptotic cell cycle regulator (4; 10; 11). Thus considerable focus has been placed on characterizing oncogenic gene signatures regulated by MYCN.
As of February 2000, literature has supported evidence for approximately 130 genetic and biological markers in neuroblastoma that hold potential prognostic value in addition to MYCN
(12). Some of the most valuable genetic markers include loss of heterozygosity on chromosome
1p in the 1p36 region, which occurs in 70% of patients, as well as variable deletions in chromosome 14q and 11q, which also strongly correlate with unfavorable outcome (4). Clinical attributes that are linked to unfavorable disease prognosis include patient age (>18months) and bilateral tumor dispersion (4). Histological analyses that are used for prognostic assessments
! 3 ! ! include evaluations of tumor stroma levels and mitosis-karyorrhexis index as part of the Shimada classification system (13; 14). Cell surface markers have also been associated with unfavorable prognosis, including the receptor tyrosine kinase TrkB and surface glycoprotein CD44 (13). It is therefore of significant importance to uncover further oncogenic factors for which a cellular mechanism can be accurately defined and linked to disease progression.
1.0.1 Current clinical therapies and challenges in neuroblastoma treatment
One of the greatest challenges of high-stage neuroblastoma treatment is the resistance to chemotherapy as well as frequent patient relapse. Current therapeutic schemes for high-risk neuroblastoma are typically comprised of multi-nodal approaches that include surgery, radiotherapy as well as immuno- and chemotherapy. The latter two approaches are typically administered as part of a combinatorial treatment regiment (6).
Common chemotherapeutic agents against neuroblastoma include alkylating agents such as melphalan, cyclophosphamide and ifosfamide, other DNA cross-linking agents including carboplatin and cisplatin, microtubule inhibitors paclitaxel and vindesine, and DNA intercalating compounds such as doxorubicin (1; 6). Differentiation therapy using 13-cis retinoid acid has also been incorporated in treatment plans after myoablative therapy and during remission (15). Such dose-intensive chemotherapy regiments followed by myoablative treatments have serious toxic side effects with long term consequences, such as hearing and vision problems, to which the pediatric patient pool is particularly sensitized (15; 16).
Immunotherapy using Anti-GD2 antibody has been utilized against disseminated tumors with positive outcomes for patients with post-surgical recurrent disease (2; 6). GD2 is a cell surface receptor with restricted tissue expression that is highly abundant on most neuroblastoma cells and can thus be used for selective therapy (1; 2). The anti-GD2 monoclonal antibody 3F8 is
! 4 ! ! cytotoxic to NB cells by activating the complement system as well as lymphocytes and granulocytes (2). While GD2-directed immunotherapies have not been found to hold long-term risks, short-term side effects include allergic reactions in children (2). Still, despite combinatorial treatments, a large number of high-risk patients relapse following standard therapies, making the discovery of novel treatment approaches for high-risk disease a necessary priority.
1.1 Cancer stem cells and neuroblastoma tumor initiating cells
1.1.0 The cancer stem cell theory
Identification of cancer stem cells
Over the past four decades, increasing understanding of tumor biology has given rise to postulations that tumor heterogeneity is based on a finite functional hierarchy of cancer cells. In the 1960s, pioneering studies by W.R. Bruce and H. Van Der Gaag quantified that only a subset of cells in a murine lymphoma had tumorigenic potential (17). This led to the hypothesis that only a fraction of cancer cells have self-renewal and proliferative abilities that allow them to initiate, maintain and facilitate the recurrence of cancer. Today, these fractions of cells are termed cancer stem cells (CSCs).
The modern cancer stem cell theory predicts that a subset of tumor-initiating cancer stem cells form the apex of a hierarchy of cells in the cancer population and are distinguishable from the rest of the population by the functional features of self-renewal and differentiation (18; 19).
Cancer stem cells are therefore a self-sustaining population. In turn, the phenotypic heterogeneity of tumors is postulated to be established by differentiation of cancer stem cells into lineage-restricted progenitor cells, which divide to give rise to the terminally differentiated cell
! 5 ! ! types comprising the bulk population (19; 20). The latter cell types do not have oncogenic potential, and hence will not establish tumors in vivo.
The experimental premise of the cancer stem cell theory is based on the ability to purify
CSCs by their physical and/or functional characteristics, such as a cell surface markers or dye exclusion (18; 21). Importantly, these cell fractions have been shown to have a higher tumor initiating frequency than the bulk cells from neoplasms. Thus, the efficient enrichment of a CSC- containing population strongly argues that tumorgenicity is not a stochastic property, but rather an inherent cellular attribute that can be selected for within a group of cancer cells (19).
A second imperative requirement to the study of CSC biology is the development of a reliable assay to quantify CSC tumorgenicity and proliferation. Current standard in vivo assays employ immunocompromised rodents such as non-obese diabetic/severe combined immunodeficiency (NOD/SCID) or NOD/SCID interleukin-2 receptor gamma chain null (Il2rg-/-) mice for xenografts of human cancer cells. NOD/SCID mice are entirely ablated in innate and acquired immunity, thus allowing for the study of tumor formation in xenograft assays of human cancer cells (22; 23).
Using the aforementioned techniques, the first cancer type upon which the CSC theory has been established is acute myeloid leukemia (AML) (24). A cancer of the hematopoietic system, AML has been found to contain a CD34(+)/CD38(-) population of cells that are enriched in tumorigenic potential versus their CD34(-)/CD38(+) counterparts. The presence of cancer stem cells has also been established in solid tumors, including breast, brain, colon and pancreatic cancers. Al-Hajj et al found that breast cancer stem cells are enriched in a CD44(+)/CD22(-) population, comprising 1%-10% of the total tumor population (25). Furthermore, as few as 200
CD44(+)/CD22(-) cells were sufficient to establish tumors in vivo within 6 months, this being a
! 6 ! !
1000-fold enrichment over the CD44(-)/CD22(+) population (25). Pancreatic CSC have been
hi enriched in a CD44(+)/!2b1 /CD133(+) population, where tumor establishment was achieved with as few as 500 cells (26).
Cancer stem cells as therapeutic targets
The cancer stem cell theory predicts that unless the cancer stem cell pool is eradicated by a given set of therapeutic approaches, the cancer will recur. The current paradigm of cancer therapy is focused on reducing the cancer burden in a patient. The success standard of solid tumor chemotherapeutics is in fact measured by quantitative parameters of tumor regression
(27). It follows that such standards of therapeutic success are irrelevant when assessing patient long-term survival unless the pool of CSCs is completely ablated. Indeed, many proponents of the cancer stem cell theory argue that the presence of CSCs in residual disease explains the frequent relapse in patients following surgery, radiation and chemo- or immunotherapies (20; 21;
28).
It is believed that CSCs are inherently more resistant to standard therapies. This feature can in part attributed to their functional similarities with stem cells of normal development and homeostasis. It is known that adult tissue-specific stem cells such as those found in the bone marrow are more resistant to apoptosis and have a slower cell cycle than their differentiated progeny, allowing them to be less sensitive to cell death induced by chemotherapeutic agents and radiation (28). This quiescent phenotype is also predicted to be shared by CSCs, leading to tumor dormancy during remission (28).
Enhanced resistance of CSCs to molecular drugs and radiation has been established for solid tumors and blood malignancies such as chronic myeloid leukemia, brain and lung cancer
(29-31). Bao et al characterized the resistance of the CSC containing the CD133(+) cell
! 7 ! ! population in glioblastoma multiforme to radiation both in vitro and in vivo, which has been attributed to an increase in DNA damage checkpoint response and repair (32). For the treatment of chronic myeloid leukemia (CML), the BCR-ABL fusion protein inhibitor imatinib has been used as the standard first line therapy since successful randomized trials in 2003 (33; 31). It has been shown that imatinib targets primarily differentiated CML progenitors, while remaining less toxic to CSCs (34). Thus imatinib treatment of CML patients must be continued indefinitely to prevent disease relapse (29). It is therefore evident that CSC-specific therapy regiments must be developed in order to eliminate the pool of oncogenic cells in residual disease and move from remission to a cure, while keeping in mind that the bulk tumor and more differentiated tumor cells must also be eradicated.
1.1.1 Isolation and characterization of neuroblastoma tumor initiating cells
Extensive metastases and frequent relapse in neuroblastoma patients following aggressive treatments gave rise to early hypotheses that neuroblastoma contains a set of cells that were inherently more resistant to therapeutic regiments and had the self-renewal, proliferation and differentiation properties of cancer stem cells. In 2007, Hansford et al isolated a highly tumorigenic population of sphere-forming cells from bone marrow aspirates of low and high-risk neuroblastoma patients with metastatic disease (35). This population, termed neuroblastoma tumor-initiating cells (NB-TICs), were successfully isolated from patients in various clinical stages of remission and relapse.
NB-TICs are isolated from bone marrow aspirates and expanded under serum-free neurosphere growth conditions containing basic fibroblast growth factor, epidermal growth factor and B27, a multi-factor neuronal growth supplement. Similar to brain, breast and colon
CSCs, NB-TICs proliferate as suspension spheres in vitro. Furthermore, NB-TICs express
! 8 ! ! neuroblastoma makers NB84 and tyrosine hydroxylase as well as the neural crest marker nestin and have genetic alterations commonly seen in neuroblastoma, further establishing this population as neuroblastoma-lineage cells (35).
NB-TICs have been found to have the self-renewal and differentiation properties characteristic of other solid-tumor CSCs. NB-TICs can be successfully differentiated into neuronal lineage cells expressing neural and glial markers, consistent with the presence of these lineages in heterogeneous neuroblastoma tumors (35). Interestingly, NB-TIC lines established from low-risk patients were more likely to differentiate under neurogenic differentiation conditions containing nerve growth factor (NGF) and neurotrophin 3 (NT3) than high-risk NB-
TIC lines. This differentiation potential may thus have potential prognostic value with respect to neuroblastoma staging and outcome. As CSC differentiation approaches are pursued as therapeutic options in other solid tumors, it is reasonable to hypothesize that the resistance of high-risk NB-TICs to differentiation signals is consistent with their heightened clonogenic potential and tumorgenicity (35; 36).
NB-TICs have an enriched tumorigenic capacity. Recent unpublished data has established that at least 1 in 4.5 NB-TICs are tumorigenic in a renal injection orthotopic
SCID/beige xenograft model of tumor initiation compared to several million cells in other models with NB Cell lines (37). In addition to forming tumors in the primary NB site (adrenal medulla), NB-TICs were found in common human metastatic sites, including the liver and kidney in SCID/beige mice (35). Furthermore, the in vivo self-renewal ability of NB-TICs has been successfully established as NB-TIC tumors could be serially re-transplanted and propagated across at least three animals (35).
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NB-TICs pose several advantages for the study of cellular signaling pathways regulating innate CSC properties. The high enrichment of tumorigenic cells in NB-TIC spheres allows for the assessment of signaling pathways unique to tumor-initiating cells, which may be not be evident in fractionated cell populations with a relatively low occurrence of TICs. Furthermore, as a number of NB-TIC lines are bone marrow-derived, putative metastatic signatures inherent to
CSCs may be identified from such NB-TIC populations. Finally, the substantial proliferative potential of NB-TICs in vitro makes them an ideal class of primary cells that can be expanded for experiments requiring large amounts of cellular material, such as small-molecule drug screens and various proteomic techniques.
1.2 Qualitative phospho-proteomics – An overview
1.2.0 An introduction to phospho-proteomics
In cellular signal transduction, over 200 unique types of post-translational modifications are known to influence protein structure and function (38). Of these, protein phosphorylation is predicted to influence one third of the mammalian proteome (39). The phosphorylation of serine, threonine or tyrosine residues in the primary sequence of a protein is accomplished by protein kinases, which are typically themselves targets of this modification in an antonymous or heteronymous manner. Phosphorylation events often have crucial biological implications as they can modify a proteins’ function and stability by influencing protein conformation and protein- protein interactions, amongst others (38). Within this spectrum of phosphorylation-mediated effects, tyrosine phosphorylation is the most tightly controlled, as this modification acts as the regulatory switch for many signaling cascades in biological systems. As such, tyrosine
! 10 ! ! phosphorylation plays a pivotal role in the regulation of cell homeostasis and well as malignant cellular states, such as cancer (40).
Recent advances in proteomics have allowed for the development of novel, large-scale techniques for the study of cellular signaling pathways. The field of phospho-proteomics has greatly advanced due to significant developments in mass spectrometry and informatics over the past two decades. A large fraction of studies in the phospho-proteomics field are based on the
enrichment of phosphate (PO4) containing residues from complex peptide or protein mixtures, such as whole cell lysates, for subsequent identification by tandem mass spectrometry. While phosphate residues may be identified by mass spectrometry without prior enrichment, enrichment steps facilitate a more efficient identification process by increasing the number of phospho sites that can be discerned by mass spectrometry. Mass spectrometry has advantages in comparison with the older techniques of phospho-protein analysis such as 32P orthophosphate kinase assays and 2-D electrophoresis, in that the positional identification of a phosphate group as part of a protein’s primary sequence can be defined (41; 42). This is a significant methodological benefit as it is known that different phospho-sites on a protein have differing effects on protein activity. Thus mass spectrometry offers a high throughput approach to locate individual phosphate moieties as part of a peptide on a proteome-wide scale.
1.2.1 Phospho-peptide and protein enrichment methods
Numerous types of phospho-serine (p-S), threonine (p-T) and tyrosine (p-Tyr) enrichment methods have been developed to survey the cellular phospho-proteome. Metal affinity-based approaches, such as Immobilized Metal Ion Affinity Chromatography (IMAC) and
Metal Oxide Affinity Chromatography (MOAC), are common techniques found in current literature (39). In IMAC enrichment, negatively charged phosphate moieties are attracted to
! 11 ! ! positively charged metal ions such as Fe(III)+ that are chelated to resin (43; 39). Similarly to
metal ions, metal oxides such as TiO2 can also be utilized to enrich for negatively charged phospho residues (40). Chemical modifications are a second cohort of diverse organic and inorganic techniques that can be utilized to isolate phospho-serine/threonine containing peptides.
- Methods such as !-elimination/Michael addition (BEMA) alter the native PO4 group and replace it with a second functional group for which different enrichment methods are available (39).
Phospho-serine and threonine residues can also be enriched by utilizing p-S/T specific antibodies, although due to the small size of p-S/T residues, generic antibodies have been more difficult to develop to these epitopes compared to p-Tyr sites (44). Grønborg and colleagues have utilized anti-p-S/T antibodies to immunoprecipitate whole proteins that were resolved by
SDS PAGE and subsequently identified by mass spectrometry (45). Olson et al adopted a similar approach in which mixtures of p-S and p-T antibodies were employed for immunoprecipitation prior to protein identification (44).
Phospho-tyrosine residues comprise only 2% of the total phospho-proteome and thus their enrichment necessitates a more selective technique for phospho-tyrosine specific studies
(41). Compared to p-S/T residues, the detection of pY moieties by MS is impacted less by the effects of neutral loss due to the expulsion of metaphosphoric acid, although the loss of phosphoric acid has recently been reported for phopsho-tyrosine containing monoanion peptides
(46).
Early methods described by Nollau and Mayer utilized Src homology 2 (SH2) domains to bind pY residues, which are SH2 domain ligands, for site-specific identification of tyrosine phosphorylated proteins (47). Based on this method, commercially available kits utilize SH2 domains for the enrichment of SH2 ligands prior to mass-spectrometry based identification. A
! 12 ! ! chemical modification protocol for pTyr residues has also been described by the Aebersold group, where phospho-tyrosine residues are modified by a series of chemical steps for subsequent capture by acetyl iodine gel affinity columns (48; 49).
The enrichment for phospho-tyrosine containing peptides using immunoaffinity reagents has been supported in large part by the availability of excellent anti-pTyr antibodies that can be utilized for peptide or protein immunoprecipitation. In 2005, Rush et al published an optimized workflow for the efficient enrichment of phospho-tyrosine peptides from trypsin-digested whole cell lysates (50). This technique is based on an immuno-precipitation approach whereby a resin- conjugated phospho-tyrosine specific antibody (pY-100) is used to enrich for pY peptides. The work-flow of the aforementioned technique is depicted in Figure 1. Rush and colleagues successfully adapted this approach to profile oncogenic signaling networks in anaplastic large cell lymphoma (ALCL) cell lines. This study stands as a benchmark for the identification of a significant number of low-abundance phospho-tyrosine residues for the purpose of global phospho-tyrosine profiling. Since 2004, this method has been cited across over 360 publications and was adopted in numerous diverse high-profile studies including global surveys of lung cancer signaling pathways, hematopoietic transformation and phosphorylation patterns of drug resistant kinases (51-53).
! 13 ! !
Figure 1: Representation of phospho-tyrosine peptide enrichment and identification procedure developed by Rush et al (50). Cells are lysed and cysteine residues reduced, alkylated, followed by protein trypsin digestion. Trypsin fragments are purified and enriched for pY-containing residues using a resin-conjugated anti-pY antibody prior to identification by LC-
MS/MS and associated database searches.
1.3 The Src kinase family: Signaling effectors and oncogenes
1.3.0 Src family kinases: Tyrosine kinase structure and function
Protein tyrosine kinases are a family of enzymes that are essential to the regulation of various cellular functions including proliferation, differentiation, migration and cell death. The mammalian genome encodes 90 unique tyrosine kinases that are differentiated by receptor and non-receptor classes (54). Non-receptor tyrosine kinases can be further clustered into subfamilies across 32 proteins that share common domain features. In humans, 10 non-receptor tyrosine kinase subfamilies have been identified which include ABL, ACK, CSK, FAK, FES, FRK, JAK,
! 14 ! !
SRC, TEC and SYK families (54).
c-Src is a member of the SRC tyrosine kinase family and is the oldest non-receptor tyrosine kinase studied in mammals. Since the initial discovery of the c-Src gene in 1976, at least
10 other Src-like tyrosine kinases have been identified in vertebrates: Yes, Fgr, Fyn, Lyn, Blk,
HCK, LCK, Yrk, Frk and Iyk (55-57). Of these, all are represented in the human genome with the exception of Yrk, which is unique to the avian clade (chicken) and Iyk, which is expressed in mice (57). Eight mammalian kinases, c-Src, Yes, Fgr, Fyn, Lyn, Blk, HCK, and LCK are associated by highly conserved primary and tertiary structure and are collectively recognized as the Src family kinases (SFKs).
The expression pattern of SFKs across different cell types varies with each member. Src,
Fyn and Yes share ubiquitous expression across all major tissue types (57). Other family members have more restricted expression predominantly in hematopoietic systems. These include Lyn and Fgr (myeloid and lymphoid), HCK (myeloid), Blk (lymphoid), and LCK
(lymphoid) (58; 57; 59). Interestingly, Lyn and LCK are also expressed in the central nervous system, such as in granule cells in the brain and spinal microglia, respectively, further highlighting the functional versatility of SFKs across different cell lineages (60; 61). The function of individual SFKs may thus dependent on the particular cellular milieu in which the kinase is expressed.
The Src family kinases share highly conserved structural features that define their activity, localization and protein-protein and protein-lipid interactions (Outlined in Figure 2).
In the N-terminus, the cysteine-rich Src Homology 4 Domain (SH4) is the site of enzyme- mediated attachment of myristoyl or palmoyl lipid groups to SFKs (62; 57; 59). Attachment of lipid groups allows for the localization of SFKs to the plasma membrane, where they may be
! 15 ! ! further segregated into lipid rafts. The Src homology 3 (SH3) domain also mediates protein localization by direct interactions with components of the cytoskeleton, such as actin and tubulin filaments, as well as substrates by binding to proline-rich sequences (63). The SH2 domain facilitates protein-protein interactions by binding to highly specific phospho-tyrosine sequences.
As with most tyrosine kinases, SFK catalytic activity is tightly regulated. This is accomplished by discrete modifications of the enzyme’s tertiary structure via the SH2 and SH3 domains (reviewed extensively in (63-66)), as well as phosphorylation of key tyrosine residues.
The catalytic or SH1 domain of all SFKs contains a conserved activation loop, which harbors an auto-phosphorylation tyrosine residue at amino acid 416 (or equivalent homologue for other
SFKs, See Figure 1) (56). Phosphorylation of tyrosine 416 is an indicator of SFK catalytic activity up-regulation (57). Conversely, the c-Src C-terminus contains a negative regulatory site at tyrosine residue 527 whose phosphorylation correlates with low activity. The tyrosine kinase
Csk phosphorylates pY527 while the phosphatases PTP!, SHP2 and PTP1B remove the phosphate moiety, thus establishing a dynamic turnover of inhibitory signals for kinase function
(63). Further modulation of kinase activity may also be accomplished by phospho-mediated modifications in SH2 domain pY sites. For example, pY215 on c-Src, which lies in the SH2 domain, has been found to be phosphorylated by HER2 to increase c-Src activity in breast cancer cell lines (67).
The src family kinases act as central effectors of various cell-signaling relays in non- malignant and malignant cells. One of the most prominent roles of SFKs is coupling of membrane receptor pathways, such tyrosine kinase receptors, interleukin receptors, integrins, G- protein coupled receptors, and immune receptors, with their respective downstream targets (57).
In this function, SFKs act upon a large cohort of substrates, including other tyrosine kinases (ex.
! 16 ! !
FAK, FGFR), serine/threonine kinases (ex. PKC), phosphatases (ex. PTPN6), adaptor proteins
(ex. SHC, CBL), and cytoskeleton/structural components (ex. ezrin, cortactin) (57). Thus, the actions of SFKs result in a diverse array of biological manifestations.
Figure 2: Structural domains of Src family kinases and their putative function in kinase interaction and activity. Adapted from Sanderson and Smith, 1999 (59)
1.3.1 Src family kinases in immune receptor signaling
In the immune system, SFKs play an essential role in relaying downstream signals of immune recognition receptors (IRR). In particular, SFKs associate with receptors that signal through an immunoreceptor tyrosine-based activation motif (ITAM), which upon receptor activation are phosphorylated and recruit effector kinases (68; 69). IRR receptors with ITAMs include the B Cell receptor (BCR), T-Cell receptor (TCR) as well as the Fc" receptor family found in a variety of immune cells such as macrophages, neutrophils and natural killer cells (70;
57; 68). Each receptor type has been found to interact with a specific subset of SFKs through their SH2 domains. The Ig!/! units of the BCR interact primarily with Lyn, Fyn, Fgr, LCK and
! 17 ! !
Blk, while the TCR "-chain and co-receptor CD3 interact with LCK and Fyn. Different sub- classes of the Fc" receptor, such as Fc"RI and FC"RIIA/B, interact with different SFKs depending on the host cell, such as Fyn and Lyn in platelets (57; 68).
In lymphocytes, the BCR signaling complex recruits tyrosine kinases following ligand activation that in turn relay signals essential for B cell survival, proliferation and differentiation.
These kinases mediate B-cell proximal signaling events, which ultimately feed into a number of signaling pathways, including Ras/Raf/MAPK and PI3K/Akt (see Results 3.0.2 Figure 6) (71;
69). Following the initial events of ligand-mediated BCR aggregation and activation, SFKs interact with the ITAM motifs of the Ig! or Ig! units of the BCR, leading to an increase in SFK activity and phosphorylation of other tyrosine resides in the ITAM by SFKs (69). This heightened phosphorylation of membrane proximal regions of Ig! and Ig! recruits and activates additional SFKs in a positive feedback loop. Subsequently, Spleen Tyrosine Kinase (SYK) binds to phosphorylated SH2 binding sites on the ITAM, leading to an increase in SYK kinase activity
(72). The close temporal and spatial activation of SFKs and SYK suggests cross-phosphorylation between these kinases following recruitment by Ig! and Ig!.
Activated SYK relays signals to a number of downstream signaling effectors including
BTK, Vav and GAB/BCAP, initiating distinct signaling axes downstream of proximal BCR events (73; 69):
1) SYK phosphorylates the kinase BTK upon binding to the adaptor molecule BLNK
(74). Syk and BTK further phosphorylate BLNK-bound Phospholipase C-"2 (PLC"2)
(75; 74). Activated PLC"2 cleaves the substrate phosphatidylinositol 4,5-bisphosphate
(PI(4,5)P2) to produce 1,4,5-trisphosphate (IP3) and diacyl glycerol (DAG), thus initiating
further signaling events through these second messengers (75). Activated downstream
! 18 ! !
pathways activated by IP3 and DAG include Ras/Raf/MAPK and PKC/IKK/NF#B,
regulating cellular transcription (69).
2) SYK phosphorylates the adaptor proteins GAB and BCAP (76; 73). Phosphorylation
of these membrane-associated scaffold proteins recruits the p85 regulatory unit of
Phosphoinositide 3-kinase (PI3K) to the plasma membrane (69). This localization allows
for the phosphorylation of the PLC" substrate PI(4,5)P2 by PI3K to generate
phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3). Generation of PIP3 by PI3K leads
to the activation of Akt pathways regulating numerous cell activities including survival,
protein synthesis and energy metabolism.
3) SYK is also an activator of the Vav guanine nucleotide exchange factor and adapter
protein, which has been implicated in the activation of the p38 and JNK1/2 via Rac/cdc42
(69). The primary effects of p38 and JNK1/2 activation are on transcriptional regulation.
As SFKs are the initial kinases that relay the BCR activation signal to downstream effectors such as SYK and BTK, they play crucial roles in both the maintenance and ablation of
BCR signals. Lyn is the best-characterized BCR proximal SFK although Fyn and Blk have also been implicated in proximal BCR signaling (77). Following activation at the BCR complex, Lyn binds to and phosphorylates CD19, a BCR co-receptor (69). The phosphorylation of CD19 by
Lyn recruits PI3K and Vav to the membrane, boosting BCR signal transmission to downstream effectors through PI3K and Vav (78). Evidently, in B cells deficient in CD19, BCR-mediated proliferative responses are ablated (79; 69).
! 19 ! !
1.3.2 Src family signaling in cancer initiation and progression
The first characterized oncogene, v-Src, is a constitutively active counterpart of the wild type tyrosine kinase c-Src. V-Src was first sequenced and characterized from the Rous-sarcoma virus (RSV), a retrovirus that induces mesenchymal tumors of spindle cells in chicken (63). v-
Src expression oncogenically transforms fibroblasts by reducing growth factor dependence and ablating density inhibition, while driving cell cycle progression, proliferation and motility (63;
80). The activation of PI3K/AKT, Jak/Stat3 and Ras/Raf/MAPK pathways by v-Src, amongst others, has been described to drive this malignant cell phenotype.
Interestingly, wildtype c-Src does not have strong inherent transforming ability when transfected into fibroblasts, nor does it appear to accumulate activating mutations in a majority of studied cancers (64). Rather, c-Src expression and activation levels positively correlate with neoplasmic progression from precancerous cells to carcinoma and finally metastasis (64; 66). To this extent, the role of SFKs has been suggested to be that of an activated facilitator of aberrant cellular phosphorylation events that drive tumor progression (63). This view is supported by studies showing the interaction between c-Src with over-expressed receptor tyrosine kinases in cancer cell lines, where c-Src acts as a substrate as well as an activator of receptor tyrosine kinases during malignant proliferation. For example, fibroblasts engineered to over-express both c-Src and EGFR undergo increased cell proliferation in vitro and tumorigenicity in vivo due to synergistic activity of both kinases in activating downstream targets of the EGFR signaling pathway (81; 82). Indeed, c-Src and other SFKs have been linked to the progression of a range of solid tumor types including breast, pancreatic, colon and ovarian cancers (83).
c-Src has also been implicated in the progression of neuro-endocrine cancers (66). In neuroblastoma, numerous studies support the role of c-Src in regulating structural features of NB
! 20 ! ! cell lines. Early studies showed that overexpression of the neuronal-specific src isoform pp60c-src led to an increase in p130-Cas phosphorylation that regulates axonal growth cone formation during NB differentiation (84). In the neuroblastoma cell line NB8, !4 !1 integrin, which is expressed in tumors and metastases, activates c-Src to facilitate cell motility (85). c-Src has also regulates NB cell adhesion by activating the Erk1/2 pathway via caspase-8 (86). Interestingly, a recent study showed that dual inhibition of c-Src and focal adhesion kinase (FAK) decreased cell attachment and viability while increasing apoptotic responses (87). Further studies are required to define the mechanism of c-Src-regulated cell viability in NB.
1.4 Rationale and aims of thesis
The current prognoses for advanced stage neuroblastoma in children above 18 months are poor. The high metastatic potential of neuroblastoma merits the study of signaling pathways that allow for the survival and proliferation of cancer stem cells in their metastatic niche. Considering that over half of patients present with distal metastases at diagnosis, the isolation and characterization of tumor initiating cells from the bone marrow is an important research avenue to identify new treatment options for metastatic disease. In this regard, high-throughput mass spectrometry-based phospho-proteomics offers an attractive platform to survey the phospho- tyrosine proteome in NB-TICs. In light of recent clinical successes of tyrosine kinase inhibitors such as Imatinib (Gleevec), surveying the phospho-tyrosine proteome will facilitate the identification of ‘druggable’ kinase effectors in NB-TICs that regulate cell survival and proliferation.
This Master’s thesis has the following aims:
! 21 ! !
Aim 1: To survey the phospho-tyrosine proteome of bone marrow derived NB-TICs. I propose that by coupling immuno-affinity based enrichment of pY peptides with mass spectrometry- based protein identification, a non-biased and inclusive list of phospho-tyrosine modified proteins in NB-TICs can be derived.
Aim 2: To identify activated signaling pathways that are candidate regulators of cell viability by bioinformatics approaches. I aim to identify key signaling effectors in NB-TICs which modulate cellular survival and proliferation and that can be targeted by small-molecule tyrosine kinase inhibitors (TKIs).
Aim 3: To test the efficacy of specific TKIs on NB-TICs and the mechanism by which TKIs reduce cell viability and proliferation in NB-TICs.
This research project offers the unique opportunity to study the mechanism of human cancer stem cell signaling using a large-scale phospho-tyrosine focused approach. Current literature offers only few applications of mass spectrometry for the study cancer stem cells. This research project is the first comprehensive survey of the phospho-tyrosine proteome of a human cancer stem cell type. Furthermore, this study is at the forefront of cancer stem cell-targeted therapeutics research as it aims to uncover specific kinase-regulated signaling pathways of cancer stem cells derived from the bone marrow for the purpose of identifying novel kinase inhibitors that can be applied clinically.!
! 22 ! !
Chapter 2: Materials and Methods
2.0 Patient samples, cell lines and culture conditions
All NB-TIC lines used in this study were derived from bone marrow aspirates or primary tumor samples obtained from patients at the Hospital for Sick Children (Protocol 1000006069).
The NB-TIC lines utilized in this study as well as the patient background are listed in Table 1
(Information obtained from Loen Hansford, The Hospital for Sick Children). Currently we have not established primary bone-marrow NB lines that lack tumor initiating cell properties and that can be expanded in vitro for mass spectrometry or biochemical analyses. Therefore, all outlined experiments were performed on bone marrow derived NB-TIC lines, with the exception of tumor-derived NB153.
NB-TIC lines NB12, NB25, NB61, and NB88R were grown at 37°C/5%CO2 in ‘NB-TIC media’ consisting of DMEM/F12 (3:1), 2% B27 supplement (Life Technologies), 20 ng/mL epidermal growth factor (EGF), 40 ng/mL basic fibroblast growth factor (bFGF/FGF2), and 100
µg/mL penicillin/streptomycin (described in (35)). NB153 was cultured in a 3:1 mixture of NB-
TIC media and human skin derived precursor (SKPs) conditioned media until confluency could be established, at which point cells were grown in NB-TIC media only. NB-TIC lines were passaged at confluency by triturating spheroids using a P1000 pipette.
! 23 ! !
NB-TIC Patient age at NB Risk NMYC Clinical state at TIC TIC isolation TIC line diagnosis group amplification isolation site morphology NB12 >18months High no Stable relapse Bone marrow spheroid NB25 disease spheroid NB61 spheroid NB88R >18months High no Progressive disease Bone marrow spheroid following multiple relapse NB122R >18months High no Relapsed disease Bone marrow Attached single cells & spheroid NB153 >18months High yes Post chemotherapy Tumor Tight spheroids Table 1: Clinical features of NB-TICs used in this study.
2.1 Mass spectrometry
2.1.0 Phospho-tyrosine enrichment and mass spectrometry
NB-TIC lines were grown to high sphere density in NB-TIC media prior to lysis.
Additionally, for the 3 NB-TIC cell lines NB12, NB25 and NB88, one supplementary analysis
was performed whereby cells were treated with 60µM pervanadate (Na3VO4) for 10 min at
37°C/5% CO2. This was done to inhibit phospho-tyrosine phosphatase (PTP) activity in order to increase basal pY levels in the cells. Following this, the same protocol was followed as with non- treated cells.
Approximately 100 x 106 NB-TIC cells were cultured for a single analysis. Subsequent steps have been adapted and modified from Rush et al and Cell Signaling Technologies (CST)
PY100 Kit (50). NB-TIC cells were collected by centrifugation (3 x 103 g, 3min) and immediately lysed at 4°C in urea buffer containing 9 M urea, 2 mM HEPES buffer (pH 8), 1 mM
!-glycerophosphate, 2.5 mM sodium pyrophosphate, and 1 mM Na3VO4 (CST). The lysate was subsequently sonicated for 3 X 30 s at 15 W and cleared by centrifugation (20 x103 g, 15 min).
! 24 ! !
The disulfide bonds in the cleared protein lysate were broken and covalently modified by carboxamidomethylation. This former was accomplished by treating the lysate with 4.5 mM dithiothreitol (DTT) for 10 min at 60°C followed by alkylation using 10 mM of iodoacetamide for 15 min at ambient temperature. The lysate was diluted from 9 M urea to 2 M to facilitate permissive conditions for trypsin digestion and was treated with 10 µg/ml trypsin/TPCK (CST) overnight at 23°C. If digested lysate was not purified the following day, it was stored at 4°C for a maximum of 2 days.
The trypsin digested peptides were purified using a Sep-Pak C18 resin in a purification column. Briefly, lysate was acidified with TFA and added to hydrated C18 resin in the column and washed in sequence with 5%, 10%, 20% and 30% of acetonitrile (MeCN) in!0.1% triflouroacetic acid (TFA). Pure peptides were eluted in 40% MeCN in!0.1% TFA and lyophilized for two days.
The dried peptides were re-suspended in IAP buffer (50 mM MOPS (pH 7.2), 50 mM
NaCl, and 10 mM sodium phosphate, CST). The solution was cleared by centrifugation (2x103 g,
5 min), adjusted to pH 7 and incubated with anti-pY100 resin (CST) over night at 4°C. The immuno-precipitation was washed twice with IAP buffer and once with water, and pY peptides were eluted with 0.15% TFA and concentrated by vacuum centrifugation.
The purified pY peptides were loaded into a Proxeon nano-LC system, separated over a reverse-phase C18 column and eluted using a MeCN/formic acid gradient. The elutions were timed over 120 min with a flow rate of 250 nL/min. Peptides underwent electrospray ionization
(ESI) into an LTQ-Orbitrap mass spectrometry system (Thermo) for parallel MS and MS/MS analyses. All relevant MS and MS/MS data acquisition parameters are outlined in Tong et al
(88): For MS scans, parent ion m/z values (400-1400 m/z) were generated by the Orbitrap
! 25 ! ! system. The automatic gain control (AGC) target was set at 500 000 with a maximal ion injection time of 500ms, resolution of 60 000 and 1 µscan. Fragment ions for MS/MS analysis in the linear ion trap were generated by collision induced dissociation (CID) using 35% normalized collision energy, where 3 MS/MS scans of the 3 most intense ions were linked to 1 MS scan in the Orbitrap. In the LTQ, MS/MS scans were made with an AGC target of 10,000 and a maximal ion injection of 100ms. An ion intensity of 1000 or greater was set as a requirement to prompt a
MS/MS spectrum. For recurring peptides, a 30 s exclusion for MS/MS was set with an exclusion mass width of 0.05 Da. The exclusion list was set at a maximum of 500. Raw MS files (.raw) were converted to mzXML files using the ReAdW software. !
The extracted mass spectra were analyzed using Sequest (ThermoFinnigan, v. 5) and
X!Tandem (v. 2007.01.01 2) search algorithms. The search algorithms were used to search the
IPI human protein database v. 3-41 (72155 entries) or v. 3.49 (74017 entries). For both Sequest and X!Tandem searches the following parameters were used: Parent ion tolerance was 5.0 PPM
(NB12, NB25, NB61, NB88, NB122), 7.0 PPM (NB122, NB12, NB88 – replicate runs), and
0.0100 Da (NB25-Vanadate, NB12-Vanadate, NB88-Vanadate). Fragment tolerance was set at
0.5 Da. Carbamidomethyl addition to Cysteine (+57) was set as a fixed modification while oxidation on methionine (+16) and phosphorylation on tyrosine (+80) were set as variable modifications. Different software and database versions were used as the samples were sequentially analyzed over a period of one and a half years in 2008/2009, with sequential updates becoming available during this period and applied to each run.
The proteomics software Scaffold2 (v. Scaffold_2_05_02) was utilized for the final compilation of phospho-peptides and proteins. In Scaffold a 95% peptide probability score cutoff was used according to the PeptideProphet statistical algorithm (89; 90). Manual validation of at
! 26 ! ! least 1 unique pY peptide was performed for each protein. Each peptide that was specifically referenced in this text was also manually validated. All phospho-proteomics data generated for
NB-TIC lines NB12, NB25, NB61, NB88, and NB122, including vanadate-treated runs, were compiled into a single Scaffold file containing a total of 305 pY proteins and was termed the
‘NB-TIC pY dataset’. Sample loading for MS and database searches were done with assistance from Mr. Paul Taylor (The Hospital for Sick Children).
2.1.1 Membrane fraction enrichment and 2D mass spectrometry
Crude membrane fraction
NB88 cells were re-suspended and swollen in hypotonic buffer (20 mM Tris buffer pH
7.4, 10 mM KCl, 1 mM PMSF, 5 mM Na3VO4), followed by lysis with dounce homogenization.
The cleared cell lysate was centrifuged (12 x 103 g, 20 min) to collect the crude membrane fraction containing intact nuclei. The crude membrane fraction was re-suspended in urea buffer
(9 M urea, 2 mM HEPES, 2.5 mM sodium pyrophosphate, 1 mM ß–glycerophosphate and 1 mM
Na3VO4, (CST). The fraction was reduced, alkylated, trypsin digested and purified prior to mass spectrometry as described previously (see 2.1.0).
Whole cell fraction
NB88 cells were lysed in urea lysis buffer (9 M urea, 2 mM HEPES, 2.5 mM sodium
pyrophosphate, 1 mM ß–glycerophosphate and 1 mM Na3VO4) and sonicated (3 x 4W for 10 s each). The cell lysate was cleared by centrifugation (20 x 103 g, 15 min, 4°C). The fraction was reduced, alkylated, trypsin digested and purified prior to mass spectrometry as described previously (see 2.1.0).
MuDPIT for membrane enriched and whole cell fractions
! 27 ! !
MuDPIT analyses were performed as described in Taylor et al 2009 (91) with the following exceptions: For the membrane-enriched fraction, approximately 60 $g of protein was analyzed using a linear ion trap LTQ mass spectrometer. For the whole cell fraction, approximately 40 µg of protein digested to peptides was analyzed using an OrbiTrap-LTQ hybrid system. For the whole cell fraction the sample was loaded using a Proxeon HPLC system and for the membrane enriched samples, the sample was manually loaded into a cation exchange/reverse phase column. All samples were subjected to an 8-cycle MudPIT. The instrument methods, including data dependant modes and dynamic exclusion criteria, were adopted from Taylor et al (91): For MS scans, parent ion m/z values were generated in the range of 400-1800 m/z. The AGC target was 500 000 with a maximal ion injection time of 500 ms, resolution of 60 000 and 1 µscan. Fragment ions for MS/MS analysis in the linear ion trap were generated by collision induced dissociation (CID) using 35% normalized collision energy. Six data-dependant MS/MS scans of the 6 most intense ions were linked to 1 high-resolution MS scan. MS/MS scans were made with an AGC target of 100 000 and a maximal ion injection of
100ms. An ion intensity of 1000 or greater was set as a requirement to prompt a MS/MS spectrum. For recurring peptides, a 30 s exclusion for MS/MS was set with an exclusion mass width of 0.05 Da. The exclusion list was set at a maximum of 500, with 1 repeat count , repeat duration of 30 s and exclusion duration of 45 s.
Raw MS files (.raw) were conversted to mzXM files using the ReAdW software. All data was analyzed using Sequest and X!Tandem (v. 2007.01.01.2 for membrane fraction or v.
TORNADO, 2009.04.01.3 for whole cell fraction). Searches were performed using the Human
International Protein Index database v. 3.41 (72155 entries) or v. 3.66 (86845 entries) for membrane and whole cell fractions, respectively. Sequest and X! Tandem were searched with a
! 28 ! ! fragment ion mass tolerance of 0.50 or 0.40 Da for membrane and whole cell fraction, respectively, and a parent ion tolerance of 2.0 or 5.0 Da for membrane or whole cell fraction, respectively. Carbamidomethyl addition to Cysteine (+57) was set as a fixed modification while oxidation on methionine (+16). Proteins were accepted based on the following criteria: At least two peptides per protein were identified with a probability threshold of 95% as derived by the
Peptide Prophet algorithm and an overall protein probability greater than 95.0% using the Protein
Prophet algorithm (89; 90).
The MuDPIT setup and sample loading was done in collaboration with Dr. Thomas
Kislinger (University of Toronto). Sample loading and database searches were done with the assistance of Mr. Paul Taylor (The Hospital for Sick Children).
1D MS analysis for membrane fraction
Approximately 60 $g of protein from the membrane-enriched fraction was loaded on a
Proxeon HPLC system and analyzed on a linear ion trap LTQ mass spectrometer. The data were processed and analyzed the same manner as stated for the MuDPIT run of the membrane- enriched fraction (see MuDPIT for membrane enriched and whole cell fractions).
2.1.2 Data collection and interpretation: GO Annotations, DAVID and Ingenuity analyses
Gene Ontology (GO) annotations
Gene Ontology (GO) annotations of all 305 proteins identified in the NB-TIC pY dataset were imported from the National Center for Biotechnology Information (NCBI) database and added to the dataset through the Scaffold2 proteomics software. Scaffold was used to generate pie charts classifying 1) Biological processes and 2) Cellular components for the pY dataset proteins according to the annotated GO terms for each protein in the dataset.
DAVID Bioinformatics Resource – Identification of KEGG-annotated pathways
! 29 ! !
The Database for Annotation, Visualization and Integrated Discovery (DAVID v. 6.7,
National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases,
NIAID) was utilized to add functional annotations to the NB-TIC pY dataset that can be exploited for pathway enrichment analyses. The list of 305 pY proteins in the NB-TIC pY dataset were entered into DAVID as gene_symbol names. Of these, DAVID could not identify 9 proteins for which a gene_symbol name or symbol could not be matched to the entered IPI protein name. These were thus excluded in subsequent analyses.
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed as part of the DAVID resource. Pathway probabilities are based on the enrichment of a pathway in the NB-TIC pY dataset as compared to the default human gene population background. KEGG signaling pathways were sorted based on highest probability according to P-value (EASE score).
Proteins which mapped to a pathway were visualized on KEGG pathway models in DAVID.
Ingenuity Pathway Analysis
The IPI protein symbols were extracted from the NB-TIC pY dataset and sent to Olena
Morozova (BC Cancer Agency) for analysis using the Ingenuity software (Ingenuity Systems).
The list was searched with default settings for enriched canonical pathways in the Ingenuity database (P<0.05). The threshold of significance for each enriched pathway was computed and corresponds a p-value of 0.05.
2.2 Cellular proliferation assays: quantifying growth response in differential growth factor media
In order to assess differences in NB-TIC proliferation in response to different growth factor treatments, we quantified the number of cells in culture over 7 d using differential growth factor and media formulations. NB12 spheres were dissociated and 10 x 103 cells were plated in
! 30 ! !
1 ml of different media conditions in a 24-well dish. The different media conditions tested were:
(1) Complete media (DMEM/F12 with growth factors EGF, bFGF, and B27 containing insulin— a neurobasal medium supplement), (2) Growth factor deficient media (DMEM/F12 only), (3)
B27 media (DMEM/F12, B27 without insulin), and (4) B27 media with bFGF and Insulin
(DMEM/F12, B27 containing insulin, bFGF). Live cell counts were made by using trypan blue on days 1, 3, 5 and 7 and expressed as the number of live cells per mm3 media.
2.3 Testing small molecule kinase inhibitor efficacy
2.3.0 Resazurin reduction cytotoxicity assays
The SFK inhibitors SU6656, dasatinib, bosutinib and PD180970 and SYK inhibitors
R406, ER27319 and BAY-61 3606 were tested on NB-TICs. Eight step dilutions of the compounds were prepared in DMSO and added directly to cells in culture. The added amount of
DMSO did not exceed 0.5%. To assess the effect of kinase inhibitors on NB-TIC viability and proliferation, we utilized the resazurin-containing reagent Alamar Blue (Invitrogen) as a chemical readout for cellular metabolic activity that can be correlated to cell growth.
NB-TIC spheres were dissociated and 1 x 104 cells were added to 96-well compartments in a total volume of 100 µl per well. For SU6656, dasatinib, boustinib, PD80970, R406 and
ER27319, NB-TICs were incubated with the 8-point dilution series for 3 d at 37°C/5% CO2. For
BAY-61 3606, cells were incubated for 7 d, with a supplement of 50 µl inhibitor diluted in NB-
TIC media at day 4. The Alamar Blue reagent was added at a 1/10 volume directly to the cell culture at day 3 for SU6656, dasatinib, boustinib, PD80970, R406 and ER27319 or day 7 for
BAY-61 3606 and the cells were incubated with the reagent for 4 h at 37°C/5% CO2. The
! 31 ! ! fluorescence readings were made at an excitation of 485 nm and an emission of 530 nm. The fluorescence output was quantified as a fraction of DMSO control. Statistical analyses, including
EC50 values and logarithmic regression curves were generated using Prism 4 software (v. 4.0c,
GraphPad Software Inc.). For NB12 morphology observation following SU6656 treatment, the same protocol was used as for BAY-61 3606 treatment of NB88 cells. All images were taken at day 7.
2.3.1 Confirming SFK and Erk1/2 kinase inhibition
In order to validate that SFK inhibitors SU6656, dasatinib, boustinib, and PD80970 inhibit the activity SFKs and their downstream effector Erk1/2, I assessed the phosphorylation status of the respective kinase domain activation loop residues Y416 (v-SRC equivalent) of
SFKs and Thr202/Tyr204 of Erk1/2. NB88 cells were treated with a dilution series of dasatinib,
boustinib and PD80970 for 10 min and SU6656 for 30 min at 37°C/5%CO2. Treated cells were washed once with HBSS and lysed for 15-20 min on ice using NP40 lysis buffer (10 mM Tris,
pH 8.0, 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 1 mM PMSF, 1 mM Na3VO4 and
COmplete Mini proteinase inhibitor tablet (Roche). The lysate was cleared by centrifugation (12 x 103 g, 10 min, 4°C). Total protein amounts were determined by a standard BCA Assay (Pierce) and 25-30 µg fractions of protein were loaded on 12% SDS gels. The resolved proteins were transferred to nitrocellulose membranes and blocked with 2% Bovine Serum Albumin (BSA,
Fisher). The membranes were probed with anti-pY416 SFK (CST), anti-Phospho-
Thr202/Tyr204 Erk1/2 (CST), anti-Src (CST), and anti-Erk K23 (Santa Cruz). Blots were subsequently probed with either mouse or rabbit HRP-conjugated secondary antibodies (Goat anti-mouse IgG HRP, Thermo, and Goat anti-rabbit IgG, Millipore). Western blots were
! 32 ! ! developed using SuperSignal West Femto Maximum Sensitivity Substrate chemiluminescence reagents (Pierce).
2.4 Assessment of concurrent SFK phosphorylation by immunoprecipitation
To assess for concurrent expression and phosphorylation of individual SFKs, I employed a reciprocal anti-pY and anti-SFK immunoprecipitation approach, where cell lysates are immunoprecipitated using the anti-pY antibody 4G10 and probed with individual SFK antibodies and vice versa in two sets of experiments. NB88 cells were collected by centrifugation (1.2 x 103 g, 3 min) and washed once with cold HBSS buffer. Cells were lysed and protein amounts were determined as outlined previously (See 2.3.1). For the anti-pY or anti-SFK immunoprecipitations, cell lysates were incubated with the following antibodies: anti-pY 4G10 antibody (Upstate), anti-LCK (CST), anti-Src (CST), anti-Lyn (CST), anti-HCK (Santa Cruz), anti-Fgr (CST). For non-specific binding controls, anti-mouse (Santa Cruz) and anti-rabbit IgG
(Santa Cruz) antibodies were utilized for immunoprecipitations in parallel. The antibodies were incubated with the cell lysate for 2 h at 4°C. Subsequently, 50% protein-A-agarose slurry P-3391
(Sigma) was added to all lysates and incubated at 4°C for an additional 1 h. The protein-A-
agarose resin was washed 3 times with cold lysis buffer and once with cold H2O. SDS-PAGE loading buffer was added directly to the resin, incubated at 95°C for 5 min and loaded on a SDS-
PAGE gel for western blotting. Immunoprecipitates were resolved on a 12% SDS gel, and processed as previously (See 2.3.1). Anti-4G10 immunoprecipitates were probed with the individual SFK antibodies listed above. Similarly, the anti-Lyn, anti-LCK, anti-Src, anti-HCK, and anti-Fgr immunoprecipitates were probed with the anti-4G10 antibody listed.
! 33 ! !
2.5 Apoptosis assays: Caspase 3/7 activation, PARP cleavage and phosphatidylserine detection
2.5.0 Caspase-3/7 activity assay
NB88 spheres were dissociated and seeded at 100 x 103 in 100 µl of NB-TIC media containing bosutinib or dasatinib in a 96-well dish. NB-TICs were treated with 5 µM bosutinib
for 0, 1 h and 25 h or with 5 µM or 10 µM dasatinib for 0 h, 1 h, 24 h and 48 h at 37°C/5%CO2.
A 1:100 dilution of caspase peptide substrate was prepared with Apo-ONE® Caspase-3/7 buffer as per manufacturer’s instructions (Promega). An equal volume of caspase peptide substrate/Apo-ONE® Caspase-3/7 buffer reagent was added to the cells, and the mixture was incubated for 4h at room temperature on a plate shaker. Caspase-3/7 activity was determined by quantifying the fluorescence signal following sample excitation at 485 nm and emission at 530 nm. All fluorescence values were corrected for the fluorescence units read from media only.
The statistical significance of increased caspase 3/7 activity was established by a standard
Student’s T-test of corrected fluorescence values using Prism 4 software.
2.5.1 Poly (ADP-ribose) polymerase (PARP) cleavage assays
NB88 spheres were dissociated and 500 x 103 cells were treated with bosutinib (0.2 µM,
2.0 µM) or dasatinib (0.2 µM, 2 µM, 10 µM or 20 µM) and vehicle only (DMSO) in 1 ml NB-
TIC media. Cells were incubated for 48 h at 37°C/5%CO2. Cellular lysate for each sample was obtained using NB40 buffer, which was subsequently resolved on SDS-PAGE, transferred to nitrocellulose and blocked with 2% BSA as described previously (See 2.3.1). Blots were probed with anti-cleaved PARP (Asp214) (Cell Signaling) and anti-Erk1/2 K23 (Santa Cruz) antibodies.
Secondary antibodies and developing reagents were utilized as previously described (See 2.3.1).
! 34 ! !
2.5.2 Detection of phosphatidylserine surface expression by annexin-V
NB88 spheres were dissociated and 800 x 103 cells were treated with 0.2 µM, 2.0 µM, 5.0
µM, and 10 µM bosutinib or 0.2 µM, 2.0 µM, 5.0 µM, 10.0 µM and 20 µM dasatinib and vehicle only (DMSO) in 1.6 ml of NB-TIC media in a 12-well dish. Bosutinib treated cells were incubated for 4 h, 8 h and 24 h while dasatinib treated cells were incubated for 24 h. 200 x 103 cells were re-suspended in annexin-V Binding Buffer (BD Biosciences). The suspension was incubated with Propidium Iodide (PI) Staining Solution and FITC conjugated Annexin-V (both
BD Biosciences) for 15 min at room temperature in the dark and analyzed by flow cytometry within 1 h of staining. The fraction of annexin-V positive, PI negative cells was quantified as early apoptotic cells and analyzed statistically for significance by 1-way ANOVA (Tukey Test) using Prism 4 software.
! 35 ! !
Chapter 3: Results
3.0 Phosphoproteomics screen of the NB-TIC phospho-tyrosine proteome
3.0.1 Results of phospho-tyrosine screening of NB-TICs
To generate a comprehensive list of phospho-tyrosine containing proteins across different
NB-TIC lines, a total of 5 bone-marrow derived lines established from three individual stage 4, non-NMYC amplified, relapsed patients were utilized for phosphoproteomics (Table 2). A sixth
NB spheroid line, NB153 was established from a post-chemotherapy tumor sample and was screened for the purpose of qualitative comparison of identified pY proteins. Two technical
(NB12) or biological (NB88, NB122) replicates were performed for at least one line established from each patient (Table 1). All NB-TIC lines were maintained in serum-free media supplemented with growth factors including EGF, basic FGF and insulin, thereby ensuring activation of cell proliferation signaling pathways mediated by these mitogens.
In addition to performing phospho-proteomics on NB-TICs grown in the above growth- factor conditions, three TB-TIC lines (NB12, NB25 and NB88) were also treated with the
general tyrosine phosphatase inhibitor pervanadate (Na3VO4). As phospho-tyrosine residues are rare and under tight regulation by tyrosine phosphatases, pervanadate treatment of live cells allows for the amplification of low-level phospho-tyrosine signals prior to cell lysis. This method has been successfully utilized in the analysis of receptor tyrosine kinase pathways by increasing global pY levels, thus markedly enhancing pY peptide detection by mass spectrometry (92; 93).
In this study, pervanadate treatment had variable effects on the number of phospho-peptides identified. It would be expected that pervanadate would increase the likelihood of detecting pY
! 36 ! ! residues that are under stringent control of phospho-tyrosine phosphatases. It should be noted that the effect of pervanadate might be limited to increasing the abundance of phospho-peptides that have already been identified in non-treated samples. As the present analysis does not assess differences in peptide stoichiometry between pervanadate treated and non-treated samples, this hypothesis cannot be confirmed without further quantitative analyses.
A total of 12 individual pY screens across 5 NB-TIC lines were pooled to derive a combined dataset of 358 pY containing proteins at a 95% peptide confidence level (Table 2).
Subsequent manual assessments of pY peptide spectra further refined this dataset to 305 proteins containing at least one pY peptide with a manually validated MS/MS spectrum. All subsequent analyses were performed with the latter set of 305 validated pY proteins, which is henceforth referred to as ‘NB-TIC pY dataset’. A list of identified phospho-peptides and scores can be found in Appendix 1, where the positions of phospho-residues cited in this text are additionally annotated according to PhosphoSite online resource (www.phosphosite.org).
Gene Ontology (GO) annotations for cellular processes and locations were added to the validated list of pY proteins in order to assess the function and localization of pY proteins across the NB-TIC pY dataset. As seen in Figure 3 (A), approximately one third of all pY proteins have been annotated to compartmentalize to biological membranes according to the GO classification system. Of these, plasma membrane localization comprises the largest fraction with
76 pY proteins or 25% of the dataset. At least 41 pY proteins were annotated to compartmentalize to components of the cellular cytoskeleton. Figure 3 (B) summarizes functional annotations across the NB TIC pY dataset. Approximately 45% of pY proteins are recognized to be involved in general metabolic processes, including macromolecular processes such as DNA replication and protein synthesis, which can be regulated by phospho-relay
! 37 ! ! signaling pathways to facilitate cell growth. Interestingly, approximately 10% of the pY dataset was annotated to be involved in immune system processes.
! 38 ! !
Source Bone Marrow Tumor Clinical Stage Relapse Post Chemo. Patient High Risk-1 High Risk-2 High Risk-3 High Risk-4 TIC line NB12 NB25 NB61 NB88 NB122 N B153 MS Runs 1 2 V 1 V 1 1 2 V 1 2 1 pY protein IDs 178 187 144 78 159 108 196 162 188 126 134 85 225 162 108 246 167 85 Total pY 358 89 protein IDs Validated pY 305 85 protein IDs Table 2. Summary of phopsho-proteomic screening of 6 NB-TIC lines. Six lines derived from four high-risk patient samples (bone marrow or tumor) were utilized for phospho-proteomic analysis. In addition to endogenous phospho-tyrosine sreening, NB12, NB25 and NB88 were also stimulated with the phosphatase inhibitor vanadate (MS Runs “V”). The phospho-tyrosine proteome of each NB-TIC line was assessed according to protocols developed by Rush et al (50).
The number of pY protein IDs is based on a 95% peptide/50%protein confidence thresholds based on the ProteinProphet and PeptideProphet algorithms (See Materials and Methods 2.1.0).
! 39 ! !
! Figure 3. Cellular distribution and function of tyrosine phosphorylated proteins identified by phospho-proteomic screening of NB-TICs. The (A) cellular localization and (B) biological process of identified proteins were annotated based on GO annotations in the NCBI protein database and graphed by Scaffold 2 Proteome Software.
! 40 ! !
3.0.2 Identification of pY signaling pathways in NB-TIC pY dataset
In order to identify phospho-tyrosine signaling pathways expressed in NB-TICs, two independent bioinformatics approaches were undertaken to analyze the NB-TIC pY dataset.
Furthermore, extensive literature reviews were performed in order to manually group pY proteins as components of distinct signaling pathways.
Ingenuity Pathway Analysis
A pathway analysis using the Ingenuity Pathway Analysis Software was undertaken in collaboration with Olena Morozova and Marco Marra (BC Cancer Agency). A total of 305 pY proteins were entered into Ingenuity, generating a list of identified pathways and respective enrichment thresholds. The ten pathways with the highest probability scores are depicted in
Figure 4. A number of receptor tyrosine kinase pathways were found to be enriched in the NB-
TIC dataset, including insulin, PDGF, and neuregulin signaling. Interestingly, the top three pathways, Fc-", BCR and leukocyte extravasation signaling, are representative of cells from leukocyte origin. The activation of such pathways in neuronal lineage cells represents a novel and unexpected result. The BCR signaling pathway in particular has been implicated in mediating cell viability of blood-borne cancers (94; 71). As the screened NB-TIC lines are derived from the bone marrow, I hypothesize that the BCR pathway may serve a niche specific signaling function in NB-TICs.
KEGG Pathway Analysis
The online bioinformatics resource DAVID (Database for Annotation, Visualization and
Integrated Discovery) was utilized to identify KEGG (Kyoto Encyclopedia of Genes and
Genomes)-annotated signaling pathways across the NB-TIC pY protein dataset (95). KEGG pathway annotations assigned 54% of pY IDs across 47 discrete signaling pathways. Table 3
! 41 ! ! depicts the highest probability pathways identified according to a EASE probability score cutoff of P>0.004.
As with the Ingenuity analysis, a number of receptor tyrosine kinase signaling pathways were enriched amongst the dataset, including ErbB (P=3.4E-9), neurotrophin (P=4.5E-9) and insulin receptor (P=2.6E-6). These results are expected as NB-TICs respond to growth factor receptor ligands insulin, EGF and basic FGF (Figure 5). The cell growth assays depicted in
Figure 5 (A) and (B) suggest that NB-TICs undergo increased proliferation in response to treatment with bFGF, EGF and insulin. Furthermore, bFGF and insulin were confirmed to be sufficient for maximal NB-TIC-proliferation in DMEM/B27 (neurosphere growth media).
Notably, the phospho-proteomics screen was able to identify the phosphorylated activating tyrosine residues in the kinase domains of the insulin receptor (pY1189/1190) and fibroblast growth factor receptor 1 (pY653/654), consistent with an activation of those receptors in response to their ligands, insulin and bFGF, respectively. This suggests that NB-TICs may respond to growth factors bFGF and insulin by activating receptor tyrosine kinase signaling pathways which could regulate cell proliferation in vitro.
Several hematopoietic-specific pathways were represented in the dataset including the
Fc-" receptor phagocytosis (P=1.9E-9), B-cell receptor (P=2.6E-9), Fc-% receptor (P=6.9E-8), and T-cell receptor (P=3.1E-6) signaling pathways (Table 3). Figure 6 depicts the BCR pathway according to KEGG annotations with the proteins identified in the NB-TIC pY dataset highlighted in red. The expression of the cell surface BCR component IgM on NB-TICs has been demonstrated (Kristen Smith and David Kaplan, unpublished data). It should be noted that not all
BCR pathway components are known to be tyrosine phosphorylated, and therefore a complete overlay given all pY proteins in the proteome is not expected. Notwithstanding this, the
! 42 ! ! comprehensive overlay of pY proteins across several nodes of the BCR pathway provides further evidence that these pathway components are functionally active and play a role in NB-TICs. In particular, the phosphorylation of BCR proximal kinases Lyn, SYK and BTK suggests tyrosine kinase regulated BCR signal propagation in NB-TICs. As previously stated, I hypothesize that the expression and phosphorylation of these sets of proteins may be reflective of bone marrow niche specific signaling pathways in NB-TICs.
! 43 ! !
Figure 4: Ten high-probability pathways identified across the NB-TIC pY protein dataset by Ingenuity Pathway Analysis. Pathway enrichment analysis was done on 305 pY proteins identified in the NB-TIC pY dataset. The threshold line (orange) serves as a probability indicator for each pathway (P>0.05), where significantly enriched pathways cross the respective threshold.
Ratio values correspond to the fraction of gene names in the pY dataset that belong to a pathway over the total number of genes represented in that pathway. Pathways are listed in an ascending order from the lowest p-value (highest probability). Ingenuity Data was generated in collaboration with Olena Morozova and Marco Marra (BC Cancer Agency).
! 44 ! !
Table 3: KEGG pathway assignments across the NB-TIC pY protein dataset. A total of 175 phospho-tyrosine proteins were grouped across 47 signaling pathways represented in the KEGG database. Depicted are the top 25 pathways (EASE probability score cutoff 4.0E-3). Notation:
Term: KEGG pathway name, Count: Number of DAVID gene IDs assigned to pathway, P-
Value: Significance for pathway enrichment based on Fisher exact test (EASE score), Fold
Enrichment: Magnitude of pathway enrichment based on population background.
! 45 ! !
Figure 5: NB-TICs respond to growth factors bFGF and insulin with an increase in proliferation. NB-TIC spheres were grown in different media conditions and proliferation was quantified over 7 days by counting live cells using tyrpan blue exclusion. Notation: Complete media: DMEM/F12 with growth factors EGF, bFGF, Insulin, and B27 (neurobasal medium supplement). No GF (growth factor) media: DMEM/F12 only. (A) NB12 cells proliferate when treated with bFGF and insulin in the presence of B27. (N=3) (B) NB12 cells respond and proliferate optimally in the presence of EGF, bFGF and Insulin (N=2)
! 46 ! !
Figure 6: NB-TICs express numerous phosphorylated effectors of BCR signaling as annotated by the KEGG database. KEGG pathway analysis was performed through DAVID software on NB-TIC pY dataset. The BCR pathway proteins that are annotated in the KEGG database and which were also identified in the pY dataset are highlighted in red. Proteins that were identified in pY dataset and that are associated with lymphocyte lineage cells are annotated with blue stars.
! 47 ! !
3.0.3 Identification of NB-TIC cell surface markers by mass spectrometry
Subsequent to the identification of hematopoietic cell specific pathways such as the Fc-" and BCR pathways in NB-TICs, I sought further evidence that NB-TICs express molecular features of hematopoietic cells. I asked whether the cell surface markers on NB-TICs reflect a mixed neural and hematopoietic phenotype. For this purpose, a crude membrane enriched protein fraction was prepared from the NB-TIC line NB88 (for details see Materials and Methods 2.1.1) and subsequently analyzed by multidimensional protein identification technology (MuDPIT).
Furthermore, a whole cell protein fraction of NB88 was analyzed by the same approach. Using this mass spectrometry-based technique, a highly complex mixture of peptides can be separated by cation exchange and reverse phase liquid chromatography prior to analysis in the mass spectrometer. Such separation over time allows for improved detection of low-abundance peptides and the identification of several hundreds to thousands of proteins per run (96; 97).
As shown in Table 4, membrane enrichment coupled to MuDPIT analysis yielded the highest number of protein identifications. This is expected as an enrichment step, such as membrane fractionation, reduces protein and peptide complexity thus allowing subsequent
MuDPIT analysis to identify moderate and low abundance peptides. These experiments identified numerous NB-TIC surface antigens that are characteristic of hematopoietic stem cells as well as mature lymphoid and myeloid progenitors (Table 4). The cluster of differentiation
(CD) markers expressed on NB-TICs include CD19 (B cell), CD20 (B cell, T cell), CD74 (B cell, T cells, macrophages), CD45 (lymphocyte), CD48 (lymphocyte) and CD80 (lymphocyte)
(98-102). The expression of these cell surface antigen markers, including CD19, CD44, CD45 and CD74, has subsequently been confirmed on a single cell level using flow-cytometry and immunostaining (Loen Hansford, Tatiana Lipman, Kristen Smith, David Kaplan, unpublished
! 48 ! ! data). These results provide strong evidence that NB-TICs express markers of hematopoietic cells. This supports the notion that bone-marrow derived NB-TICs may be of mixed neural and hematopoietic linage and that the expression of hematopoietic signaling pathways is indeed a functional attribute of NB-TICs.
! 49 ! !
Sample Membrane Enriched Whole Cell Fraction LC-MS/MS 1-D MuDPIT MuDPIT # protein/peptide 420/1803 1530/7176 768/4282 IDs Protein # unique Spectral Protein # unique Spectral Protein # unique Spectral peptides counts peptides counts peptides counts IPI Database CD44 6 7 CD44 6 34 CD20 3 12 designated CD CD20 7 7 CD20 8 14 CD48 4 6 antigens CD74 4 5 CD48 9 15 CD70 2 2 CD45 4 4 CD45 11 13 CD70 3 4 CD74 4 13 CD40 2 4 CD97 4 6 CD82 3 3 CD70 3 7 CD48 2 3 CD300A 4 7 CD97 2 2 CD40 2 4 CD166 4 5 CD46 3 4 CD59 3 4 CD81 2 4 CD80 2 2 IPI Database Lyn 3 3 Lyn 7 9 - - - designated Src Fgr 2 2 HCK 4 6 Family Kinases Fgr 5 6 Yes1 3 6 Table 4: Membrane and whole cell fraction proteomics identifies surface markers characteristic of hematopoietic lineage cells in NB-TICs. NB88 crude membrane enriched and whole cell fractions were analyzed by 1D or 2D (MuDPIT) LC-MS/MS approaches. Expressed proteins were identified at a 95% protein and peptide confidence level based on the
ProteinProphet and PeptideProphet algorithms, respectively (90; 89). Table depicts only surface markers annotated as cluster of differentiation (CD) of proteins according on the International
Protein Index (IPI) database. Identified mammalian src family kinases are also listed.
! 50 ! !
3.1 Identification of phosphorylated protein kinases in NB-TICs
3.1.0 Expression of tyrosine phosphorylated protein kinases in the NB-TIC pY dataset
Protein kinases are central effectors of cellular signaling pathways and I therefore sought to identify serine/threonine and tyrosine kinases expressed in NB-TICs. Manual assessment of protein function across the NB-TIC pY dataset identified a total of 36 protein kinases, including
15 tyrosine phosphorylated serine/threonine kinases and 21 tyrosine kinases (Figure 7).
Functional analysis of the NB-TIC pY dataset using GO molecular function annotation through the DAVID software also indicated that approximately 12% of the proteins in the dataset were protein kinases, indicating that kinase activity is one of the most enriched functions.
As it is predicted that the human genome encodes approximately 90 tyrosine kinases, it is noteworthy that the phospho-proteomic analysis of NB-TICs captured 23% of these genes on a protein level with phospho-tyrosine post-translational modifications (54). As tyrosine kinases are a comparatively low abundance family of enzymes, the identification of a large fraction of these kinases highlights the effectiveness of tyrosine immuno-precipitation in screening the tyrosine modified cellular kinome. Figure 7 also highlights that at least 73% of all listed kinases and 62% of all tyrosine kinases were identified across at least 3 NB-TIC lines. This suggests that these kinases have conserved cellular functions inherent to all NB-TICs, thus potentially defining a cancer stem cell-specific phenotypic property. For example, tyrosine kinases such as FLT3 expressed in AML cancer stem cells have already been implicated in AML cancer stem cell functions such as engraftment to the bone marrow niche (103). The present data thus establishes a rationale for targeting tyrosine kinases for further studies of cell survival and proliferation pathways in bone marrow derived NB-TICs.
! 51 ! !
Since I observed enrichment of hematopoietic signaling pathways, I next validated the expression of tyrosine kinases known to be central effectors of B-Cell receptor, T-Cell receptor and Fc-" receptor proximal events. As previously noted, BCR signaling initiation is facilitated through the tyrosine kinases Lyn, Syk and BTK (69). Furthermore, the tyrosine kinases LCK and
Fgr are known to associate with ITAM motifs of the TCR and Fc-" receptors, respectively. The concurrent expression of Lyn, BTK, Syk, LCK and Fgr were validated using western blotting with kinase-specific antibodies (Figure 8).
! 52 ! !
Figure 7. Protein kinases identified by phosphoproteomics across NB-TIC lines. Across 5
NB-TIC lines, a total of 36 protein kinases were identified at a 95% peptide/50% protein confidence interval based on the ProteinProphet and PeptideProphet algorithms (90; 89). Among these were 15 serine/threonine kinases and 21 tyrosine kinases including 7 members of the Src kinase family (SFK): Lyn, Fgr, LCK, Yes, Src, HCK, Blk (unlerlined).
NB NAM NB CCRF NB NAM 75 TIC 75 75
50 50 50 Lyn LCK Fgr
NAM NB NAM NB 100 100 75 ! 75 ! ! Syk BTK
Figure 8: NB-TICs express effector kinases Lyn, LCK, Fgr, SYK, and BTK downstream of the B-Cell, T-Cell and Fc-" receptors. Effector kinases are also expressed in Burkitt’s
! 53 ! !
Lymphoma and Acute Lymphoblastic Leukemia, which were used as a control for expression levels. Whole cell lysate of NB-TIC line NB88 (“NB”) and Burkitt lymphoma cell line
NAMALWA (“NAM”) or acute lymphoblastic leukemia line CCRF-HSB-2 (“CCRF”) were probed with polyclonal antibodies to Lyn, LCK, Fgr, Syk and BTK kinases for the detection of endogenous kinase expression.
! 54 ! !
3.0.1 Identification of activated Src family kinases in NB-TICs
As part of the identified set of 21 tyrosine kinases, phospho-proteomics identified 6 members of src family kinases (SFK): Lyn, LCK, HCK, Fgr, Src, and Yes (Figure 7).
Furthermore, membrane enrichment coupled to MuDPIT also identified Lyn, Fgr, Yes1 and
HCK protein expression in NB88 at high confidence (Table 4). SFKs have been implicated in the progression of numerous cancers, including neuroblastoma (86; 87; 104). Importantly, SFKs play crucial roles in the relay of signals downstream of the B-cell receptor, which have been implicated in cancer cell survival (see Introduction 1.3.1). I therefore focused on SFKs as putative signaling effectors regulating NB-TIC cell viability (94).
As shown in Table 5, phosphoproteomics identified over 20 unique pY peptides of 6 Src family members, including known phosphorylation sites in the activation loop and the C- terminus which correlate with increased and decreased kinase activity, respectively.
Furthermore, numerous phosphorylation sites in the SFK SH2 domains were reproducibly identified.
Src family kinases are conserved in mammals both in structure and function. These family members retain high primary sequence conservation in the catalytic domain, particularity the activation loop regulating kinase function. In order to confirm that individual SFKs were in an activated state, amino acid differences around the activating pY site pY416 (or equivalent) must be present to generate unique m/z values and MS/MS spectra of the activation loop peptide for each SFK. A protein sequence alignment of human SFKs Lyn, HCK, LCK, BLK, Src, Fgr and Yes was performed using the ClustalW multiple sequence alignment program (European
Bioinformatics Institute, EMBL-EBI). As shown in Figure 9, each trypsin fragment containing the activation loop pY site has at least one amino acid difference in the primary sequence
! 55 ! ! between all SFKs with the exception of HCK/ Lyn and Src/Yes, which share identical sequences.
Thus the phosphoproteomics screen successfully identified at least 5 SFKs in an activated state based on pY phosphorylation patterns. Furthermore, the individual activation loop tryptic peptides generated high quality MS/MS spectra indicating the presence of Y416 (or equivalent) phosphorylation in SFKs with high confidence (Figure 10).
In order to verify the expression and phosphorylation of individual SFKs, a reciprocal immuno-precipitation approach was pursued. In this method, all pY proteins were immuno- precipitated with a pan pY antibody (4G10) from NB-TIC lysate, and the precipitated proteins were probed with individual monoclonal SFK antibodies on western blots. Subsequently, individual SFK antibodies were used for immuno-precipitation, followed by probing of pY signals using 4G10 antibody by western blotting. Figure 11 depicts the expression and phosphorylation of SFKs Lyn, LCK, HCK, Fgr and Src in NB-TIC line NB88. I conclude that
NB-TICs express at least 5 SFKs, and that the kinases are in a phosphorylation state which correlates with heightened catalytic function, thus suggesting their active role as signaling effectors in NB-TICs.
! 56 ! !
SFK Sequence pY Site Domain pY site TIC line feature Lyn SLDNGGpYYISPR . pY 192 SH2 domain NB12, NB88, NB61, NB25 VIEDNEpYTAREGAK. pY 396 Activation Activating NB12, NB88, NB122, NB25, VIEDNEpYTAR. loop NB61 VENCPDELpYDIMK. pY 472 Kinase NB12, NB88, NB25, NB61 domain EKAEERPTFDYLQSVLDDFYTATEGQpYQQQP. pY507 C-terminus Inhibiting NB12 Src LIEDNEpYTARQGAK. pY 416 Activation Activating NB12 LIEDNEpYTAR. loop KEPEERPTFEYLQAFLEDYFTSTEPQpYQPGENL. pY 529 C-terminus Inhibiting NB12 LCK LIEDNEpYTAREGAK. pY 394 Activation Activating NB12, MB88, NB122, NB25, LIEDNEpYTAR. loop NB61 NLDNGGPpYISPR. pY 192 SH2 domain Inhibiting NB12, NB88, NB122, NB25 IRNLDNGGPpYISPR. HCK TETSASPHCPVpYVPDPTSTIKPGPNSHNSNTPGIR. pY 50 N-terminus NB88 TLDNGGFpYISPR. pY 208 SH2 domain NB88 VIEDNEpYTAREGAK. pY 410 Activation Activating NB88 loop FGR SYGAADHpYGPDPTK. pY 34 N-terminus NB12, NB88 KLDMGGpYYITTR. pY 208, SH2 domain NB12, NB88, NB25, NB61 KLDMGGYpYITTR. p Y209 LIKDDEpYNPCQGSK. pY 412 Activation NB88 Loop BLK CLDEGGYpYISPR. pY 187, SH2 domain NB12, NB122, NB25, NB61 CLDEGGpYYISPR. pY 188 IIDSEpYTAQEGAK. pY 389 Activation NB12 Loop Yes LIEDNEpYTAR. pY 426 Activation Activating NB12, NB61 Loop KLDNGGpYYITTR pY 222, SH2 domain NB12, NB61 KLDNGGYpYITTR pY 223 Table 5. SFK peptides identified by phosphoproteomics across NB-TIC lines NB12, NB88,
NB122R, NB61 and NB25. Phospho-peptides were identified at a minimum 90% peptide confidence using the PeptideProphet algorithm and individual spectra were manually verified
(90; 89). All trypsin miss-cut variants identified are listed. Individual domain and pY site features were derived from PhosphoSite resource (www.phosphosite.org). pY sites in activation loop associated with activity status based on primary sequence homology were noted in italics
(also see Figure 9).
! 57 ! !
Figure 9. Sequence alignment of activation loops in mammalian SFKs Lyn, HCK, LCK,
BLK, Src, Yes and Fgr depicts non-identical sequence homology in tryptic fragment containing pY416 residue for all SFKs except Src/Yes and HCK/Lyn. ClustalW sequence alignment of mammalian SFKs shows strong sequence homology in the activation loop trypsin fragment, which harbors the activating pY site 416 (yellow), across all SFKs. Lyn and HCK as well as Src and Yes1 share identical primary sequences in the tryptic fragments, while other
SFKs differ by at least 1 amino acid.
! 58 ! !
SFK MS/MS Spectra Sequence
LCK/ LIEDNEpYTAREGAK HCK
Src/ LIEDNEpYTARQGAK Yes
Lyn VIEDNEpYTAREGAK
Fgr LIKDDEpYNPCQGSK
BLK IIDSEpYTAQEGAK
Figure 10: Representative MS/MS spectra of SFK activation loop tryptic fragments suggest that individual SFKs are in an activated state in NB-TICs. Selected spectra were derived from NB-TIC pY dataset and depict tyrosine phosphorylation of activating tyrosine site pY416
(or homologous equivalent) for each SFK.
! 59 ! !
Figure 11: Reciprocal IP approach confirms expression and phosphorylation of SFKs Lyn,
HCK, Fgr, Src and LCK in NB-TICs. (A) Whole cell lysate of NB-TIC line NB88 was immunoprecipitated with polyclonal antibodies against Lyn, HCK, Fgr, Src or LCK and the precipitate was probed with 4G10 antibody detecting tyrosine phosphorylation. (B) Whole cell lysate of NB88 was immunoprecipitated with 4G10 antibody and the lysate was probed with individual polyclonal antibodies detecting SFKs Lyn, HCK, Fgr, Src and LCK. Non-specific mouse and rabbit IgG control antibodies were used for immunoprecipitations alongside (A) and
(B) to control for reagent non-specificity (not shown).
! 60 ! !
3.2: Inhibition of NB-TIC cell growth by Src and SYK kinase inhibitors
3.2.0 Small molecule SFK inhibitors reduce NB-TIC cell growth
In order to address if inhibiting SFK activity affects NB-TIC growth and viability I utilized small molecular kinase inhibitors of SFKs in vitro. SFK inhibitors such as dasatinib
(BMS-354825) and saracatinib (AZD-0530) have gained considerable interest for the treatment of leukemia and solid tumors over the past five years (105-107). I therefore sought a small molecule inhibitor approach as a first line of functional assessment of SFK activity in regulating
NB-TIC growth and/or viability. A total of 6 SFK inhibitors were tested on NB-TICs using a treatment period of 3 days to assess acute effects on cell viability and growth by resazurin reduction, a measure of metabolic activity. Figure 12 depicts the effect of SFK inhibitors
SU6656, PD180970, bosutinib and dasatinib on 3 bone marrow derived NB-TIC lines. All SFK inhibitors were highly potent in decreasing NB-TIC growth in the high nanomolar to low micromolar concentration range within 3 days. This effect was consistent across NB-TIC lines
NB12, NB88 and NB122R, suggesting that SFK inhibitors impede on the growth NB-TICs from different patients.
Interestingly, the SFK inhibitor SU6656 was observed to have a significant effect on NB-
TIC morphology following a 7-day incubation period. Individual NB-TICs appear grossly enlarged compared to non-treated cells when treated with 1-5µM of SU6656 (See Figure 13).
Cells still retain the ability to form spheres, albeit with enlarged cells. The enlarged morphology has been proposed to be due to polyploidy which is suggested to be a product of endomitosis
(108; 109). This striking phenotype has previously been observed in SU6656-treated leukemic cell lines, B-cells, megakaryocytes and bone marrow derived hematopoietic progenitors (108;
110; 109). It is notable that this phenotype has been hypothesized to be due to the off-target
! 61 ! ! inhibition of Aurora Kinase B, a regulator of mitotic events and cell ploidy, by SU6656 (109).
The expression of Aurora Kinase B has been confirmed in 3 NB-TIC lines (data not shown), and therefore it cannot be excluded that the effect observed may be a product of similar SU6656 cross-reactivity with Aurora Kinase B in NB-TICs. Notwithstanding this curious phenotype,
SU6656 was still found to be the least potent tested small molecule inhibitor in reducing NB-TIC growth (Figure 12) and SFK activity (Figure 14).
! 62 ! !
! Figure 12: Small molecule SFK inhibitors SU6656, PD18970, bosutinib (SKI-606) and dasatinib (BMS-354825) potently inhibit NB-TIC growth. NB-TIC lines NB12, NB88 and
NB122 were treated with an 8-point dilution series (log $M) of SU6656, PD18970, Bosutinib or
Dasatinib over a period of three days. Cellular growth was assessed by resazurin reduction and cell growth values were expressed as a fraction of control treated cells (DMSO).
! 63 ! !
! Figure 13: SFK inhibitor SU6656 induces enlargement of cell volume in individual NB-
TICs.! NB-TIC line NB88 was treated with 1:3 dilutions of SU6656 or DMSO and cellular morphology was observed at day 7. NB-TIC cells appear to have a grossly enlarged morphology at 1.7µM and 5µM concentration at day 7 following treatment. At 15µM SU6656 no sphere formation or cell proliferation is observed. !
! 64 ! !
3.2.1 Validation of SFK inhibition and assessing MAPK activity downstream of SFKs
To determine whether SFK inhibitors were functioning by decreasing SFK kinase activity, I assessed SFK phosphorylation in cells treated with those drugs. To test this, I employed a phospho-SFK antibody that recognizes the activation loop tyrosine residue 416 (or equivalent) of Src, Lyn, Fyn, LCK, Yes, and HCK when phosphorylated. As shown in Figure
14, SFK inhibitors dasatinib, bosutinib, SU6656 and PD180970 potently inhibited SFK activity in the NB-TIC line NB88 within 15 or 30 minutes of treatment as expected. For dasatinib, the apparent differences observed between inhibitor concentrations required for inhibition of NB-
TIC growth and in vitro inhibition of SFK activity may be attributed to the fact that growth assays were performed over a 3-day period. The duration of SFK inhibition by dasatinib was only tested at 15 min and may not last as long as 3 days due to inhibitor stability, thus leading to lower observed efficacy.
Numerous signaling effectors downstream of SFKs have been implicated in mediating cellular viability and growth. Src is known to activate the Ras/Raf/MAPK pathway leading to cellular proliferation (62). I therefore tested the effect of SFK inhibition on MAPK/ERK1/2 activity using the SFK inhibitors dasatinib and PD180970 (Figure 15). Inhibition of SFK activity decreased the levels of active MAPK/ERK1/2 as measured by phosphorylation of residues
Thr202/Tyr204 in the MAPK/ERK1/2 activation loop. Both, PD180970 and dasatinib treatment reduced MAPK/ERK1/2 activity as expected. Preliminary results using bosutinib and SU6656 also suggest a decrease in pY MAPK/ERK1/2 levels corresponding to SFK inhibition (data not shown). It worthy to note that the potent inhibition of SFK activity by dasatinib and PD180970 is not matched by an equally potent inhibition of MAPK/ERK1/2 activity. This could potentially be reconciled by the fact that the ERK1/2 activity was done after an acute treatment with SFK
! 65 ! ! inhibitors, and that there is a temporal delay in ERK1/2 activity downstream of SFKs that requires a longer treatment. Furthermore, other upstream pathways, which may not be regulated by SFKs, feed into ERK1/2 therefore potentially reducing the effect of SFK inhibition on
ERK1/2 activity.
It is important to note that all SFK inhibitors analyzed herein are ATP competitive inhibitors and are known to have off-target effects on other receptor and non-receptor protein kinases. We cannot preclude that the cellular and molecular effects can at least in part be attributed to secondary inhibitor effects on other kinases. For example, a recent analysis of the global target profile of the SFK inhibitor bosutinib in CML lines and K562 cells established that bosutinib has a target profile that spans Abl, TEC and STE kinase families in addition to SFKs
SFKs in vitro (111). In light of this, validation of the role of SFK activity in NB-TIC cell growth was derived through individual SFK knockdowns by shRNA. Data obtained by our collaborators
Kim Blakely and Jason Moffat (University of Toronto) indicate that individual knockdown of
Lyn, Src, Fyn, BLK, and Yes reduces cell growth of NB-TIC lines NB88 and NB122 by at least
50% for each SFK. As individual knockdown of SFK did not lead to complete inhibition of cell growth, it is hypothesized that the function of some SFKs may be redundant, leading to residual growth phenotypes. These results are consistent with the notion that SFKs play a role in optimal cell viability and proliferation in NB-TICs.
! 66 ! !
Figure 14: Src family kinase inhibitors reduces phosphorylation in the activating kinase domain of the SFKs. NB88 cells were treated with (A) Bosutinib, (B) Dasatinib, (C) PD190870
(D) SU6656 or vehicle (DMSO) for 15 minutes (A, B, C) or 30 minutes (D) at 37°C/5%CO2 and cellular lysates probed with phospho-specific antibody recognizing the activation loop pY site
416 (or equivalent) and anti c-Src only antibody.
! 67 ! !
Figure 15: SFKs inhibitors PD180970 and dasatinib decreases levels of active ERK1/2.
NB88 cells were treated with (A) PD180970 and (B) dasatinib for 15 minutes at 37°C/5%CO2 and lysates were probed with anti-pThr/202/pY204 Erk1/2 and anti-Erk1/2 antibodies to test for reduction in active Erk1/2 levels.
! 68 ! !
3.2.2 Inhibitors of BCR effector kinase SYK reduce NB-TIC growth
The BCR pathway has proximal effector kinases, including SFKs, SYK and BTK, which mediate downstream signals from the receptor. The tyrosine kinase SYK has been shown to be essential to functional BCR signaling and has also been implicated as a putative therapeutic target in numerous hematopoietic malignancies including chronic lymphocytic leukemia, diffuse large cell B lymphoma, and most recently acute myeloid leukemia (112-115). I found that SYK kinase was expressed in 5 bone marrow derived NB-TIC lines and was activated based on the phosphorylation of activation loop residues pY525/pY526 in the NB-TIC pY dataset. I next assessed if NB-TICs are responsive to SYK kinase inhibition by small molecule inhibitors. The
ATP competitive SYK inhibitors R406 and BAY 61-3606 as well as the non-kinase competitive
Syk activity inhibitor ER27319, were potent inhibitors of NB-TIC growth (Figure 16). These results suggest that SYK inhibitors may be developed further for targeting of NB-TICs. This has particular significance in light of ongoing clinical trials with R788, a pro-drug of R406, in peripheral T-cell lymphoma (116).
! 69 ! !
! Figure 16: SYK Kinase inhibitors Rigel 406, ER 25319 and BAY 61-3606 inhibit NB-TIC growth in vitro. (A) NB-TIC lines NB12 and NB88 were treated with Rigel R406 and ER27319 for 3 days. (B) NB-TIC lines NB12 and NB88 were treated with BAY 61-3606 for 7 days with a feed at day 4 (see methods). Cellular growth was assessed by resazurin reduction and cell growth values were expressed as a fraction of DMSO control treated cells.
! 70 ! !
3.3 SFK inhibitors bosutinib and dasatinib induce apoptosis in NB-TICs
3.3.0 SFK inhibitor bosutinib potently induces apoptosis in NB-TICs
The results presented in 3.2.0 suggest that SFKs may be novel therapeutic targets for neuroblastoma progression. The SFK inhibitor bosutinib is currently in phase II trials for metastatic breast cancer. Dasatinib is already in clinical use for the treatment of CML and several phase II studies are ongoing for small cell lung, breast, prostate and pancreatic cancers amongst others. In order to further address the applicability of these two SFK inhibitors for the treatment of neuroblastoma, it is important to define the mechanism by which bosutinib and dasatinib inhibit NB-TIC growth.
Bosutinib has been shown to induce apoptosis in cell lines derived from non-small cell lung cancer and CML, albeit only moderately for the latter (117-119). I therefore characterized the effect of bosutinib on several nodes of the apoptotic pathway in NB-TICs. The activation of effector caspases -3, -6, -7, which are a family of proteolytic enzymes, is an important hallmark feature of apoptosis (120). The activation of these effector caspases following cytochrome-c release from the mitochondrion induces a relay of downstream effects resulting in cell death, including caspase-mediated cleavage of proteins involved in DNA-damage repair such as Poly-
(ADP-ribose) polymerase (PARP) (121; 122).
I tested apoptotic induction in NB-TICs by measuring caspase-3/7 activity using a fluorophore-conjugated synthetic recognition peptide for proteolytic cleavage by caspase-3 and -
7. As can be seen in Figure 17, bosutinib treatment of NB88 induced caspase 3/7 activity within
24 hours of treatment and apoptosis was further sustained for 48 hours. I subsequently tested cleavage of the caspase-3 target PARP by western blotting with an antibody to cleaved PARP.
Cleavage of the 116kDa PARP protein by caspase-3 results in 24kDa and 89kDa cleavage
! 71 ! ! products. As expected, an increase in 89kDa PARP cleavage product was observed after 24h with increasing bosutinib concentrations (Figure 18). Taken together, these results indicated an activation of effector caspase activity in NB-TICs as induced by bosutinib exposure, consistent with the activation of apoptotic cell death pathways.
In order to identify the fraction of cells undergoing apoptotic cell death versus necrosis, I assayed phosphatidylserine exposure on the cell surface of NB-TICs. Phosphatidylserine is a plasma membrane lipid that is actively sequestered within the cytoplasmic (inner) leaflet of the membrane. During early apoptosis, phosphatidylserine is also present on the outer leaflet on the membrane, while the overall membrane structure and permeability remains intact. Annexin-V is a high-affinity phospholipid binding protein that can be conjugated to a fluorophore, allowing for the detection of its binding to phosphatidylserine to the outer membrane leaflet by flow cytometry. I utilized Annexin-V mediated detection of phosphatidylserine on the cellular surface of intact cells to quantify the fraction of cells in early apoptosis.
Figure 19 depicts the percentage of cells that had Annexin-V bound to the outer membrane leaflet while otherwise retaining intact membrane structure. After 24h, a 10µM treatment of bosutinib induced early apoptosis in nearly 30% of cells with an intact membrane.
Furthermore, 10µM of bosutinib significantly induced apoptosis in approximately 20% of intact cells within 8 hours of treatment. This rapid response suggests that bosutinib is a potent apoptosis-inducing agent in NB-TICs, inhibiting cell growth by reducing cell viability through apoptosis.
! 72 ! !
Figure 17: Bosutinib increases Caspase 3/7 activity in NB88 cells. NB88 cells were dissociated and treated with 5µM of Bosutinib or DMSO (vehicle). Caspase 3/7 activity was measured by fluorescence output of Asp-Glut-Val-Asp peptide sequence cleavage at indicated time intervals.
! 73 ! !
Figure 18: Bosutinib induces Poly ADP-ribose polymerase (PARP) cleavage in NB-TICs.
Dissociated NB88 cells were treated with indicated concentrations of Bosutinib for 48h. Cell lysate was collected and probed by western blotting for PARP cleavage at Asp214.
!
! 74 ! !
! 75 ! !
Figure 19: Bosutinib potently induces early apoptosis in NB-TICs as indicated by the exposure of phosphatidylserine lipids on the cell surface. (A) Cells in early apoptosis are quantified as the fraction of cells staining positive for annexin-V and negative for propidium iodide (PI) using flow cytometry. (B) Representative flow cytometry images of NB88 treated with DMSO, 2µM, 5.0µM and 10.0µM depicting cell population shift into early apoptosis
(second quadrant).
! 76 ! !
3.3.1 Clinically utilized SFK inhibitor dasatinib is a moderate inducer of apoptotic responses in NB-TICs
Similar to the response observed with bosutinib, I hypothesized that the SFK inhibitor dasatinib would also induce apoptotic events in NB-TICs. Numerous articles have shown that dasatinib drives malignant cells to undergo apoptosis, including CML, CLL, and prostate cancer
(123-125). I assessed induction of apoptosis by quantifying caspase-3 activity, PARP cleavage and annexin-V binding of cell surface phosphatidylserine.
As shown in Figure 20, dasatinib treatment increased caspase-3/7 activity in NB-TICs. A significant increase in caspase-3/7 activity was observed at both 5µM and 10µM doses of dasatinib. Consistent with these results, dasatinib was also found to induce PARP cleavage in a concentration-dependant manner (Figure 21). NB-TIC treatment with dasatinib also led to phosphatidylserine exposure on the outer surface of the plasma membrane as detected by annexin-V binding. As seen in Figure 20, dasatinib significantly increased the number of NB-
TICs in early apoptosis with a treatment concentration of 5µM. At 24 hours, the maximum number of cells in early apoptosis detected using annexin-V was approximately 15%.
These results indicate that while dasatinib does induce apoptotic responses in NB-TICs, the increase in these responses is moderate compared to bosutinib treatment. PARP cleavage was observed at lower concentrations of bosutinib, and a larger fraction of cells were found to be in early apoptosis using bosutinib when compared to dasatinib at equivalent doses. While dasatinib may be a weaker apoptosis-inducing agent, the aforementioned results are consistent with the notion that the growth inhibition observed could at least in part be attributed to cell death by apoptosis.
! 77 ! !
Figure 20: Dasatinib increases caspase 3/7 activity in NB88 cells. NB88 cells were dissociated and treated with 5uM or 10uM of Dasatinib or DMSO (vehicle). Caspase 3/7 Activity was measured by caspase 3/7 cleavage of fluorophore-conjugated Asp-Glut-Val-Asp peptide resulting in correlating fluorescence. Activity was measured on indicated time intervals.
Figure 21: Dasatinib induces Poly ADP-ribose polymerase (PARP) cleavage in NB-TICs.
NB-TIC line NB88 treated with indicated concentrations of dasatinib for 48h and probed by western blotting for PARP cleavage at Asp214.
! 78 ! !
Figure 22: Dasatinib induces early apoptosis in NB-TICs as indicated by surface exposure of phosphatidylserine. Early apoptosis is indicated by positive annexin V staining of
! 79 ! ! phosphatidylserine lipids on the cell surface with negative PI staining of cytoplasm. (A)
Percentage of cells in early apoptosis at indicated concentrations after 24h dasatinib treatment.
(B) Representative flow cytometry images of NB88 treated with DMSO, 5.0µM, 10.0µM and
20.0µM dasatinib depicting cell population shift into early apoptosis (second quadrant) and necrosis (fourth quadrant).
! 80 ! !
Chapter 4: Discussion
4.0 Hematopoietic signaling and NB-TICs from bone marrow metastases
Preamble
This study represents the first analysis of signaling pathways in human cancer stem cells using a phosphoproteomics approach. NB-TICs from bone marrow metastases were found to express activated effectors of hematopoietic signaling pathways, including the B-cell receptor signaling cascade. Furthermore, the tyrosine kinases SFKs and SYK, which are essential mediators of BCR proximal signaling, were found to be activated across NB-TIC lines. These results were unexpected, since neuroblastoma is thought to arise from neural crest progenitors.
The identification of active lymphocyte signaling pathways in neuroblastoma is unprecedented and opens the door for new approaches to neuroblastoma therapies.
4.0.0 Molecular features of immune system cells expressed in NB-TICs
This study is the first to identify hematopoietic signaling pathways and cell surface markers in NB-TICs. A number of other approaches have subsequently supported these observations by identifying additional molecular features of immune system cells in NB-TIC lines from different patients. An array of different methods employed by our collaborators and our laboratory including whole transcriptome shotgun sequencing (Olena Morozova, Marco
Marra, BC Cancer Agency), genome-wide RNA interference screening (Kim Blakely, Jason
Moffat, University of Toronto) and flow cytometry of membrane markers (Loen Hansford,
Tatiana Lipman, Kristen Smith, David Kaplan, Hospital for Sick Children) independently confirmed the expression of transcription factors and surface proteins characteristic of lymphoid
! 81 ! ! and myeloid cells in NB-TICs. Examples of these include the hematopoietic transcription factors
Pax5 and Ikaros as well as lymphoid markers such as CD20, CD38, and CD44.
I found that NB-TICs express a cohort of surface molecules important to B-cell signaling initiation, maintenance and termination. Consistent with the hypothesis that NB-TICs express functional BCR complexes, NB-TICs have been found to contain Ig gene rearrangements and express the BCR surface component IgM (L. Hansford, K. Smith, D. Kaplan, The Hospital for
Sick Children). This study also identified phosphorylated BCR accessory receptors CD19 and
CD22, which act as positive and negative regulators of BCR signaling, respectively (77). Flow cytometry confirmed the expression of CD19, CD22 as well as CD45, a receptor phosphatase and positive regulator of BCR signaling, on NB-TICs (L. Hansford and D. Kaplan, Hospital for
Sick Children) (69). Together, these results support the hypothesis that NB-TICs contain a set of
B cell related hematopoietic lineage markers that may function as part of a BCR signaling cascade.
4.0.1 Targeting the BCR signaling pathway through SYK tyrosine kinase inhibition
Tonic or chronic BCR signaling has been implicated in the promotion of cell survival in chronic lymphoid leukemia (CLL) (126; 127). SYK is a central effector kinase that binds to the
BCR components Ig!/!, thereby relaying downstream signaling leading to cell survival (69).
SYK has thus been a rational target to moderate aberrant signals in BCR-driven neoplasia (128).
Activated SYK has been shown to be important for the growth of diffuse large cell B lymphoma
(DLBCL), non-Hodgkin’s lymphoma, and most recently acute myeloid leukemia (AML) cells
(128; 114).
Across the NB-TIC pY dataset and the MuDPIT data, SYK kinase was found to be expressed in all bone marrow derived NB-TIC lines tested. I identified 7 of a total of 11 pY sites
! 82 ! ! for SYK in the PhosphoSite database (www.phosphosite.org), including the kinase domain activation loop pY residues 525/526 and the SYK regulatory site pY353 (129). Furthermore, I observed a novel pY residue on the C terminal region of SYK at position Y629. In light of the phosphorylation sites observed, I asked whether SYK activity plays a role in bone marrow- derived NB-TIC growth in vitro by utilizing three inhibitors of SYK kinase activity: R406, BAY
61-3606, and ER27319. Each SYK inhibitor reduced NB-TIC growth when applied at in high nano-molar to low micro-molar concentrations. Furthermore, preliminary data suggests that inhibiting the expression of SYK using shRNA interference leads to a reduction in cell growth to approximately 35-50% of wildtype in three NB-TIC cell lines (K. Blakely, J. Moffat, University of Toronto).
Cumulatively, these data support my hypothesis that SYK is a viable target in NB-TICs to reduce cellular survival and proliferation in vitro. While using SYK inhibitors to target NB is a novel concept, the pro-drug of the SYK inhibitor R406 has already shown encouraging efficacy in phase I/II clinical trials for CLL and non-Hodgkin lymphoma (128). In vivo studies using xenograft models with NB-TICs of the pro-drug of R406 are therefore necessary to assess whether this SYK inhibitor is a feasible option for future clinical trials in NB patients.
4.0.2 Hematopoietic signaling pathways in NB-TICs: NB-TIC pY dataset identifies the
CD19/Lyn signaling pathway
Mass spectrometry-coupled phospho-proteomics has the distinct advantage that the phospho-tyrosine proteome of cells can be surveyed on the scale of individual pY residues rather than only on the whole protein level. Individual pY sites on proteins can be identified on a large, pathway-wide scale allowing for the inference of functional states of proteins. This allows for the
! 83 ! ! culmination of an additional layer of information from the dataset which can be manually overlain onto existing protein-based pathway analyses such as Figure 6 (Results).
Phospho-proteomics identified BCR signaling components and their respective phosphorylation sites in NB-TICs, enabling the generation of a signaling model of BCR- proximal events based on the pY dataset (Figure 23). The B-cell surface molecule CD19 has been suggested to play a crucial role in BCR signal amplification in part through its association with Lyn and PI3K (130). Lyn associates with mouse CD19 mouse on pY513 (human homolog
531), leading to baseline level of Lyn activity prior to BCR engagement (131). Following BCR
Ig!/Ig! ligation, Lyn becomes fully activated by auto-phosphorylation of the activation loop pY396. Activated Lyn then phosphorylates tyrosine residues on CD19. PI3K binds pY sites such as pY500 on CD19, leading to the recruitment of PI3K to the membrane to generate the lipid
second messenger PIP3(3,4,5) (69; 132). Lyn also phosphorylates Syk on regulatory sites Y352, leading to the trans-autophosphorylation of SYK on activation loop sites Y525/Y526 (133).
Subsequently, activated SYK phosphorylates BTK on Y551 and BTK further trans- autophosphorylates Y223 leading to full kinase activation (134; 135). The aforementioned PI3K
product PIP3 has been implicated in mediating BTK localization to the membrane and activation
(69). Both BTK and SYK are involved in the subsequent activation of PLC"2 through he adaptor protein BLNK (not shown) by phosphorylation of pY759 and pY1217 among others, leading to activation of PLC"2 and downstream BCR signal propagation (135; 136).
I hypothesize that these signaling effectors and phosphorylation sites identified in NB-
TICs are similar to phosphorylation events in proximal BCR signaling in B-cells. These results are being confirmed by shRNA-mediated knockdown of individual SFKs, assessing changes in pY levels of putative SFK substrates. Furthermore, relevant protein-protein interactions are to be
! 84 ! ! validated by co-immunoprecipitation of SFKs and their predicted substrates in NB-TIC lines
(See Future Directions 4.2.0).
Figure 23: Proposed tyrosine phosphorylation relay model downstream of BCR accessory receptor CD19 and Lyn in NB-TICs. The model depicts literature cited signaling events downstream of the BCR leading to PLC" activation and signal propagation. All depicted proteins and tyrosine phosphorylation sites (red) were identified across the NB-TIC pY dataset. Phospho- tyrosine residues were annotated according to PhosphoSite Database (www.phosphosite.org).
References for phosphorylation and binding events are cited in the text.
! 85 ! !
4.0.3 How did NB-TICs acquire hematopoietic features? The hypothesis of cell fusion
Despite their hematopoietic features, numerous assessments confirm that NB-TICs are bona fide neuroblastoma cells: They express neuroblastoma and neural crest progenitor cell surface markers NB85, tyrosine hydroxalase (TH), fibronectin and nestin, have chromosomal aberrations typical of neuroblastoma, and give rise to tumors with immature neuroblasts (35).
Thus the question arises how and when neuroblastoma tumor initiating cells acquired molecular features of hematopoietic cells.
It can be hypothesized that hematopoietic characteristics are acquired by NB-TICs as part of fusion events with bone marrow derived cells (BMDCs). Indeed, this study and others found that NB-TICs express the cell surface antigen CD44, a proposed solid tumor cancer stem cell marker, which has also been implicated in cell fusion events, such as osteoclastogenesis (137;
138). The idea that cancer stem cells may evolve by cell fusion is not new. This hypothesis has been proposed by Bjerkvig et al and Dittmar et al to explain numerous features of some cancer stem cell types, such as aneuploidy and acquired resistance to toxic agents, respectively (139;
140). In one proposed model, chronic inflammations in tumors recruit circulating immune cells, which are suggested to fuse to cancer stem cells, allowing for the acquisition of novel traits such as higher proliferative potential (139). Indeed, tumor-associated BMDCs such as macrophages are known to have fusogenic potential and produce hybrid cells that express genes from both parental cells (141).
Hybrid hematopoietic/cancer cells that are derived from myeloid fusion events have already been described for numerous cancers in mice, including melanoma and sarcoma, where hybrid cells displayed characteristics of both parental cell lineages (142-144). For example, the fusion of mouse B lymphocytes with malignant plasmacytoma cells produced hybrid cells that
! 86 ! ! secrete monoclonal immunoglobulin and had increased metastatic potential (145). Importantly, a number of studies on human renal cell carcinoma, intestinal adenoma and lung cancer samples support the existence of tumor/bone marrow hybrid cells in primary tumors and metastatic sites in humans, albeit definitive proof of fusion events is still outstanding and will likely remain inaccessible for human cancers (142; 146; 141).
For neuroblastoma, I hypothesize that a fraction NB-TICs metastasize from the primary tumor to the bone marrow to form small micro-metastases. Upon fusion to bone marrow resident cells NB-TICs acquire hematopoietic phenotypes, such as hematopoietic signaling pathways and cell surface receptors that will allow them to optimally proliferate in the bone marrow niche. Lu and Kang also propose that hybrid cancer cells reside in niches that are supportive of their newly attained phenotypes (147). This would lead to the large neuroblastoma bone marrow burden that is observed in many bone marrow aspirates from which NB-TICs are derived. The hypothesized temporal sequence of events in NB-TIC development is supported by transcriptome studies of the tumor-derived NB-TIC line NB153, which was found to have no hematopoietic features, suggesting that they are indeed acquired only following metastasis (O. Morozova, M. Marra, BC
Cancer Agency).
Despite this hypothesis it may be inquired if cultured NB-TICs are a pool of cells that contain distinct cell populations, some with hematopoietic and others with neuronal cell properties. It may also be questioned if cell fusion events are occurring during the establishment of NB-TIC lines in vitro. The most recent data produced by the Kaplan group suggests that the
NB-TICs mixed lineage phenotype is not a product of culture conditions, as primary bone marrow aspirates from NB patients have been found to contain cells that concurrently express hematopoietic and neural crest lineage markers. It is believed that these unique cells comprise
! 87 ! ! the bulk of the NB-TIC population in vitro as well. Furthermore, flow cytometry based analysis established the co-expression of hematopoietic and neural crest markers on individual cells, further supporting the notion that NB-TICs are comprised of mixed lineage cells, rather than distinct cell populations in culture.
4.1 Src family effector kinases: Novel therapeutic targets in neuroblastoma
4.1.0 The role of Src family kinases in neuroblastoma and NB-TICs
This study suggests that multiple SFKs are concurrently expressed and activated in NB-
TIC lines although the data does not indicate phosphorylation stoichiometries. SFKs play important roles in diverse tissues and cells and have been implicated in the progression of numerous solid tumor types. However the specific functions of many SFKs in the progression of neuroblastoma remains ill-defined. To date there has been no published functional characterization of SFKs LCK, HCK, Fgr and Yes1 in neuroblastoma. This study is the first to attribute a functional role of these SFKs as well as c-Src and Lyn in mediating NB cell viability.
Early studies of SFKs in NB suggested that the expression of neuronal c-Src isoform pp60c-srcN correlates with good prognosis NB (148). The levels of Fyn mRNA have also been correlated predominantly with low-stage tumors and active Fyn was shown to induce NB cell line differentiation in vitro (149). Lyn transcript expression has also been implicated in neuronal differentiation of NB cell lines (150). A more recent body of work characterized the role of c-Src in NB cell adhesion, motility and viability. Recently, Beierle and colleagues determined that the dual inhibition of c-Src and FAK led to detachment and a reduction in cell survival in NB lines
(151). Wu et al also established that activated FAK and Src are required for NB cell motility induced by a subset of integrin receptors (152).
! 88 ! !
This study suggests that the role of activated SFKs in bone marrow-derived NB-TICs is to mediate cell survival and growth. Mass spectrometry and western blotting experiments demonstated the concurrent expression and activation of SFKs. Importantly, it was shown that the ATP competitive SFK inhibitors SU6656, PD180970, bosutinib and dasatinib are potent inhibitors of SFK activity in NB-TICs and that short-term treatment with these compounds impaired NB-TIC growth in vitro. To account for the impact of inhibitor off-target effects, multiple SFK inhibitors were utilized and individual knockdowns of SFKs Src, Lyn, LCK, Fyn,
BLK, and Yes were performed in preliminary experiments (K. Blakely, J. Moffat, University of
Toronto) using shRNA interference. Preliminary data suggest a decrease in the viability and growth of NB-TIC lines by at least 50% following shRNA-mediated knockdown. Taken together, these results bring forth strong evidence of the essential role of SFKs in NB-TIC growth.
It should be noted that the function of several SFKs in pathways such as BCR signaling is believed to be redundant. For example, Blk and Fyn appear to respond similar to Lyn downstream of BCR activation (77). It will thus be necessary to address if double- or triple SFK knockdown experiments will show a stronger phenotype than individual SFK knockdowns. As
SFK inhibitors are thought to target multiple SFK members, the presented inhibitor results can thus be compared to the expected phenotype of multiple SFK knockdowns.
In conclusion, I suggest that the role of SFKs in cell growth may be specific to the TIC pool of NB cells, rather than bulk tumor cells or established cell lines, which would reconcile our findings with the aforementioned observations of SFKs in NB cell lines. Indeed, NB-TICs have been found to differ significantly from established neuroblastoma cell lines in metastatic potential and tumorigenicity in mice, with an unsorted tumor-and metastasis initiation frequency
! 89 ! ! of 4.5 cells rather than the ten to hundreds of thousands for established lines (L. Hansford, unpublished data), as well as cytotoxic responses to compound libraries (K. Smith and D.
Kaplan, unpublished data). In other cancer stem cell models, the proteins that regulate cell survival have also been found to differ between the cancer stem cells versus the bulk population.
For example, in CML, inhibition of BCR-ABL by imatinib ablates the progenitor pool of cells while the cancer stem cells are less sensitive (31). Our study holds the distinct advantage of identifying druggable signaling pathway modulators that are specific to neuroblastoma tumor initiating cells and which can be exploited further for novel therapeutic development.
4.1.1 Treatment of neuroblastoma bone marrow metastatic disease: Utilization of SFK inhibitors dasatinib and bosutinib
Dasatinib
Of the four SFK inhibitors tested on NB-TICs, two compounds, bosutinib (SKI-606) and dasatinib (BMS-354825), were focused on for further study as they are currently used either clinically or are in clinical trials for blood-borne and solid tumor cancers. I found that dasatinib is a potent inhibitor of SKFs in NB-TICs. Erk1/2 is a known downstream effector of SFK signaling and SFK inhibition by dasatinib was accompanied by a decrease in Erk1/2 activity as assessed by western blotting. Similar results were found by Konig and colleagues in CML
CD34(+) progenitor cells, where dasatinib was found to have anti-proliferative effects which were accompanied by a reduction of phospho-SFK and phospho-ERK1/2 levels in growth factor- deficient media (123). Dumka et al obtained similar results in CML cell lines, where dasatinib potently decreased Erk1/2 phosphorylation (153). Interestingly, that study found that dasatinib treatment led to an increase in p38 MAPK (MAPK14) activity and that p38 activation was
! 90 ! ! essential for the cytotoxic effects of dasatinib (154; 153). While the expression and activity of p38 MAPK (MAPK14) in NB-TICs was confirmed by phospho-proteomics and western blotting
(data not shown), the role of p38 activity in dasatinib inhibition of NB-TIC growth remains to be determined.
Dasatinib induces apoptosis in a number of solid tumor cell lines including ovarian cancer, breast cancer and melanoma (155-157). Recent studies have also shown an in vitro anti- proliferative effect of dasatinib on established NB cell lines as well as Ewing Sarcoma, a pediatric and early adulthood neuroectodermal tumor. Interestingly, this study did not find induction of apoptosis as measured by annexin-V staining of either the neuroblastoma or Ewing sarcoma cell lines (158). A second study that tested the effect of dasatinib on neuroblastoma cell line growth in vitro and in vivo did however detect an increase in apoptotic effector activity such as caspase-3/7 in the NB cell line SH-EP (159). These different results may be due to the different neuroblastoma cell lines used in each study. Accordingly, the in vivo efficacy of tumor reduction in dasatinib-treated animals was found to be highly variable with respect to treatment protocol and cell line (159).
The results in this study support the notion that the growth inhibitory effect of dasatinib on NB-TICs can at least in part be attributed to an induction of apoptosis. Unlike previous studies, our study quantified the effect of dasatinib on NB-TICs using three independent readouts for apoptosis induction, including caspase activation, PARP cleavage, and phosphatidylserine exposure on the outer membrane. It should be noted, however, that dasatinib may also drive anti- proliferative effects by inducing senescence or necrosis of NB-TICs. The former effect of dasatinib has previously been shown in the NB cell line SH-EP (159). Future experiments on
! 91 ! !
NB-TICs may substantiate NB-TIC senescence in addition to apoptosis following dasatinib treatment.
The co-administration of dasatinib with other chemotherapy agents such as Ifosfamide and Etoposide is already being tested in phase I/II clinical trials for various recurrent solid tumors including neuroblastoma, in children and young adults (160). In a recent study Snead and colleagues observed that acute dasatinib treatment committed BCR-Abl positive CML cells to an apoptotic fate within 4 hours of exposure, even with subsequent removal of the compound from the culture (161). It remains to be determined if a similar acute response could be generated in
NB-TICs. This would prove highly advantageous for future clinical development for neuroblastoma whereby intermittent or decreased dosing may allow for increased drug tolerability in pediatric patients (162). Our study thus supports the development of dasatinib as a novel agent for neuroblastoma therapy by targeting the NB-TIC population believed to be responsible for recurrent disease.
Bosutinib
Bosutinib (SKI-606) is a third generation BCR/Abl and SFK inhibitor currently in phase
III clinical trials for CML, phase II clinical trials for advanced metastatic breast cancer and phase
I involving other advanced solid tumors (163; 66). The anti-proliferative effect of bosutinib has been established in vitro for many primary cell cultures and cell lines including those derived from primitive and committed CML progenitors, non-small cell lung cancer, and breast cancer
(164; 119; 118; 165). The in vivo efficacy of bosutinib has also been successfully confirmed in xenograft studies using colon, breast, pancreatic, and CML cells lines (164-167). My results indicate that bosutinib is also a potent SFK inhibitor and anti-proliferative agent for NB-TICs.
! 92 ! !
Sakuma and colleagues found that lung adenocarcinoma cells underwent apoptosis following treatment with bosutinib as determined by an increase in caspase 3/7 activity (168).
Furthermore, bosutinib increased PARP cleavage in pancreatic cell line xenografts following treatment in vivo (167). My study also showed that bosutinib is a potent inducer of apoptosis in
NB-TICs, supporting previous observations in other solid tumors. After 24h, approximately one third of the quantified NB-TIC population was found to be in early apoptosis.
Unlike dasatinib, there is no precedent for the efficacy of bosutinib on neuroblastoma cells. My study is the first assessment of SFK inhibition by bosutinib and the respective effect on cell growth and mechanism of toxicity in neuroblastoma cells. Considering that bosutinib is being currently tested clinically for numerous metastatic solid tumors, its utilization in clinical trials for metastatic neuroblastoma may also be feasible pending further in vivo testing in mice.
As preliminary clinical results in adults have shown encouraging pharmacokinetic and safety profiles, the adaptation of bosutinib to pediatric patients may be a promising avenue for further drug development for neuroblastoma (169).
4.2 Future Directions: Characterization and exploitation of the hematopoietic phenotype of NB-TICs
Preamble
This thesis identified unique molecular features of NB-TICs, which allowed for the targeting of novel signaling effectors and pathways in neuroblastoma. I propose that by targeting the B cell receptor signaling pathway and affiliated Src family kinases, the growth of tumor initiating population of neuroblastoma cells can be prevented. With the goal of developing SFK inhibitors to standard neuroblastoma treatments, there are important scientific questions that need
! 93 ! ! to be addressed in order to conclusively establish the link between the B cell receptor pathway, the SFKs and cellular survival as implicated in the progression of metastatic neuroblastoma. The experiments proposed herein will serve to address these questions in order to link hematopoietic signaling pathways to the survival of cancer stem cells derived from a hematopoietic niche. This may establish a basis upon which to question if similar relationships are present in other cancer stem cells, such as those in breast cancer, which are known to metastasize to the bone marrow
(170).
4.2.0 Validating BCR signaling: The link between SFK, SYK and B cell membrane receptors
The next stage in characterizing the putative BCR signaling pathway in NB-TICs is to validate functional interactions between the respective components. As outlined in the proposed signaling cascade in Figure 23, an extensive set of connections comprised of kinase-substrate interactions have previously been described in B-cells and can be probed in NB-TICs by immunoprecipitation approaches. An interaction network between BCR co-receptors, scaffolding proteins and kinases may also be generated on a high-throughput platform by utilizing proteomic approaches. Coupling co-immunoprecipitation to protein identification by tandem mass spectrometry will allow for the determination of upstream and downstream interaction partners for a particular kinase or receptor, thus validating its position in the pathway.
Functional upstream and downstream relationships between BCR pathway kinases, such as SFK, SYK and BTK, and phosphatases, such as CD45 and SHP2, can be assessed biochemically through knockdown approaches (69). The effect on the phosphorylation level of putative downstream targets can subsequently be tested by using phospho-specific antibodies.
Such functional relationships, in addition to identification of direct interactions, will allow for
! 94 ! ! the construction of an accurate pathway model of BCR signaling in NB-TICs. These approaches will be essential in order to ascertain the role of the activated SFKs in the BCR signaling pathway in NB-TICs.
4.2.1 Testing hematopoietic signatures in non-bone marrow derived NB-TIC lines
The hematopoietic signaling pathways identified by phospho-proteomics have been established from a set of five bone marrow derived NB-TIC lines. It remains to be determined if these hematopoietic features are also conserved in NB-TICs derived from tumor specimen of other biopsy sites such as brain and liver metastases. The hypothesis would be that if the expression of hematopoietic surface markers and signaling pathways is a niche-specific adaptation of NB-TICs, then only bone marrow derived NB-TICs should express them.
I suggest that a microarray or transcriptome sequencing approach should be employed to ascertain the presence or absence of hematopoietic features in non-bone marrow derived NB-
TICs. Microarray and shotgun transcriptome sequencing are appropriate methods for this purpose as non-bone marrow derived NB-TIC lines are slow-growing in vitro, making them unsuitable for a phospho-tyrosine enrichment approach, where typically 100 x106 NB-TICs are required for a successful phospho-tyrosine enrichment analysis. However, a new mass spectrometry-based phospho-proteomics technique based on selected reaction monitoring (SRM, also known as multiple reaction monitoring, or MRM) has been developed and applied to measure SFK activation in complex, minute samples including tumor specimens (171). This kind of emerging methodology may facilitate the targeted measurement of hematopoietic features in the more slowly growing and minute non-bone-marrow-derived NB-TICs without prior enrichment steps that require large protein amounts.
! 95 ! !
I was able to expand a single non-bone marrow derived line, NB153, for one phospho- proteomics attempt, however due to the low yield of pY proteins, all comparisons remain of a preliminary, qualitative nature only. For NB153 I observed the expression and phosphorylation of the kinases FAK, PI3K, GSK3!, PRP4, p38MAPK, MAPK1/3, as well as the SFKs LCK and
Fyn.
4.2.2 Testing the efficacy of SFK inhibitors dasatinib and bosutinib in vivo
The SFK inhibitors dasatinib and bosutinib were potent anti-proliferative agents for NB-
TICs in vitro. To begin to determine their efficacy in in vivo, we have initiated studies in xenograft models in mice. Such studies are particularly important for testing bosutinib in vivo, as there is currently no pre-clinical data testing its effect on NB cell lines.
Two NB-TIC tumorigenesis models have been developed in NOD/SCID or SCID/beige mice, where cells are injected either into the mammary fat-pad or the adrenal fat pad (orthotopic model) (35). Together with Natalie Grinstein (The Hospital for Sick Children) I have helped develop and optimize a third injection protocol for tumor establishment in the sub-dermal skin of
NOD/SCID mice. The advantage of this protocol is that NB-TICs grow robustly in this site, and the tumors can be readily measured. Bosutinib and dasatinib have typically been delivered to rodents orally, reflecting the current clinical administration of the compounds (165; 167; 172).
Therefore, the effect of either SFK inhibitor will be assessed on NB-TIC tumor establishment and growth using this method. Furthermore, combinatorial treatments will be delivered with bosutinib or dasatinib, including for example, a currently utilized front-line neuroblastoma chemotherapy drug, such as irinotecan. Together, these experiments predispose the further development of dasatinib and bosutinib as a candidate compound for clinical trials in neuroblastoma patients.
! 96 ! !
4.2.3 Assessing the role of Insulin receptor and Fibroblast growth factor receptor pathways in NB-TICs
Phosphoproteomics identified peptides from the insulin receptor (IR) and fibroblast growth factor receptor (FGFR). Furthermore, NB-TICs appear to respond to insulin and FGF in culture media in a B27 background. As receptor tyrosine kinases (RTKs) are known to play crucial roles in the progression of numerous solid tumor malignancies such as lung, breast, and pancreatic cancer, I propose future studies to determine if these receptors play a role in NB-TIC proliferation (173). Confirmation of expression and phosphorylation of these receptor tyrosine kinases in bone marrow derived NB-TICs can be done by phospho- and protein specific antibodies by western blotting. Furthermore, knockdown experiments of IR and FGFR can be pursued, where cell viability and proliferation can be measured by counting of live cells using trypan blue. If a phenotype can be established, the differences in activation of putative downstream signaling effectors can be monitored in RTK knockdown versus wildtype cells by phospho-protein specific antibodies and western blotting. Together, this may elucidate putative pathways downstream of these RTKs which may potentially feed into effector kinases shared by hematopoietic signaling pathways, such as SFKs, Ras/Erk1/2 and PLC".
! 97 ! !
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Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
! 113 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
Biological Protein name Protein accession numbers Peptide sequence pY site cited Best Best Best X! Peptide start Peptide stop Other Proteins sample in text SEQUEST SEQUEST Tandem index index XCorr score DCn score log(e) score NB12-2 - 101 kDa protein IPI00792810 ALPAQVDDPPEPVYANIERQPR 2.65 0.6 0 259 280 NB12 - 101 kDa protein IPI00792810 ALPAQVDDPPEPVYANIER 3.91 0.634 8.96 259 277 NB12-2 - 101 kDa protein IPI00792810 ALPAQVDDPPEPVYANIER 3.87 0.627 5.8 259 277 NB25-V - 101 kDa protein IPI00792810 ALPAQVDDPPEPVYANIER 3.81 0.675 7.19 259 277 NB88-V - 101 kDa protein IPI00792810 ALPAQVDDPPEPVYANIER 3.8 0.663 9.68 259 277 NB25-V - 101 kDa protein IPI00792810 ALPAQVDDPPEPVYANIER 3.53 0.685 2 259 277 NB88-2 - 101 kDa protein IPI00792810 ALPAQVDDPPEPVYANIER 3.52 0.615 6.08 259 277 NB25 - 101 kDa protein IPI00792810 ALPAQVDDPPEPVYANIER 2.83 0.595 4.72 259 277 NB61 - 61 kDa protein IPI00879128 RSAAYVRNILDNAVAK 1.99 0.317 0 255 270 NB88-2 - 61 kDa protein IPI00879128 RSAAYVRNILDNAVAK 1.63 0.328 0 255 270 ABCF2 ATP-binding cassette sub-family F NB12-2 member 2 IPI00005045 EVPIPEHIDIYHLTR 2.58 0.354 0 135 149 NB88-V ABI1 Isoform 1 of Abl interactor 1 IPI00431025 TLEPVKPPTVPNDYMTSPAR 1.99 0.513 1.09 200 219 IPI00216969,IPI00221171,IPI00 ABL1 Isoform IA of Proto-oncogene tyrosine- 329488,IPI00383728,IPI007195 NB88 protein kinase ABL1 87,IPI00745593,IPI00855759 LMTGDTYTAHAGAK 3.98 0.652 6.74 387 400 IPI00216969,IPI00221171,IPI00 ABL1 Isoform IA of Proto-oncogene tyrosine- 329488,IPI00383728,IPI007195 NB122-2 protein kinase ABL1 87,IPI00745593,IPI00855759 LMTGDTYTAHAGAK 3.38 0.649 5.96 387 400 IPI00216969,IPI00221171,IPI00 ABL1 Isoform IA of Proto-oncogene tyrosine- 329488,IPI00383728,IPI007195 NB88-2 protein kinase ABL1 87,IPI00745593,IPI00855759 LMTGDTYTAHAGAK 3.32 0.643 6.26 387 400 IPI00216969,IPI00221171,IPI00 ABL1 Isoform IA of Proto-oncogene tyrosine- 329488,IPI00383728,IPI007195 NB12 protein kinase ABL1 87,IPI00745593,IPI00855759 LMTGDTYTAHAGAK 3.04 0.56 5.1 387 400 IPI00216969,IPI00221171,IPI00 ABL1 Isoform IA of Proto-oncogene tyrosine- 329488,IPI00383728,IPI007195 NB12-2 protein kinase ABL1 87,IPI00745593,IPI00855759 LMTGDTYTAHAGAK 2.75 0.493 5.36 387 400 IPI00329488,IPI00221171,IPI008 ABL1 Isoform IA of Proto-oncogene tyrosine- 55759,IPI00719587,IPI00383728,I NB122 protein kinase ABL1 IPI00216969 LMTGDTYTAHAGAK 2.69 0.507 0 387 400 PI00745593 IPI00216969,IPI00221171,IPI00 ABL1 Isoform IA of Proto-oncogene tyrosine- 329488,IPI00383728,IPI007195 NB61 protein kinase ABL1 87,IPI00745593,IPI00855759 LMTGDTYTAHAGAK 2.31 0.368 2.04 387 400 NB88-2 ACLY ATP-citrate synthase IPI00021290 TTDGVYEGVAIGGDRYPGSTFMDHVLR 3.55 0.484 0 677 703 NB61 ACLY ATP-citrate synthase IPI00021290 TTDGVYEGVAIGGDRYPGSTFMDHVLR 3.03 0.48 0 677 703 NB25-V ACTB Actin, cytoplasmic 1 IPI00021439,IPI00021440 TTGIVMDSGDGVTHTVPIYEGYALPHAILR 6.66 0.759 0 148 177 NB25 ACTB Actin, cytoplasmic 1 IPI00021439,IPI00021440 TTGIVMDSGDGVTHTVPIYEGYALPHAILR 5.44 0.632 5.64 148 177 NB88-V ACTB Actin, cytoplasmic 1 IPI00021439,IPI00021440 TTGIVMDSGDGVTHTVPIYEGYALPHAILR 4.71 0.682 8.28 148 177 NB88-2 ACTB Actin, cytoplasmic 1 IPI00021439,IPI00021440 TTGIVMDSGDGVTHTVPIYEGYALPHAILR 4.28 0.624 4.52 148 177 NB12-2 ACTB Actin, cytoplasmic 1 IPI00021439,IPI00021440 TTGIVMDSGDGVTHTVPIYEGYALPHAILR 4.18 0.56 3.82 148 177 NB61 ACTB Actin, cytoplasmic 1 IPI00021439,IPI00021440 TTGIVMDSGDGVTHTVPIYEGYALPHAILR 3.92 0.553 1.68 148 177 NB25-V ACTB Actin, cytoplasmic 1 IPI00021439,IPI00021440 TTGIVMDSGDGVTHTVPIYEGYALPHAILR 3.37 0.614 7.72 148 177 NB12 ACTB Actin, cytoplasmic 1 IPI00021439,IPI00021440 TTGIVMDSGDGVTHTVPIYEGYALPHAILR 3.34 0.659 3.52 148 177 NB25-V ACTB Actin, cytoplasmic 1 IPI00021439,IPI00021440 KDLYANTVLSGGTTMYPGIADR 5.04 0.671 11.7 291 312 NB88-2 ACTB Actin, cytoplasmic 1 IPI00021439,IPI00021440 KDLYANTVLSGGTTMYPGIADR 4.61 0.615 3.44 291 312 NB12-2 ACTB Actin, cytoplasmic 1 IPI00021439,IPI00021440 KDLYANTVLSGGTTMYPGIADR 3.92 0.48 3.74 291 312 NB12 ACTB Actin, cytoplasmic 1 IPI00021439,IPI00021440 KDLYANTVLSGGTTMYPGIADR 3.6 0.67 13.1 291 312 NB122-2 ACTB Actin, cytoplasmic 1 IPI00021439,IPI00021440 KDLYANTVLSGGTTMYPGIADR 3.05 0.392 0 291 312 NB25-V ACTB Actin, cytoplasmic 1 IPI00021439,IPI00021440 IWHHTFYNELR 3.99 0.575 1.82 85 95 NB25 ACTB Actin, cytoplasmic 1 IPI00021439,IPI00021440 IWHHTFYNELR 2.98 0.485 1.62 85 95 NB88-V ACTB Actin, cytoplasmic 1 IPI00021439,IPI00021440 IWHHTFYNELR 2.97 0.481 1.47 85 95 NB12 ACTB Actin, cytoplasmic 1 IPI00021439,IPI00021440 IWHHTFYNELR 2.69 0.535 2.74 85 95 NB12-2 ACTB Actin, cytoplasmic 1 IPI00021439,IPI00021440 HQGVMVGMGQKDSYVGDEAQSKR 4.39 0.711 1.96 40 62 NB12-2 ACTB Actin, cytoplasmic 1 IPI00021439,IPI00021440 DSYVGDEAQSKR 2.76 0.227 1.36 51 62 NB25-V ACTB Actin, cytoplasmic 1 IPI00021439,IPI00021440 DSYVGDEAQSKR 2.55 0.0315 3.8 51 62 NB88-V ACTB Actin, cytoplasmic 1 IPI00021439,IPI00021440 DSYVGDEAQSKR 1.77 0.208 2.89 51 62 NB88 ACTB Actin, cytoplasmic 1 IPI00021439,IPI00021440 DSYVGDEAQSKR 1.6 0.252 1.74 51 62 NB12-2 ACTB Actin, cytoplasmic 1 IPI00021439,IPI00021440 DSYVGDEAQSK 2.72 0.204 5.22 51 61 NB88 ACTB Actin, cytoplasmic 1 IPI00021439,IPI00021440 DSYVGDEAQSK 2.58 0.0323 5.82 51 61 NB88-2 ACTB Actin, cytoplasmic 1 IPI00021439,IPI00021440 CDVDIRKDLYANTVLSGGTTMYPGIADR 3.54 0.539 3.29 285 312 NB12-2 ACTB Actin, cytoplasmic 1 IPI00021439,IPI00021440 CDVDIRKDLYANTVLSGGTTMYPGIADR 3.53 0.602 2.82 285 312 NB12 ACTB Actin, cytoplasmic 1 IPI00021439,IPI00021440 CDVDIRKDLYANTVLSGGTTMYPGIADR 2.87 0.516 4.55 285 312 IPI00219065,IPI00219066,IPI00 AGL Isoform 5 of Glycogen debranching 328318,IPI00514126,IPI008787 NB88 enzyme 98 EAMSAYNSHEEGR 2.27 0.568 5.01 562 574
114 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
IPI00219065,IPI00219066,IPI00 AGL Isoform 5 of Glycogen debranching 328318,IPI00514126,IPI008787 NB12 enzyme 98 EAMSAYNSHEEGR 2.05 0.414 5.74 562 574 IPI00219065,IPI00219066,IPI00 AGL Isoform 5 of Glycogen debranching 328318,IPI00514126,IPI008787 NB12-2 enzyme 98 EAMSAYNSHEEGR 2.05 0.785 3.62 562 574 NB88 AHCY Adenosylhomocysteinase IPI00012007 SKFDNLYGCR 3.18 0.667 1.43 187 196 NB12 AHCY Adenosylhomocysteinase IPI00012007 SKFDNLYGCR 2.86 0.616 3.49 187 196 NB12-2 AHCY Adenosylhomocysteinase IPI00012007 SKFDNLYGCR 2.86 0.539 1.09 187 196 NB88-2 AHCY Adenosylhomocysteinase IPI00012007 SKFDNLYGCR 2.75 0.582 3.15 187 196 NB122 AHCY Adenosylhomocysteinase IPI00012007 SKFDNLYGCR 1.82 0.627 1.15 187 196 AMICA1 Isoform 2 of Junctional adhesion NB25-V molecule-like precursor IPI00183703,IPI00385063 HIYSPIIVR 2.11 0.519 2.41 337 345 NB12-2 ANXA1 Annexin A1 IPI00218918 ALYEAGERR 2.29 0.4 0 205 213 NB88 ANXA1 Annexin A1 IPI00218918 ALYEAGERR 1.86 0.261 1.21 205 213 NB12-2 ANXA1 Annexin A1 IPI00218918 ALYEAGER 1.61 0.552 0 205 212 IPI00334627,IPI00418169,IPI00 NB88 ANXA2P2 Putative annexin A2-like protein 455315 LSLEGDHSTPPSAYGSVK 4.9 0.536 5.64 11 28 IPI00334627,IPI00418169,IPI00 NB12-2 ANXA2P2 Putative annexin A2-like protein 455315 LSLEGDHSTPPSAYGSVK 4.3 0.424 4.85 11 28 IPI00334627,IPI00418169,IPI00 NB25-V ANXA2P2 Putative annexin A2-like protein 455315 LSLEGDHSTPPSAYGSVK 3.95 0.45 3.42 11 28 IPI00334627,IPI00418169,IPI00 NB12 ANXA2P2 Putative annexin A2-like protein 455315 LSLEGDHSTPPSAYGSVK 3.91 0.596 4.24 11 28 IPI00334627,IPI00418169,IPI00 NB88-V ANXA2P2 Putative annexin A2-like protein 455315 LSLEGDHSTPPSAYGSVK 3.9 0.598 6.77 11 28 IPI00334627,IPI00418169,IPI00 NB25-V ANXA2P2 Putative annexin A2-like protein 455315 LSLEGDHSTPPSAYGSVK 3.8 0.636 1.77 11 28 NB122 ANXA2P2 Putative annexin A2-like protein IPI00334627 LSLEGDHSTPPSAYGSVK 2.04 0.515 0 11 28 IPI00455315,IPI00418169 NB88-V ANXA6 annexin VI isoform 2 IPI00002459 NKPLFFADKLYK 3.06 0.6 0 593 604 NB88 ANXA6 annexin VI isoform 2 IPI00002459 NKPLFFADKLYK 2.48 0.416 2.96 593 604 NB12 ANXA6 annexin VI isoform 2 IPI00002459 LIVGLMRPPAYCDAK 3.81 0.651 4.4 85 99 NB12-2 ANXA6 annexin VI isoform 2 IPI00002459 LIVGLMRPPAYCDAK 3.74 0.536 1.8 85 99 NB88-2 ANXA6 annexin VI isoform 2 IPI00002459 LIVGLMRPPAYCDAK 3.66 0.561 2.34 85 99 NB25-V ANXA6 annexin VI isoform 2 IPI00002459 LIVGLMRPPAYCDAK 3.64 0.641 0 85 99 NB88 ANXA6 annexin VI isoform 2 IPI00002459 LIVGLMRPPAYCDAK 3.04 0.549 0 85 99 NB25 ANXA6 annexin VI isoform 2 IPI00002459 LIVGLMRPPAYCDAK 2.96 0.463 2.05 85 99 NB88-V ANXA6 annexin VI isoform 2 IPI00002459 LIVGLMRPPAYCDAK 2.43 0.281 0 85 99 NB122 ANXA6 annexin VI isoform 2 IPI00002459 LIVGLMRPPAYCDAK 2.39 0.493 0 85 99 NB25-V ANXA6 annexin VI isoform 2 IPI00002459 LIVGLMRPPAYCDAK 2.21 0.212 1.85 85 99 NB122-2 ANXA6 annexin VI isoform 2 IPI00002459 LIVGLMRPPAYCDAK 1.75 0.455 0 85 99 NB12-2 APLP2 Isoform 1 of Amyloid-like protein 2 IPI00031030 MQNHGYENPTYK 2.93 0.682 0 745 756 IPI00006608,IPI00219183,IPI00 219185,IPI00219186,IPI002191 87,IPI00394658,IPI00412568,IP APP Isoform APP770 of Amyloid beta A4 I00412681,IPI00412924,IPI0090 NB12-2 protein (Fragment) 9502 MQQNGYENPTYK 3.32 0.61 2.35 752 763 IPI00219186,IPI00412924,IPI003 94658,IPI00219185,IPI00219187,I APP Isoform APP770 of Amyloid beta A4 PI00219183,IPI00909502,IPI0041 NB122 protein (Fragment) IPI00006608 MQQNGYENPTYK 1.71 0.404 0 752 763 2681,IPI00412568 ARD1A N-terminal acetyltransferase complex NB12-2 ARD1 subunit homolog A IPI00013184 YYADGEDAYAMKR 1.95 0.732 0 137 149 NB88-V ARIH2 Protein ariadne-2 homolog IPI00007304,IPI00792023 THGSEYYECSR 3.03 0.898 2.92 332 342 IPI00902655 IPI00007304,IPI00792023,IPI00 NB88-2 ARIH2 Protein ariadne-2 homolog 902655 THGSEYYECSR 3 0.725 1.7 332 342 IPI00007304,IPI00792023,IPI00 NB88 ARIH2 Protein ariadne-2 homolog 902655 THGSEYYECSR 2.55 0.777 1.44 332 342 ASH1L Probable histone-lysine N- NB61 methyltransferase ASH1L IPI00020546 AGLYSDVYK 1.13 0.505 0 1992 2000 ATIC Bifunctional purine biosynthesis protein NB88 PURH IPI00289499 VCMVYDLYK 2.83 0.495 0 286 294 ATP1A1 Isoform Long of Sodium/potassium- IPI00006482,IPI00414005,IPI00 NB12-2 transporting ATPase subunit alpha-1 646182 GIVVYTGDR 1.94 0.287 1.14 256 264 ATP1A1 Isoform Long of Sodium/potassium- NB122 transporting ATPase subunit alpha-1 IPI00006482 GIVVYTGDR 1.7 0.459 0 256 264 IPI00646182,IPI00414005
NB88 ATP6V1E1 Vacuolar proton pump subunit E 1 IPI00003856 IMEYYEKK 2.68 0.37 0 53 60 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB25-V resistanceprotein 1 872386 VGQGYVYEAAQPEQDEYDIPR 4.23 0.659 6.8 218 238
115 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB122-2 resistanceprotein 1 872386 RPGPGTLYDVPRER 2.33 0.508 1.38 398 411 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB25-V resistanceprotein 1 872386 RPGPGTLYDVPRER 2.31 0.403 0 380 393 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB25-V resistanceprotein 1 872386 RPGPGTLYDVPRER 2.24 0.445 1.6 380 393 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB12 resistanceprotein 1 872386 RPGPGTLYDVPRER 2.05 0.513 1.43 398 411 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB88 resistanceprotein 1 872386 RPGPGTLYDVPRER 1.96 0.394 1.05 398 411 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB12-2 resistanceprotein 1 872386 RPGPGTLYDVPRER 1.8 0.362 0 398 411 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB122 resistanceprotein 1 872386 RPGPGTLYDVPR 2.6 0.486 0 398 409 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB25-V resistanceprotein 1 872386 RPGPGTLYDVPR 2.48 0.633 1.52 380 391 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen NB25 resistanceprotein 1 IPI00641339 RPGPGTLYDVPR 2.46 0.56 0 398 409 IPI00872386,IPI00645994 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB12 resistanceprotein 1 872386 RPGPGTLYDVPR 2.41 0.579 1.77 398 409 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB25-V resistanceprotein 1 872386 RPGPGTLYDVPR 2.25 0.561 0 380 391 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB122-2 resistanceprotein 1 872386 RPGPGTLYDVPR 2.13 0.576 0 398 409 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen NB61 resistanceprotein 1 IPI00641339 RPGPGTLYDVPR 2.11 0.451 0 398 409 IPI00872386,IPI00645994 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB12-2 resistanceprotein 1 872386 RPGPGTLYDVPR 1.97 0.621 2.06 398 409 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB88-2 resistanceprotein 1 872386 RPGPGTLYDVPR 1.56 0.483 0 398 409 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB88 resistanceprotein 1 872386 RPGPGTLYDVPR 1.4 0.466 1.18 398 409 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB25-V resistanceprotein 1 872386 HLLAPGPQDIYDVPPVR 5.01 0.677 11.6 239 255 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB122-2 resistanceprotein 1 872386 HLLAPGPQDIYDVPPVR 4.47 0.697 4.57 257 273 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB88-2 resistanceprotein 1 872386 HLLAPGPQDIYDVPPVR 4.38 0.683 4.49 257 273 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB12-2 resistanceprotein 1 872386 HLLAPGPQDIYDVPPVR 4.29 0.723 10.5 257 273 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB88-V resistanceprotein 1 872386 HLLAPGPQDIYDVPPVR 4.27 0.714 8.08 239 255 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB25-V resistanceprotein 1 872386 HLLAPGPQDIYDVPPVR 4.14 0.655 2.35 239 255 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB12 resistanceprotein 1 872386 HLLAPGPQDIYDVPPVR 3.66 0.683 9.96 257 273
116 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB88 resistanceprotein 1 872386 HLLAPGPQDIYDVPPVR 3.42 0.647 9.14 257 273 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB122 resistanceprotein 1 872386 HLLAPGPQDIYDVPPVR 3.18 0.688 5.06 257 273 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB25-V resistanceprotein 1 872386 GLPPSNHHAVYDVPPSVSK 1.86 0.802 1.4 296 314 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB25-V resistanceprotein 1 872386 GLLPSQYGQEVYDTPPMAVK 3.35 0.624 6.31 256 275 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB12 resistanceprotein 1 872386 GLLPSQYGQEVYDTPPMAVK 3.22 0.534 3.52 274 293 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB25-V resistanceprotein 1 872386 AQQGLYQVPGPSPQFQSPPAK 3.59 0.643 5.21 123 143 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB12-2 resistanceprotein 1 872386 AQQGLYQVPGPSPQFQSPPAK 3.53 0.524 2.14 141 161 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB12 resistanceprotein 1 872386 AQQGLYQVPGPSPQFQSPPAK 3.5 0.615 5.55 141 161 BCAR1 cDNA FLJ55515, highly similar to Breast cancer anti-estrogen IPI00641339,IPI00645994,IPI00 NB122-2 resistanceprotein 1 872386 AQQGLYQVPGPSPQFQSPPAK 3.41 0.468 3.77 141 161 BCR Isoform 1 of Breakpoint cluster region NB88-V protein IPI00004497 KGHGQPGADAEKPFYVNVEFHHER 3.16 0.536 0 163 186 NB12-2 BLK BLK protein (Fragment) IPI00554756 IIDSEYTAQEGAK pY 389 3.34 0.64 5.64 384 396 NB25-V BLK BLK protein (Fragment) IPI00554756 CLDEGGYYISPR pY 187 4.31 0.616 6.62 181 192 NB12-2 BLK BLK protein (Fragment) IPI00554756 CLDEGGYYISPR pY 188 3.79 0.696 6.72 181 192 NB12 BLK BLK protein (Fragment) IPI00554756 CLDEGGYYISPR pY 188 3.6 0.64 7.2 181 192 NB122-2 BLK BLK protein (Fragment) IPI00554756 CLDEGGYYISPR pY 187 3.58 0.592 5.92 181 192 NB25-V BLK BLK protein (Fragment) IPI00554756 CLDEGGYYISPR pY 188 3.45 0.624 5.15 181 192 NB61 BLK BLK protein (Fragment) IPI00554756 CLDEGGYYISPR pY 188 3.22 0.655 4.24 181 192 NB12 BTK Tyrosine-protein kinase BTK IPI00029132 YVLDDEYTSSVGSKFPVR pY 551 4.18 0.649 6 545 562 NB12-2 BTK Tyrosine-protein kinase BTK IPI00029132 YVLDDEYTSSVGSKFPVR pY 551 4.08 0.471 5.13 545 562 NB25-V BTK Tyrosine-protein kinase BTK IPI00029132 YVLDDEYTSSVGSKFPVR pY 551 3.54 0.425 5.11 545 562 NB88-2 BTK Tyrosine-protein kinase BTK IPI00029132 VVALYDYMPMNANDLQLR pY 223 4.79 0.638 7.39 219 236 NB88-V BTK Tyrosine-protein kinase BTK IPI00029132 VVALYDYMPMNANDLQLR pY 223 4.56 0.673 9.44 219 236 NB25-V BTK Tyrosine-protein kinase BTK IPI00029132 VVALYDYMPMNANDLQLR pY 223 3.81 0.594 4.33 219 236 NB12-2 BTK Tyrosine-protein kinase BTK IPI00029132 LKYPVSQQNK pY 375 2.3 0.334 1.59 373 382 NB88 BTK Tyrosine-protein kinase BTK IPI00029132 LKYPVSQQNK pY 375 2.15 0.46 0 373 382 NB88-2 BTK Tyrosine-protein kinase BTK IPI00029132 KVVALYDYMPMNANDLQLR pY 223 5.67 0.599 11.8 218 236 NB88-V BTK Tyrosine-protein kinase BTK IPI00029132 KVVALYDYMPMNANDLQLR pY 223 4.86 0.703 16.2 218 236 NB25-V BTK Tyrosine-protein kinase BTK IPI00029132 KVVALYDYMPMNANDLQLR pY 223 4.61 0.683 0 218 236 NB12 BTK Tyrosine-protein kinase BTK IPI00029132 KVVALYDYMPMNANDLQLR pY 223 4.28 0.665 13.7 218 236 NB25 BTK Tyrosine-protein kinase BTK IPI00029132 KVVALYDYMPMNANDLQLR pY 223 3.77 0.688 8.77 218 236 NB12-2 BTK Tyrosine-protein kinase BTK IPI00029132 KVVALYDYMPMNANDLQLR pY 325 3.6 0.585 2.55 218 236 NB122-2 BTK Tyrosine-protein kinase BTK IPI00029132 KVVALYDYMPMNANDLQLR pY 225 3.36 0.482 1.55 218 236 NB61 BTK Tyrosine-protein kinase BTK IPI00029132 KVVALYDYMPMNANDLQLR pY 223 2.51 0.64 1.77 218 236 NB88 BTK Tyrosine-protein kinase BTK IPI00029132 HYVVCSTPQSQYYLAEK pY 344 4.76 0.689 11.7 333 349 NB12 BTK Tyrosine-protein kinase BTK IPI00029132 HYVVCSTPQSQYYLAEK pY 344 4.7 0.638 7.85 333 349 NB88-2 BTK Tyrosine-protein kinase BTK IPI00029132 HYVVCSTPQSQYYLAEK pY 344 4.55 0.656 4.62 333 349 NB12-2 BTK Tyrosine-protein kinase BTK IPI00029132 HYVVCSTPQSQYYLAEK pY 344 4.46 0.599 5.25 333 349 NB88-V BTK Tyrosine-protein kinase BTK IPI00029132 HYVVCSTPQSQYYLAEK pY 344 4.02 0.752 13 333 349 NB25 BTK Tyrosine-protein kinase BTK IPI00029132 HYVVCSTPQSQYYLAEK pY 344 3.41 0.614 4.7 333 349 NB122-2 BTK Tyrosine-protein kinase BTK IPI00029132 HYVVCSTPQSQYYLAEK pY 345 2.97 0.561 0 333 349 NB25 BTK Tyrosine-protein kinase BTK IPI00029132 HLFSTIPELINYHQHNSAGLISR pY 361 4.15 0.568 0 350 372 NB25-V BTK Tyrosine-protein kinase BTK IPI00029132 HLFSTIPELINYHQHNSAGLISR pY 361 3.07 0.585 2.24 350 372 NB12-2 BTK Tyrosine-protein kinase BTK IPI00029132 HLFSTIPELINYHQHNSAGLISR pY 361 2.44 0.42 0 350 372 NB88-V BTK Tyrosine-protein kinase BTK IPI00029132 HLFSTIPELINYHQHNSAGLISR pY 361 2.43 0.459 0 350 372 MQEGSEVYSNPCLEENKPGIVYASLNHSVIGL NB25-V BTLA B- and T-lymphocyte attenuator IPI00376243 NSR 4.21 0.712 0 236 270 MQEGSEVYSNPCLEENKPGIVYASLNHSVIGL NB61 BTLA B- and T-lymphocyte attenuator IPI00376243 NSR 2.68 0.429 0 236 270 BTN2A2 Isoform 1 of Butyrophilin subfamily NB88-V 2 member A2 IPI00013576 SQFSPAVFVYKGGRER 2.19 0.279 0 73 88 BTN2A2 Isoform 1 of Butyrophilin subfamily NB88 2 member A2 IPI00013576 SQFSPAVFVYKGGRER 2.13 0.338 0 73 88
117 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
BTN2A2 Isoform 1 of Butyrophilin subfamily NB122-2 2 member A2 IPI00013576 SQFSPAVFVYKGGRER 2.08 0.374 0 73 88 NB25-V C11orf59 UPF0404 protein C11orf59 IPI00016670 ALNGAEPNYHSLPSAR 2.67 0.57 0 32 47 NB12-2 C2orf71 Uncharacterized protein FLJ34931 IPI00397879 REPQEQPNLLQQLLQYTVSK 1.94 0.454 0 258 277 NB88 C2orf71 Uncharacterized protein FLJ34931 IPI00397879 REPQEQPNLLQQLLQYTVSK 1.93 0.298 0 258 277 NB122 C2orf71 Uncharacterized protein FLJ34931 IPI00397879 REPQEQPNLLQQLLQYTVSK 1.86 0.477 0 258 277 NB12 C2orf71 Uncharacterized protein FLJ34931 IPI00397879 REPQEQPNLLQQLLQYTVSK 1.85 0.403 0 258 277 NB12 CALM1;CALM2;CALM3 Calmodulin IPI00075248 VFDKDGNGYISAAELR 4.83 0.655 9.82 92 107 NB88-V CALM1;CALM2;CALM3 Calmodulin IPI00075248 VFDKDGNGYISAAELR 4.78 0.694 11.7 92 107 NB25-V CALM1;CALM2;CALM3 Calmodulin IPI00075248 VFDKDGNGYISAAELR 4.41 0.589 8.96 92 107 NB88 CALM1;CALM2;CALM3 Calmodulin IPI00075248 VFDKDGNGYISAAELR 4.29 0.561 3.32 92 107 NB25 CALM1;CALM2;CALM3 Calmodulin IPI00075248 VFDKDGNGYISAAELR 4.18 0.66 5 92 107 NB25-V CALM1;CALM2;CALM3 Calmodulin IPI00075248 VFDKDGNGYISAAELR 3.35 0.537 4.41 92 107 NB12-2 CALM1;CALM2;CALM3 Calmodulin IPI00075248 VFDKDGNGYISAAELR 3.24 0.614 3.55 92 107 NB122-2 CALM1;CALM2;CALM3 Calmodulin IPI00075248 VFDKDGNGYISAAELR 3.22 0.572 2.16 92 107 NB88-2 CALM1;CALM2;CALM3 Calmodulin IPI00075248 VFDKDGNGYISAAELR 3.19 0.584 2.01 92 107 NB122 CALM1;CALM2;CALM3 Calmodulin IPI00075248 VFDKDGNGYISAAELR 3.16 0.602 1.17 92 107 LPPGEQCEGEEDTEYMTPSSRPLRPLDTSQSS NB122-2 CBL E3 ubiquitin-protein ligase CBL IPI00027269 R 3.33 0.639 1.06 686 718 LPPGEQCEGEEDTEYMTPSSRPLRPLDTSQSS NB88-2 CBL E3 ubiquitin-protein ligase CBL IPI00027269 R 3.29 0.556 1.2 686 718 LPPGEQCEGEEDTEYMTPSSRPLRPLDTSQSS NB12-2 CBL E3 ubiquitin-protein ligase CBL IPI00027269 R 2.8 0.635 0 686 718 LPPGEQCEGEEDTEYMTPSSRPLRPLDTSQSS NB88-V CBL E3 ubiquitin-protein ligase CBL IPI00027269 R 2.23 0.509 0 686 718 LPPGEQCEGEEDTEYMTPSSRPLRPLDTSQSS NB25-V CBL E3 ubiquitin-protein ligase CBL IPI00027269 R 2.17 0.663 1.7 686 718 LPPGEQCEGEEDTEYMTPSSRPLRPLDTSQSS NB12 CBL E3 ubiquitin-protein ligase CBL IPI00027269 R 1.88 0.468 0 686 718 LPPGEQCEGEEDTEYMTPSSRPLRPLDTSQSS NB61 CBL E3 ubiquitin-protein ligase CBL IPI00027269 R 1.87 0.529 0 686 718 LPPGEQCEGEEDTEYMTPSSRPLRPLDTSQSS NB25-V CBL E3 ubiquitin-protein ligase CBL IPI00027269 R 1.43 0.438 3.48 686 718 NB88-2 CBL E3 ubiquitin-protein ligase CBL IPI00027269 IKPSSSANAIYSLAARPLPVPK 1.97 0.373 0 664 685 NB88 CBL E3 ubiquitin-protein ligase CBL IPI00027269 IKPSSSANAIYSLAARPLPVPK 1.87 0.55 3.34 664 685 NB25-V CBL E3 ubiquitin-protein ligase CBL IPI00027269 IKPSSSANAIYSLAARPLPVPK 1.87 0.635 4.6 664 685 NB88-V CBL E3 ubiquitin-protein ligase CBL IPI00027269 IKPSSSANAIYSLAARPLPVPK 1.49 0.25 3.38 664 685 NB25 CBL E3 ubiquitin-protein ligase CBL IPI00027269 IKPSSSANAIYSLAARPLPVPK 1.39 0.368 2.62 664 685 NB12 CBL E3 ubiquitin-protein ligase CBL IPI00027269 IKPSSSANAIYSLAARPLPVPK 1.09 0.351 1.68 664 685 NB12-2 CBL E3 ubiquitin-protein ligase CBL IPI00027269 IKPSSSANAIYSLAAR 4.08 0.569 0 664 679 NB25-V CBL E3 ubiquitin-protein ligase CBL IPI00027269 IKPSSSANAIYSLAAR 3.15 0.608 0 664 679 CBLB Isoform Long of E3 ubiquitin-protein NB25-V ligase CBL-B IPI00292856 TSQDYDQLPSCSDGSQAPARPPKPRPR 0 0 4.09 885 911 CBLB Isoform Long of E3 ubiquitin-protein NB88-V ligase CBL-B IPI00292856 TSQDYDQLPSCSDGSQAPARPPKPR 2.94 0.492 0 885 909 CBLB Isoform Long of E3 ubiquitin-protein NB25-V ligase CBL-B IPI00292856 TSQDYDQLPSCSDGSQAPARPPKPR 2.62 0.627 0 885 909 CCDC50 Isoform 1 of Coiled-coil domain- NB25-V containing protein 50 IPI00383423,IPI00872183 AYADSYYYEDGGMKPR 4.24 0.773 2.77 271 286 CCDC50 Isoform 1 of Coiled-coil domain- NB88-V containing protein 50 IPI00383423,IPI00872183 AYADSYYYEDGGMKPR 3.95 0.774 4.96 139 154 CCDC50 Isoform 1 of Coiled-coil domain- NB88 containing protein 50 IPI00383423,IPI00872183 AYADSYYYEDGGMKPR 3.83 0.743 5.8 139 154 GQPGPNHEEDADSYENMDNPDGPDPAWGGG NB122-2 CD19 B-lymphocyte antigen CD19 IPI00305031,IPI00442499 GR pY 531 5.55 0.854 4.42 518 549 GQPGPNHEEDADSYENMDNPDGPDPAWGGG NB88 CD19 B-lymphocyte antigen CD19 IPI00305031,IPI00442499 GR pY 531 5.33 0 7.04 518 549 GQPGPNHEEDADSYENMDNPDGPDPAWGGG NB88-2 CD19 B-lymphocyte antigen CD19 IPI00305031,IPI00442499 GR pY 531 4.08 0.946 4.82 518 549 GQPGPNHEEDADSYENMDNPDGPDPAWGGG NB12-2 CD19 B-lymphocyte antigen CD19 IPI00305031,IPI00442499 GR pY 531 4.03 0.907 2.68 518 549 GQPGPNHEEDADSYENMDNPDGPDPAWGGG NB25-V CD19 B-lymphocyte antigen CD19 IPI00305031,IPI00442499 GR pY 531 3.72 0 4.8 518 549 NB88 CD19 B-lymphocyte antigen CD19 IPI00305031,IPI00442499 GILYAAPQLR pY 508 2.82 0.482 3.68 505 514 NB12 CD19 B-lymphocyte antigen CD19 IPI00305031,IPI00442499 GILYAAPQLR pY 508 2.67 0.436 1.64 505 514 NB25-V CD19 B-lymphocyte antigen CD19 IPI00305031 GILYAAPQLR pY 508 2.61 0.355 0 505 514 IPI00442499 NB122 CD19 B-lymphocyte antigen CD19 IPI00305031 GILYAAPQLR pY 508 2.43 0.458 0 505 514 IPI00442499 NB88-2 CD19 B-lymphocyte antigen CD19 IPI00305031,IPI00442499 GILYAAPQLR pY 508 1.97 0.297 0 505 514 NB122-2 CD19 B-lymphocyte antigen CD19 IPI00305031,IPI00442499 GILYAAPQLR pY 508 1.94 0.331 0 505 514 NB12-2 CD19 B-lymphocyte antigen CD19 IPI00305031,IPI00442499 GILYAAPQLR pY 508 1.86 0.346 1.39 505 514 NB12 CD19 B-lymphocyte antigen CD19 IPI00305031,IPI00442499 EATSLGSQSYEDMR pY 500 2.19 0.764 1.85 491 504
118 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
CD22 Isoform CD22-alpha of B-cell receptor IPI00218390,IPI00295133,IPI00 NB88-V CD22 precursor 867562 TGDAESSEMQRPPPDCDDTVTYSALHKR 2.92 0.429 1.47 598 625 CD300A Isoform 2 of CMRF35-like molecule NB12-2 8 IPI00054095 IAAQRPREEEPDSDYSVIR 3.52 0.566 2.74 166 184 CD300A Isoform 2 of CMRF35-like molecule NB88 8 IPI00054095 IAAQRPREEEPDSDYSVIR 3 0.54 4.62 166 184 CD300A Isoform 2 of CMRF35-like molecule NB25-V 8 IPI00054095 IAAQRPREEEPDSDYSVIR 3 0.659 1.55 166 184 CD300A Isoform 2 of CMRF35-like molecule NB25-V 8 IPI00054095 IAAQRPREEEPDSDYSVIR 2.85 0.607 0 166 184 CD300A Isoform 2 of CMRF35-like molecule NB88-V 8 IPI00054095 IAAQRPREEEPDSDYSVIR 2.75 0.523 3.92 166 184 CD300A Isoform 2 of CMRF35-like molecule NB12 8 IPI00054095 IAAQRPREEEPDSDYSVIR 2.05 0.348 4.17 166 184 CD300A Isoform 2 of CMRF35-like molecule NB61 8 IPI00054095 IAAQRPREEEPDSDYSVIR 1.94 0.312 2.46 166 184 CD300A Isoform 2 of CMRF35-like molecule NB122 8 IPI00054095 IAAQRPREEEPDSDYSVIR 1.87 0.23 1.27 166 184 CD300A Isoform 2 of CMRF35-like molecule NB88-V 8 IPI00054095 EVEVEYSTVASPR 3.24 0.54 3.92 137 149 CD300A Isoform 2 of CMRF35-like molecule NB25-V 8 IPI00054095 EELHYASVVFDSNTNR 3.35 0.535 1.85 150 165 CD300A Isoform 2 of CMRF35-like molecule NB88-V 8 IPI00054095 EEEPDSDYSVIR 2.14 0.613 3.77 173 184 NB88 CD37 Leukocyte antigen CD37 IPI00003400 NLDHVYNR 2 0.603 0 269 276 NB12 CD37 Leukocyte antigen CD37 IPI00003400 NLDHVYNR 1.79 0.736 0 269 276 NB88-2 CD37 Leukocyte antigen CD37 IPI00003400 NLDHVYNR 1.74 0.369 0 269 276 NB25-V CD82 CD82 antigen IPI00020446,IPI00385291 HVHSEDYSKVPKY 3.76 0.685 2.29 255 267 NB12-2 CD82 CD82 antigen IPI00020446 HVHSEDYSKVPKY 2.9 0.681 0 255 267 IPI00385291 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 NB88 DKFZp686L20222 908424 IGEGTYGVVYKGR 3.7 0.231 4.85 10 22 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 NB122 DKFZp686L20222 908424 IGEGTYGVVYKGR 3.54 0.236 0 10 22 CDC2 Putative uncharacterized protein NB88-V DKFZp686L20222 IPI00026689,IPI00073536 IGEGTYGVVYKGR 3.12 0.263 2.96 10 22 IPI00908424 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 NB61 DKFZp686L20222 908424 IGEGTYGVVYKGR 2.82 0.263 1.59 10 22 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 NB12-2 DKFZp686L20222 908424 IGEGTYGVVYKGR 2.68 0.301 0 10 22 CDC2 Putative uncharacterized protein NB25-V DKFZp686L20222 IPI00026689,IPI00073536 IGEGTYGVVYKGR 1.96 0.554 1.55 10 22 IPI00908424 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 NB88-2 DKFZp686L20222 908424 IGEGTYGVVYKGR 1.83 0.365 0 10 22 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 NB25 DKFZp686L20222 908424 IGEGTYGVVYKGR 1.73 0.42 0 10 22 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 NB12 DKFZp686L20222 908424 IGEGTYGVVYKGR 1.59 0.486 0 10 22 CDC2 Putative uncharacterized protein IPI00031681,IPI00260318,IPI000 NB25-V DKFZp686L20222 IPI00026689,IPI00073536 IGEGTYGVVYK 3.43 0.439 5.35 10 20 23503,IPI00908424 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 IPI00031681,IPI00260318,IPI000 NB12 DKFZp686L20222 908424 IGEGTYGVVYK 3.27 0.406 6.74 10 20 23503 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 IPI00031681,IPI00260318,IPI000 NB122-2 DKFZp686L20222 908424 IGEGTYGVVYK 3.19 0.364 6.92 10 20 23503 CDC2 Putative uncharacterized protein IPI00031681,IPI00260318,IPI000 NB88-V DKFZp686L20222 IPI00026689,IPI00073536 IGEGTYGVVYK 3.05 0.28 4.8 10 20 23503,IPI00908424 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 IPI00031681,IPI00260318,IPI000 NB88 DKFZp686L20222 908424 IGEGTYGVVYK 2.98 0.338 6.11 10 20 23503 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 IPI00031681,IPI00260318,IPI000 NB88-2 DKFZp686L20222 908424 IGEGTYGVVYK 2.95 0.311 5.24 10 20 23503 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 IPI00031681,IPI00260318,IPI000 NB61 DKFZp686L20222 908424 IGEGTYGVVYK 2.93 0.359 6.3 10 20 23503 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 IPI00031681,IPI00260318,IPI000 NB12-2 DKFZp686L20222 908424 IGEGTYGVVYK 2.66 0.357 6.11 10 20 23503 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 IPI00031681,IPI00260318,IPI000 NB122 DKFZp686L20222 908424 IGEGTYGVVYK 2.17 0.338 2.96 10 20 23503 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 IPI00031681,IPI00260318,IPI000 NB25 DKFZp686L20222 908424 IGEGTYGVVYK 1.48 0 2.96 10 20 23503 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 NB122-2 DKFZp686L20222 908424 IEKIGEGTYGVVYKGR 4.64 0.655 2.66 7 22 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 NB88 DKFZp686L20222 908424 IEKIGEGTYGVVYKGR 3.47 0.708 1.6 7 22
119 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 NB12 DKFZp686L20222 908424 IEKIGEGTYGVVYKGR 3.27 0.654 1.52 7 22 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 NB122 DKFZp686L20222 908424 IEKIGEGTYGVVYKGR 3.27 0.443 1.85 7 22 CDC2 Putative uncharacterized protein NB25-V DKFZp686L20222 IPI00026689,IPI00073536 IEKIGEGTYGVVYKGR 3 0.638 3.36 7 22 IPI00908424 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 NB25 DKFZp686L20222 908424 IEKIGEGTYGVVYKGR 2.74 0.686 0 7 22 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 NB88-2 DKFZp686L20222 908424 IEKIGEGTYGVVYKGR 2.47 0.415 0 7 22 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 NB12-2 DKFZp686L20222 908424 IEKIGEGTYGVVYKGR 2.19 0.317 0 7 22 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 NB61 DKFZp686L20222 908424 IEKIGEGTYGVVYK 4.27 0.567 4.66 7 20 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 NB122 DKFZp686L20222 908424 IEKIGEGTYGVVYK 4.02 0.636 0 7 20 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 NB88-2 DKFZp686L20222 908424 IEKIGEGTYGVVYK 4.01 0.609 6.08 7 20 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 NB12-2 DKFZp686L20222 908424 IEKIGEGTYGVVYK 3.99 0.594 4.25 7 20 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 NB122-2 DKFZp686L20222 908424 IEKIGEGTYGVVYK 3.93 0.551 4.74 7 20 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 NB25 DKFZp686L20222 908424 IEKIGEGTYGVVYK 3.84 0.584 0 7 20 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 NB12 DKFZp686L20222 908424 IEKIGEGTYGVVYK 3.7 0.575 6.01 7 20 CDC2 Putative uncharacterized protein NB25-V DKFZp686L20222 IPI00026689,IPI00073536 IEKIGEGTYGVVYK 3.64 0.491 5.96 7 20 IPI00908424 CDC2 Putative uncharacterized protein NB88-V DKFZp686L20222 IPI00026689,IPI00073536 IEKIGEGTYGVVYK 3.45 0.534 4.85 7 20 IPI00908424 CDC2 Putative uncharacterized protein IPI00026689,IPI00073536,IPI00 NB88 DKFZp686L20222 908424 IEKIGEGTYGVVYK 2.51 0.625 0 7 20 CDC2 Putative uncharacterized protein NB25-V DKFZp686L20222 IPI00026689,IPI00073536 IEKIGEGTYGVVYK 1.91 0.404 3.4 7 20 IPI00908424 IPI00023503,IPI00031681,IPI00 NB61 CDK3 Cell division protein kinase 3 260318 VEKIGEGTYGVVYK 3.53 0.233 4.05 35 48 IPI00023503,IPI00031681,IPI00 NB88 CDK3 Cell division protein kinase 3 260318 VEKIGEGTYGVVYK 3.34 0.581 5.03 35 48 IPI00023503,IPI00031681,IPI00 NB122-2 CDK3 Cell division protein kinase 3 260318 VEKIGEGTYGVVYK 2.89 0.492 3 35 48 IPI00023503,IPI00031681,IPI00 NB12 CDK3 Cell division protein kinase 3 260318 VEKIGEGTYGVVYK 2.79 0.587 3.09 35 48 IPI00023503,IPI00031681,IPI00 NB25-V CDK3 Cell division protein kinase 3 260318 VEKIGEGTYGVVYK 2.78 0.505 3.05 35 48 IPI00023503,IPI00031681,IPI00 NB88-V CDK3 Cell division protein kinase 3 260318 VEKIGEGTYGVVYK 2.77 0.542 2.49 35 48 IPI00023503,IPI00031681,IPI00 NB25 CDK3 Cell division protein kinase 3 260318 VEKIGEGTYGVVYK 2.47 0.597 4.6 35 48 IPI00023503,IPI00031681,IPI00 IPI00026689,IPI00908424,IPI000 NB88 CDK3 Cell division protein kinase 3 260318 IGEGTYGVVYK 3.57 0.403 5.38 38 48 73536 IPI00023503,IPI00031681,IPI00 IPI00026689,IPI00908424,IPI000 NB25-V CDK3 Cell division protein kinase 3 260318 IGEGTYGVVYK 3.43 0.439 5.35 38 48 73536 IPI00023503,IPI00031681,IPI00 IPI00026689,IPI00908424,IPI000 NB12 CDK3 Cell division protein kinase 3 260318 IGEGTYGVVYK 3.27 0.406 6.74 38 48 73536 IPI00023503,IPI00031681,IPI00 IPI00026689,IPI00908424,IPI000 NB88-V CDK3 Cell division protein kinase 3 260318 IGEGTYGVVYK 3 0.374 4.27 38 48 73536 IPI00023503,IPI00031681,IPI00 IPI00026689,IPI00908424,IPI000 NB61 CDK3 Cell division protein kinase 3 260318 IGEGTYGVVYK 2.94 0.466 3.96 38 48 73536 IPI00023503,IPI00031681,IPI00 IPI00026689,IPI00908424,IPI000 NB122-2 CDK3 Cell division protein kinase 3 260318 IGEGTYGVVYK 2.88 0.402 5.72 38 48 73536 IPI00023503,IPI00031681,IPI00 IPI00026689,IPI00908424,IPI000 NB25 CDK3 Cell division protein kinase 3 260318 IGEGTYGVVYK 2.61 0.348 0 38 48 73536 NB25 CDK5 Cell division protein kinase 5 IPI00023530 IGEGTYGTVFK 2.7 0.529 2.57 10 20 NB12 CDK5 Cell division protein kinase 5 IPI00023530 IGEGTYGTVFK 2.66 0.538 0 10 20 NB122-2 CDK5 Cell division protein kinase 5 IPI00023530 IGEGTYGTVFK 2.5 0.537 3.04 10 20 NB88-2 CDK5 Cell division protein kinase 5 IPI00023530 IGEGTYGTVFK 2.46 0.411 1.35 10 20 NB12-2 CDK5 Cell division protein kinase 5 IPI00023530 IGEGTYGTVFK 2.37 0.328 1.27 10 20 NB25-V CDK5 Cell division protein kinase 5 IPI00023530 IGEGTYGTVFK 2.28 0.286 0 10 20 NB61 CDK5 Cell division protein kinase 5 IPI00023530 IGEGTYGTVFK 1.5 0.348 0 10 20 IPI00014197,IPI00787933,IPI00 NB25-V CDV3 Isoform 1 of Protein CDV3 homolog 795684 LQLDNQYAVLENQK 3.44 0.561 3.06 239 252
120 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
IPI00014197,IPI00787933,IPI00 NB12 CDV3 Isoform 1 of Protein CDV3 homolog 795684 KTPQGPPEIYSDTQFPSLQSTAK 5.35 0.457 4.08 181 203 IPI00014197,IPI00787933,IPI00 NB25-V CDV3 Isoform 1 of Protein CDV3 homolog 795684 KTPQGPPEIYSDTQFPSLQSTAK 5.27 0.533 2.2 181 203 NB12-2 CDV3 Isoform 1 of Protein CDV3 homolog IPI00014197,IPI00795684 KTPQGPPEIYSDTQFPSLQSTAK 5.13 0.618 3.62 181 203 IPI00787933 NB88 CDV3 Isoform 1 of Protein CDV3 homolog IPI00014197,IPI00795684 KTPQGPPEIYSDTQFPSLQSTAK 5.01 0.537 4.48 181 203 IPI00787933 NB88-2 CDV3 Isoform 1 of Protein CDV3 homolog IPI00014197,IPI00795684 KTPQGPPEIYSDTQFPSLQSTAK 4.94 0.566 2.82 181 203 IPI00787933 NB122-2 CDV3 Isoform 1 of Protein CDV3 homolog IPI00014197,IPI00795684 KTPQGPPEIYSDTQFPSLQSTAK 4.65 0.51 2.24 181 203 IPI00787933 IPI00014197,IPI00787933,IPI00 NB25-V CDV3 Isoform 1 of Protein CDV3 homolog 795684 KTPQGPPEIYSDTQFPSLQSTAK 4.22 0.474 3.89 181 203 NB122 CDV3 Isoform 1 of Protein CDV3 homolog IPI00014197,IPI00795684 KTPQGPPEIYSDTQFPSLQSTAK 3.49 0.43 2.28 181 203 IPI00787933 NB25 CDV3 Isoform 1 of Protein CDV3 homolog IPI00014197,IPI00795684 KTPQGPPEIYSDTQFPSLQSTAK 3.36 0.519 2.77 181 203 IPI00787933 IPI00014197,IPI00787933,IPI00 NB88-V CDV3 Isoform 1 of Protein CDV3 homolog 795684 KTPQGPPEIYSDTQFPSLQSTAK 3.19 0.633 2.96 181 203 NB61 CDV3 Isoform 1 of Protein CDV3 homolog IPI00014197 KTPQGPPEIYSDTQFPSLQSTAK 2.46 0.257 0 181 203 IPI00787933,IPI00795684 NB12-2 CENTD1 Centaurin-delta-1 IPI00292471 MQDIPIYANVHK 2.93 0.394 2.14 71 82 NB25-V CENTD1 Centaurin-delta-1 IPI00292471 MQDIPIYANVHK 2.18 0.359 0 71 82 NB12 CENTD1 Centaurin-delta-1 IPI00292471 MQDIPIYANVHK 1.96 0.293 1.27 71 82 NB12-2 CFL1 Cofilin-1 IPI00012011 YALYDATYETKESK 4.39 0.675 5.8 82 95 NB88-V CFL1 Cofilin-1 IPI00012011 YALYDATYETKESK 4.33 0.712 3.32 82 95 NB88 CFL1 Cofilin-1 IPI00012011 YALYDATYETKESK 3.97 0.679 4.68 82 95 NB12-2 CFL1 Cofilin-1 IPI00012011 YALYDATYETK 3.36 0.62 8.19 82 92 NB12-2 CFL1 Cofilin-1 IPI00012011 LTGIKHELQANCYEEVKDR 5.07 0.578 0 128 146 NB61 CFL1 Cofilin-1 IPI00012011 LTGIKHELQANCYEEVKDR 4.33 0.456 2.07 128 146 NB88 CFL1 Cofilin-1 IPI00012011 LTGIKHELQANCYEEVKDR 4.08 0.61 4.46 128 146 NB88-V CFL1 Cofilin-1 IPI00012011 LTGIKHELQANCYEEVKDR 3.61 0.588 2.59 128 146 NB122 CFL1 Cofilin-1 IPI00012011 LTGIKHELQANCYEEVKDR 2.52 0.245 0 128 146 NB12-2 CFL1 Cofilin-1 IPI00012011 HELQANCYEEVKDR 4.81 0.745 6.48 133 146 NB88-V CFL1 Cofilin-1 IPI00012011 HELQANCYEEVKDR 4.71 0.667 5.04 133 146 NB25-V CFL1 Cofilin-1 IPI00012011 HELQANCYEEVKDR 4.63 0.698 4.96 133 146 NB88-2 CFL1 Cofilin-1 IPI00012011 HELQANCYEEVKDR 4.6 0.583 1.62 133 146 NB88 CFL1 Cofilin-1 IPI00012011 HELQANCYEEVKDR 4.52 0.772 1.68 133 146 NB12 CFL1 Cofilin-1 IPI00012011 HELQANCYEEVKDR 3.95 0.62 4.74 133 146 NB122-2 CFL1 Cofilin-1 IPI00012011 HELQANCYEEVKDR 3.17 0.618 0.959 133 146 NB122 CFL1 Cofilin-1 IPI00012011 HELQANCYEEVKDR 2.62 0.598 0 133 146 CFTR Cystic fibrosis transmembrane conductance regulator ATP-binding cassette NB122-2 sub-family C member 7 IPI00302383 RLSLVPDSEQGEAILPR 0 0 5.35 735 751 CFTR Cystic fibrosis transmembrane conductance regulator ATP-binding cassette NB12-2 sub-family C member 7 IPI00302383 RLSLVPDSEQGEAILPR 0 0 5.8 735 751 CFTR Cystic fibrosis transmembrane conductance regulator ATP-binding cassette NB88-2 sub-family C member 7 IPI00302383 RLSLVPDSEQGEAILPR 0 0 7.01 735 751 CHD9 Isoform 1 of Chromodomain-helicase- NB12 DNA-binding protein 9 IPI00383105 QMIQQYEMYFR 1.73 0.705 0 946 956 NB12-2 CLTC Isoform 1 of Clathrin heavy chain 1 IPI00024067 FLRENPYYDSR 3.36 0.503 0 893 903 NB88 CLTC Isoform 1 of Clathrin heavy chain 1 IPI00024067 FLRENPYYDSR 3.04 0.494 1.72 893 903 NB88 CLTC Isoform 1 of Clathrin heavy chain 1 IPI00024067 ENPYYDSR 1.55 0.667 0 896 903 NB12-2 CLTC Isoform 1 of Clathrin heavy chain 1 IPI00024067 ENPYYDSR 1.16 0.406 1.42 896 903 NB88-2 CLTC Isoform 1 of Clathrin heavy chain 1 IPI00024067 ENPYYDSR 1.14 0.716 0 896 903 NB12-2 CLTC Isoform 1 of Clathrin heavy chain 1 IPI00024067 CYDEKMYDAAK 2.5 0.826 2.28 1205 1215 NB88 CLTC Isoform 1 of Clathrin heavy chain 1 IPI00024067 CYDEKMYDAAK 2.33 0.826 1.82 1205 1215 NB88 CLTC Isoform 1 of Clathrin heavy chain 1 IPI00024067 ALEHFTDLYDIKR 4.79 0.394 2.6 626 638 NB88-V CLTC Isoform 1 of Clathrin heavy chain 1 IPI00024067 ALEHFTDLYDIKR 4.15 0.449 4.28 626 638 NB88-2 CLTC Isoform 1 of Clathrin heavy chain 1 IPI00024067 ALEHFTDLYDIKR 3.58 0.463 0 626 638 NB12-2 CLTC Isoform 1 of Clathrin heavy chain 1 IPI00024067 ALEHFTDLYDIKR 3.4 0.61 0 626 638 NB12-2 CNNM4 Metal transporter CNNM4 IPI00418426 FDEHNKYYAR 2.52 0.385 0 590 599 NB122 CNTNAP4 Cell recognition protein CASPR4 IPI00216250 GHCSSYGKLCR 1.16 0.458 0 963 973 NB12-2 COPA Isoform 1 of Coatomer subunit alpha IPI00295857 VKGNNVYCLDR 3.2 0.491 1.08 573 583 NB88 COPA Isoform 1 of Coatomer subunit alpha IPI00295857 VKGNNVYCLDR 2.68 0.524 0 573 583 NB122 COPA Isoform 1 of Coatomer subunit alpha IPI00295857 VKGNNVYCLDR 1.72 0.516 0 573 583 NB88 COPA Isoform 1 of Coatomer subunit alpha IPI00295857 GNNVYCLDR 2.15 0.617 2.03 575 583 NB12-2 COPA Isoform 1 of Coatomer subunit alpha IPI00295857 GNNVYCLDR 1.87 0.38 0 575 583 NB88-V COPB1 Coatomer subunit beta IPI00295851 LVTEMGTYATQSALSSSRPTKK 2.45 0.56 3.31 514 535 NB88-V COPB1 Coatomer subunit beta IPI00295851 LVTEMGTYATQSALSSSRPTK 2.01 0.389 1.85 514 534 NB88-V CORO1A Coronin-1A IPI00010133 HVFGQPAKADQCYEDVR 4 0.687 1.66 13 29 NB88-2 CORO1A Coronin-1A IPI00010133 HVFGQPAKADQCYEDVR 3.77 0.567 0 13 29 NB88 CORO1A Coronin-1A IPI00010133 HVFGQPAKADQCYEDVR 3.71 0.725 0 13 29 NB12-2 CORO1A Coronin-1A IPI00010133 HVFGQPAKADQCYEDVR 3.23 0.616 0 13 29 NB88 CORO1A Coronin-1A IPI00010133 ADQCYEDVR 3.03 0.678 2.1 21 29
121 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
NB12-2 CORO1A Coronin-1A IPI00010133 ADQCYEDVR 2.07 0.412 1.74 21 29 NB88-2 CORO1A Coronin-1A IPI00010133 ADQCYEDVR 2.04 0.67 1.66 21 29 NB12-2 CRKL Crk-like protein IPI00004839 RVPCAYDK 2 0.622 3.66 246 253 NB25-V CRKL Crk-like protein IPI00004839 RVPCAYDK 1.92 0.221 3.19 246 253 NB88 CRKL Crk-like protein IPI00004839 RVPCAYDK 1.78 0.392 4.6 246 253 NSNSYGIPEPAHAYAQPQTTTPLPAVSGSPGAA NB25-V CRKL Crk-like protein IPI00004839 ITPLPSTQNGPVFAK 5.54 0.575 0 194 241 NSNSYGIPEPAHAYAQPQTTTPLPAVSGSPGAA NB88-V CRKL Crk-like protein IPI00004839 ITPLPSTQNGPVFAK 4.42 0.806 5 194 241 NSNSYGIPEPAHAYAQPQTTTPLPAVSGSPGAA NB61 CRKL Crk-like protein IPI00004839 ITPLPSTQNGPVFAK 3.95 0.65 1.49 194 241 NSNSYGIPEPAHAYAQPQTTTPLPAVSGSPGAA NB88-2 CRKL Crk-like protein IPI00004839 ITPLPSTQNGPVFAK 3.9 0.476 0 194 241 NSNSYGIPEPAHAYAQPQTTTPLPAVSGSPGAA NB12-2 CRKL Crk-like protein IPI00004839 ITPLPSTQNGPVFAK 3.83 0.524 0 194 241 NSNSYGIPEPAHAYAQPQTTTPLPAVSGSPGAA NB25 CRKL Crk-like protein IPI00004839 ITPLPSTQNGPVFAK 3.76 0.743 4.07 194 241 NSNSYGIPEPAHAYAQPQTTTPLPAVSGSPGAA NB122 CRKL Crk-like protein IPI00004839 ITPLPSTQNGPVFAK 3.66 0.649 3.17 194 241 NSNSYGIPEPAHAYAQPQTTTPLPAVSGSPGAA NB122-2 CRKL Crk-like protein IPI00004839 ITPLPSTQNGPVFAK 3.56 0.494 0 194 241 NSNSYGIPEPAHAYAQPQTTTPLPAVSGSPGAA NB25-V CRKL Crk-like protein IPI00004839 ITPLPSTQNGPVFAK 3.33 0.673 3.72 194 241 NSNSYGIPEPAHAYAQPQTTTPLPAVSGSPGAA NB12 CRKL Crk-like protein IPI00004839 ITPLPSTQNGPVFAK 2.82 0.565 4.05 194 241 IPI00219727,IPI00219875,IPI002 19730,IPI00845519,IPI00219870,I PI00848265,IPI00219869,IPI0021 9728,IPI00219725,IPI00182540,IP I00219732,IPI00219873,IPI00419 482,IPI00219726,IPI00219731,IPI NB25-V CTNND1 Isoform 1AB of Catenin delta-1 IPI00182469 LNGPQDHSHLLYSTIPR 3.45 0.29 0 85 101 00219868,IPI00219872 IPI00219727,IPI00219875,IPI002 19730,IPI00845519,IPI00219870,I PI00848265,IPI00219869,IPI0021 9728,IPI00219725,IPI00182540,IP I00219732,IPI00219873,IPI00419 482,IPI00219726,IPI00219731,IPI NB88 CTNND1 Isoform 1AB of Catenin delta-1 IPI00182469 LNGPQDHSHLLYSTIPR 3.1 0.524 0 85 101 00219868,IPI00219872 IPI00182469,IPI00182540,IPI00 219725,IPI00219726,IPI002197 27,IPI00219728,IPI00219730,IP I00219731,IPI00219732,IPI0021 9868,IPI00219869,IPI00219870, IPI00219872,IPI00219873,IPI00 219875,IPI00419482,IPI008455 NB88-V CTNND1 Isoform 1AB of Catenin delta-1 19,IPI00848265 LNGPQDHSHLLYSTIPR 2.26 0.447 2.85 85 101 CYFIP2 Isoform 2 of Cytoplasmic FMR1- NB12-2 interacting protein 2 IPI00719600,IPI00789699 CNEQPNRVEIYEK 3.14 0.449 1.12 98 110 CYFIP2 Isoform 2 of Cytoplasmic FMR1- NB88-2 interacting protein 2 IPI00719600,IPI00789699 CNEQPNRVEIYEK 2.98 0.61 2.08 98 110 CYFIP2 Isoform 2 of Cytoplasmic FMR1- NB88 interacting protein 2 IPI00719600,IPI00789699 CNEQPNRVEIYEK 2.26 0.455 1 98 110 CYFIP2 Isoform 2 of Cytoplasmic FMR1- NB12 interacting protein 2 IPI00719600,IPI00789699 CNEQPNRVEIYEK 1.75 0.544 1.25 98 110 DAPP1 Isoform 2 of Dual adapter for phosphotyrosine and 3-phosphotyrosine and NB88-V 3-phosphoinositide IPI00004301,IPI00384620 KVEEPSIYESVR 3.53 0.538 5.41 132 143 DAPP1 Isoform 2 of Dual adapter for phosphotyrosine and 3-phosphotyrosine and NB25-V 3-phosphoinositide IPI00004301,IPI00384620 KVEEPSIYESVR 3.49 0.247 4.72 132 143 DAPP1 Isoform 2 of Dual adapter for phosphotyrosine and 3-phosphotyrosine and NB12-2 3-phosphoinositide IPI00004301,IPI00384620 KVEEPSIYESVR 3.34 0.489 0 132 143 DAPP1 Isoform 2 of Dual adapter for phosphotyrosine and 3-phosphotyrosine and NB25-V 3-phosphoinositide IPI00004301 KVEEPSIYESVR 3.34 0.227 0 132 143 IPI00384620 DAPP1 Isoform 2 of Dual adapter for phosphotyrosine and 3-phosphotyrosine and NB88 3-phosphoinositide IPI00004301,IPI00384620 KVEEPSIYESVR 3.31 0.56 4.14 132 143
122 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
DAPP1 Isoform 2 of Dual adapter for phosphotyrosine and 3-phosphotyrosine and NB88-2 3-phosphoinositide IPI00004301,IPI00384620 KVEEPSIYESVR 3.27 0.0779 4.49 132 143 DAPP1 Isoform 2 of Dual adapter for phosphotyrosine and 3-phosphotyrosine and NB122 3-phosphoinositide IPI00004301 KVEEPSIYESVR 3 0.3 0 132 143 IPI00384620 DAPP1 Isoform 2 of Dual adapter for phosphotyrosine and 3-phosphotyrosine and NB61 3-phosphoinositide IPI00004301,IPI00384620 KVEEPSIYESVR 2.84 0.448 2.7 132 143 DAPP1 Isoform 2 of Dual adapter for phosphotyrosine and 3-phosphotyrosine and NB122-2 3-phosphoinositide IPI00004301,IPI00384620 KVEEPSIYESVR 2.75 0.426 4.85 132 143 DAPP1 Isoform 2 of Dual adapter for phosphotyrosine and 3-phosphotyrosine and NB12 3-phosphoinositide IPI00004301,IPI00384620 KVEEPSIYESVR 2.65 0.49 3.51 132 143 IPI00101968,IPI00396437,IPI00 NB12-2 DBNL Isoform 3 of Drebrin-like protein 456925,IPI00910112 FQDVGPQAPVGSVYQK 5.28 0.63 5.34 149 164 IPI00101968,IPI00396437,IPI00 NB25-V DBNL Isoform 3 of Drebrin-like protein 456925 FQDVGPQAPVGSVYQK 4.45 0.632 6.14 149 164 IPI00910112 IPI00328309,IPI00794202,IPI00 NB25-V DCP1B mRNA-decapping enzyme 1B 871404 KITSSSAIYDNPNLIKPIPVKPSENQQQR 0 0 3.34 183 211 IPI00220503,IPI00789792,IPI00 NB88 DCTN2 dynactin 2 789999 VHQLYETIQR 2.58 0.485 2.96 314 323 NB12-2 DDB1 DNA damage-binding protein 1 IPI00293464 TVPLYESPR 2.17 0.544 1.14 714 722 NB88-2 DDB1 DNA damage-binding protein 1 IPI00293464 TVPLYESPR 1.76 0.517 1.68 714 722 NB122 DDB1 DNA damage-binding protein 1 IPI00293464 TVPLYESPR 1.68 0.481 0 714 722 NB88 DDB1 DNA damage-binding protein 1 IPI00293464 HVKTYEVSLR 2.48 0.588 0 189 198 NB88 DDX1 ATP-dependent RNA helicase DDX1 IPI00293655 VWYHVCSSR 2.79 0.346 0 626 634 DDX20 Probable ATP-dependent RNA NB88 helicase DDX20 IPI00005904 LQTEAQEDDWYDCHR 3.06 0.537 2.28 746 760 DDX20 Probable ATP-dependent RNA NB12 helicase DDX20 IPI00005904 LQTEAQEDDWYDCHR 2.96 0.703 4.46 746 760 DDX20 Probable ATP-dependent RNA NB25-V helicase DDX20 IPI00005904 LQTEAQEDDWYDCHR 2.8 0.705 4.89 746 760 NB12-2 DDX3X ATP-dependent RNA helicase DDX3X IPI00215637 SDYDGIGSRGDR 1.99 0.409 0 102 113 NB25-V DDX3X ATP-dependent RNA helicase DDX3X IPI00215637 SDYDGIGSR 3.02 0.614 4.62 102 110 NB88 DDX3X ATP-dependent RNA helicase DDX3X IPI00215637 SDYDGIGSR 2.29 0.388 4.72 102 110 NB12-2 DDX3X ATP-dependent RNA helicase DDX3X IPI00215637 SDYDGIGSR 1.75 0.324 1.96 102 110 NB12-2 DDX3X ATP-dependent RNA helicase DDX3X IPI00215637 GRSDYDGIGSRGDR 3.15 0.451 1.85 100 113 NB88 DDX3X ATP-dependent RNA helicase DDX3X IPI00215637 GRSDYDGIGSR 3.74 0.598 5.47 100 110 NB12-2 DDX3X ATP-dependent RNA helicase DDX3X IPI00215637 GRSDYDGIGSR 3.29 0.588 4.55 100 110 NB25-V DDX3X ATP-dependent RNA helicase DDX3X IPI00215637 GRSDYDGIGSR 3.28 0.597 5.22 100 110 NB12-2 DDX3X ATP-dependent RNA helicase DDX3X IPI00215637 DKDAYSSFGSR 1.93 0.396 0 65 75 DDX5 Probable ATP-dependent RNA helicase NB12-2 DDX5 IPI00017617 STCIYGGAPK 2.85 0.614 0 198 207 DDX5 Probable ATP-dependent RNA helicase NB88 DDX5 IPI00017617 STCIYGGAPK 2.69 0.572 1.38 198 207 DDX5 Probable ATP-dependent RNA helicase NB12 DDX5 IPI00017617 STCIYGGAPK 2.48 0.661 1.32 198 207 DDX5 Probable ATP-dependent RNA helicase NB88-2 DDX5 IPI00017617 STCIYGGAPK 2.33 0.669 2.36 198 207 NB12-2 DHX9 ATP-dependent RNA helicase A IPI00844578 GANLKDYYSR 2.78 0.515 0 142 151 NB88-2 DHX9 ATP-dependent RNA helicase A IPI00844578 GANLKDYYSR 2.11 0.427 0 142 151 IPI00030876,IPI00783474,IPI00 NB88 DIAPH1 diaphanous 1 isoform 2 852685,IPI00884341 NDYEARPQYYK 3.06 0.49 5.04 413 423 DNAJA1 DnaJ homolog subfamily A member NB88 1 IPI00012535 HYNGEAYEDDEHHPR 5.04 0.743 4.22 375 389 DNAJA1 DnaJ homolog subfamily A member NB88-V 1 IPI00012535 HYNGEAYEDDEHHPR 4.21 0.699 5.05 375 389 DOCK2 Isoform 1 of Dedicator of cytokinesis NB88 protein 2 IPI00022449 IKEEMSKDQPDYAMYSR 3.35 0.64 0 198 214 NB12-2 DOK1 Isoform 1 of Docking protein 1 IPI00015287 VKEEGYELPYNPATDDYAVPPPR 5.36 0.718 5.18 393 415 NB88-V DOK1 Isoform 1 of Docking protein 1 IPI00015287 VKEEGYELPYNPATDDYAVPPPR 3.42 0.655 4.6 393 415 NB12 DOK1 Isoform 1 of Docking protein 1 IPI00015287 VKEEGYELPYNPATDDYAVPPPR 3.28 0.707 7.17 393 415 NB25-V DOK1 Isoform 1 of Docking protein 1 IPI00015287 SHNSALYSQVQK 4.84 0.688 1.72 443 454 NB88-V DOK1 Isoform 1 of Docking protein 1 IPI00015287 SHNSALYSQVQK 4.63 0.736 10.1 443 454 NB12 DOK1 Isoform 1 of Docking protein 1 IPI00015287 SHNSALYSQVQK 4.3 0.791 9.55 443 454 NB12-2 DOK1 Isoform 1 of Docking protein 1 IPI00015287 SHNSALYSQVQK 4.03 0.655 7 443 454 NB88-2 DOK1 Isoform 1 of Docking protein 1 IPI00015287 SHNSALYSQVQK 3.82 0.713 6.57 443 454 NB25-V DOK1 Isoform 1 of Docking protein 1 IPI00015287 SHNSALYSQVQK 3.69 0.657 5.96 443 454 NB122-2 DOK1 Isoform 1 of Docking protein 1 IPI00015287 SHNSALYSQVQK 3.48 0.665 6.89 443 454
123 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
NB88 DOK1 Isoform 1 of Docking protein 1 IPI00015287 SHNSALYSQVQK 3.38 0.665 3.96 443 454 NB61 DOK1 Isoform 1 of Docking protein 1 IPI00015287 SHNSALYSQVQK 2.58 0.57 2.74 443 454 NB122 DOK1 Isoform 1 of Docking protein 1 IPI00015287 SHNSALYSQVQK 2.12 0.709 0 443 454 NB25-V DOK1 Isoform 1 of Docking protein 1 IPI00015287 LTDPKEDPIYDEPEGLAPVPPQGLYDLPREPK 2.97 0.436 1 353 384 NB25-V DOK1 Isoform 1 of Docking protein 1 IPI00015287 LTDPKEDPIYDEPEGLAPVPPQGLYDLPREPK 2.71 0.631 7.43 353 384 NB12-2 DOK1 Isoform 1 of Docking protein 1 IPI00015287 KKPLYWDLYEHAQQQLLK 2.13 0.377 0 333 350 NB12-2 DOK1 Isoform 1 of Docking protein 1 IPI00015287 IAPCPSQDSLYSDPLDSTSAQAGEGVQR 4.54 0.742 6.01 305 332 NB25-V DOK2 Docking protein 2 IPI00022602,IPI00398771 GQEGEYAVPFDAVAR 3.82 0.673 6.64 294 308 NB12-2 DOK2 Docking protein 2 IPI00022602,IPI00398771 GQEGEYAVPFDAVAR 3.48 0.547 2.27 294 308 NB12 DOK2 Docking protein 2 IPI00022602,IPI00398771 GQEGEYAVPFDAVAR 2.75 0.658 8.37 294 308 NB25-V DOK2 Docking protein 2 IPI00022602 GQEGEYAVPFDAVAR 2.58 0.528 0 294 308 IPI00398771 ASGPPGNEHLYENLCVLEASPTLHGGEPEPHEG NB88-V DOK3 Isoform 1 of Docking protein 3 IPI00156649 PGSR 4.38 0.388 2.74 388 424 ASGPPGNEHLYENLCVLEASPTLHGGEPEPHEG NB25-V DOK3 Isoform 1 of Docking protein 3 IPI00156649 PGSR 4.26 0.662 4.37 388 424 ASGPPGNEHLYENLCVLEASPTLHGGEPEPHEG NB12-2 DOK3 Isoform 1 of Docking protein 3 IPI00156649 PGSR 2.94 0.492 1.59 388 424 IPI00000352,IPI00014344,IPI00 DYRK1B Isoform 1 of Dual specificity 182717,IPI00215873,IPI002192 tyrosine-phosphorylation-regulated kinase 50,IPI00219251,IPI00219252,IP NB88-2 1B I00332215 IYQYIQSR 3.09 0.471 1.32 270 277 DYRK1B Isoform 1 of Dual specificity IPI00219252,IPI00215873,IPI001 tyrosine-phosphorylation-regulated kinase 82717,IPI00219250,IPI00332215,I NB122 1B IPI00000352,IPI00014344 IYQYIQSR 1.79 0.346 0 270 277 PI00219251 NB122 E2F7 Isoform 1 of Transcription factor E2F7 IPI00414604 NQYGWHGRHSLPK 1.57 0.315 0 205 217 NB12 EEF1A1 Elongation factor 1-alpha 1 IPI00396485 STTTGHLIYK 3.09 0.459 3.25 21 30 NB25-V EEF1A1 Elongation factor 1-alpha 1 IPI00396485 STTTGHLIYK 3 0.36 1.96 21 30 NB88-V EEF1A1 Elongation factor 1-alpha 1 IPI00396485 STTTGHLIYK 2.82 0.287 1.36 21 30 NB88 EEF1A1 Elongation factor 1-alpha 1 IPI00396485 STTTGHLIYK 2.76 0.451 2.49 21 30 NB25-V EEF1A1 Elongation factor 1-alpha 1 IPI00396485 STTTGHLIYK 2.35 0.32 3.41 21 30 NB122-2 EEF1A1 Elongation factor 1-alpha 1 IPI00396485 EHALLAYTLGVK 3 0.604 6.8 135 146 NB88-2 EEF1A1 Elongation factor 1-alpha 1 IPI00396485 EHALLAYTLGVK 2.98 0.568 4.52 135 146 NB25-V EEF1A1 Elongation factor 1-alpha 1 IPI00396485 EHALLAYTLGVK 2.95 0.686 4.23 135 146 NB12-2 EEF1A1 Elongation factor 1-alpha 1 IPI00396485 EHALLAYTLGVK 2.85 0.629 3.92 135 146 NB88-V EEF1A1 Elongation factor 1-alpha 1 IPI00396485 EHALLAYTLGVK 2.67 0.683 3.68 135 146 NB88 EEF1A1 Elongation factor 1-alpha 1 IPI00396485 EHALLAYTLGVK 2.66 0.728 4.26 135 146 NB25 EEF1A1 Elongation factor 1-alpha 1 IPI00396485 EHALLAYTLGVK 2.63 0.64 3.72 135 146 NB61 EEF1A1 Elongation factor 1-alpha 1 IPI00396485 EHALLAYTLGVK 2.58 0.627 0.959 135 146 NB25-V EEF1A1 Elongation factor 1-alpha 1 IPI00396485 EHALLAYTLGVK 2.29 0.658 4.52 135 146 NB12 EEF1A1 Elongation factor 1-alpha 1 IPI00396485 EHALLAYTLGVK 2.21 0.568 4.59 135 146 NB12-2 EEF2 Elongation factor 2 IPI00186290 EDLYLKPIQR 1.86 0.407 0 440 449 EIF3D Eukaryotic translation initiation factor NB12-2 3 subunit D IPI00006181 VADWTGATYQDKR 2.67 0.561 0 42 54 EIF3D Eukaryotic translation initiation factor NB88 3 subunit D IPI00006181 VADWTGATYQDKR 2.39 0.316 1 42 54 EIF3D Eukaryotic translation initiation factor NB88-2 3 subunit D IPI00006181 NLAMEATYINHNFSQQCLR 3.41 0.504 0 311 329 EIF3D Eukaryotic translation initiation factor NB12-2 3 subunit D IPI00006181 NLAMEATYINHNFSQQCLR 2.77 0.658 3.31 311 329 ENO1 Isoform alpha-enolase of Alpha- NB88 enolase IPI00465248 AAVPSGASTGIYEALELRDNDKTR 1.47 0.649 1.66 33 56 ENO1 Isoform alpha-enolase of Alpha- NB25-V enolase IPI00465248 AAVPSGASTGIYEALELRDNDKTR 1.45 0.546 0 33 56 NB12 EPB41L2 Band 4.1-like protein 2 IPI00015973 VTEGTIREEQEYEEEVEEEPRPAAK 6.1 0.674 2.77 762 786 NB88-2 EPB41L2 Band 4.1-like protein 2 IPI00015973 VTEGTIREEQEYEEEVEEEPRPAAK 5.78 0.581 2.16 762 786 NB12-2 EPB41L2 Band 4.1-like protein 2 IPI00015973 VTEGTIREEQEYEEEVEEEPRPAAK 5.51 0.598 3.4 762 786 NB88-V EPB41L2 Band 4.1-like protein 2 IPI00015973 VTEGTIREEQEYEEEVEEEPRPAAK 5.5 0.694 4.66 762 786 EPHB1 Isoform 1 of Ephrin type-B receptor 1 IPI00219245,IPI00748333,IPI003 NB88 precursor IPI00008315 EAVYSDKLQHYSTGR 2.27 0.651 0 572 586 34334,IPI00219244 EPS8 Epidermal growth factor receptor NB12-2 kinase substrate 8 IPI00290337 HIDRNYEPLKTQPK 2.89 0.49 0 520 533 EPS8 Epidermal growth factor receptor NB88 kinase substrate 8 IPI00290337 HIDRNYEPLKTQPK 2.08 0.318 0 520 533 NB88-V EZR Ezrin IPI00746388 LQDYEEKTK 2.92 0.496 0 350 358 NB88-2 EZR Ezrin IPI00746388 LQDYEEKTK 2.9 0.563 0 350 358 NB12-2 EZR Ezrin IPI00746388 LQDYEEKTK 2.88 0.607 1.82 350 358 NB25 EZR Ezrin IPI00746388 LQDYEEKTK 2.43 0.581 0 350 358 NB88 EZR Ezrin IPI00746388 LQDYEEKTK 2.1 0.582 1.21 350 358 NB25-V EZR Ezrin IPI00746388 LQDYEEKTK 2.1 0.46 0 350 358 NB12 EZR Ezrin IPI00746388 LQDYEEKTK 2.03 0.683 0 350 358 NB88-2 F11R Junctional adhesion molecule A IPI00001754 VIYSQPSAR 1.36 0.529 0 278 286
124 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
NB25 FAIM3 Fas apoptotic inhibitory molecule 3 IPI00023119 SQNNIYSACPR 3.58 0.7 0 310 320 NB25-V FAIM3 Fas apoptotic inhibitory molecule 3 IPI00023119 SQNNIYSACPR 3.48 0.519 5.85 310 320 NB88-V FAIM3 Fas apoptotic inhibitory molecule 3 IPI00023119 SQNNIYSACPR 3.46 0.572 4.89 310 320 NB12 FAIM3 Fas apoptotic inhibitory molecule 3 IPI00023119 SQNNIYSACPR 3.43 0.707 4.59 310 320 NB25-V FAIM3 Fas apoptotic inhibitory molecule 3 IPI00023119 SQNNIYSACPR 3.33 0.672 3.77 310 320 NB12-2 FAIM3 Fas apoptotic inhibitory molecule 3 IPI00023119 SQNNIYSACPR 3.13 0.627 5.25 310 320 NB88-2 FAIM3 Fas apoptotic inhibitory molecule 3 IPI00023119 SQNNIYSACPR 3.09 0.731 3.01 310 320 NB88 FAIM3 Fas apoptotic inhibitory molecule 3 IPI00023119 SQNNIYSACPR 3.07 0.657 5.66 310 320 FAM120A Isoform D of Constitutive NB12 coactivator of PPAR-gamma-like protein 1 IPI00039626 HTPLYER 1.12 0.588 0 427 433 NB88-2 FASN Fatty acid synthase IPI00026781 HGLYLPTR 2.34 0.438 1.77 1044 1051 FBXL10 Isoform 1 of JmjC domain- NB88 containing histone demethylation protein 1B IPI00185326 YYETPEAQR 0.831 0.799 0 159 167 FCHSD2 Isoform 2 of FCH and double SH3 NB25-V domains protein 2 IPI00410639 EIQISPSPKPHASLPPLPLYDQPPSSPYPSPDKR 3.94 0.654 0 584 617 IPI00827702 FCHSD2 Isoform 2 of FCH and double SH3 NB88-V domains protein 2 IPI00410639,IPI00827702 EIQISPSPKPHASLPPLPLYDQPPSSPYPSPDKR 2.4 0.519 1.27 584 617 NB122 FCRL5 FLJ00333 protein (Fragment) IPI00168766 NKGSPIIYSEVK 1.91 0.342 0 407 418 NB25-V FCRL5 FLJ00333 protein (Fragment) IPI00168766 GENVVYSEVR 3.02 0.635 1.92 379 388 NB88 FCRL5 FLJ00333 protein (Fragment) IPI00168766 GENVVYSEVR 2.95 0.43 3.59 379 388 NB88-V FCRL5 FLJ00333 protein (Fragment) IPI00168766 GENVVYSEVR 2.88 0.542 2.85 379 388 NB122 FCRL5 FLJ00333 protein (Fragment) IPI00168766 GENVVYSEVR 2.22 0.57 3.43 379 388 FER Proto-oncogene tyrosine-protein kinase NB88 FER IPI00029263 QEDGGVYSSSGLK 2.14 0.615 1.6 708 720 FGFR1 Isoform 1 of Basic fibroblast growth NB122-2 factor receptor 1 IPI00005142 RPPGLEYCYNPSHNPEEQLSSK pY 585 4.23 0.562 2.57 577 598 FGFR1 Isoform 1 of Basic fibroblast growth NB122 factor receptor 1 IPI00005142 RPPGLEYCYNPSHNPEEQLSSK pY 585 3.3 0.579 0 577 598 FGFR1 Isoform 1 of Basic fibroblast growth NB12 factor receptor 1 IPI00005142 DIHHIDYYKK pY 653 2.89 0.432 2.92 647 656 FGFR1 Isoform 1 of Basic fibroblast growth NB88 factor receptor 1 IPI00005142 DIHHIDYYKK pY 653/ 654 2.8 0.606 0 647 656 FGFR1 Isoform 1 of Basic fibroblast growth NB122-2 factor receptor 1 IPI00005142 DIHHIDYYKK pY 653 2.79 0.504 0 647 656 FGFR1 Isoform 1 of Basic fibroblast growth NB122 factor receptor 1 IPI00005142 DIHHIDYYKK pY 653 2.42 0.56 0 647 656 FGFR1 Isoform 1 of Basic fibroblast growth NB122-2 factor receptor 1 IPI00005142 DIHHIDYYK pY 653 2.57 0.541 0 647 655 FGR Proto-oncogene tyrosine-protein kinase NB12 FGR IPI00016871 SYGAADHYGPDPTK pY 34 3.45 0.716 1.77 27 40 FGR Proto-oncogene tyrosine-protein kinase NB88 FGR IPI00016871 SYGAADHYGPDPTK pY 34 3.29 0.669 4.85 27 40 FGR Proto-oncogene tyrosine-protein kinase NB88 FGR IPI00016871 LIKDDEYNPCQGSK pY 412 4.51 0.603 4.96 406 419 FGR Proto-oncogene tyrosine-protein kinase NB88 FGR IPI00016871 KLDMGGYYITTR pY 208 3.93 0.593 5.32 202 213 FGR Proto-oncogene tyrosine-protein kinase NB12-2 FGR IPI00016871 KLDMGGYYITTR pY 209 3.92 0.626 6 202 213 FGR Proto-oncogene tyrosine-protein kinase NB88-V FGR IPI00016871 KLDMGGYYITTR pY 208 3.89 0.58 6.03 202 213 FGR Proto-oncogene tyrosine-protein kinase NB88-2 FGR IPI00016871 KLDMGGYYITTR pY 209 3.87 0.626 4.8 202 213 FGR Proto-oncogene tyrosine-protein kinase NB12 FGR IPI00016871 KLDMGGYYITTR pY 208 3.76 0.644 2.85 202 213 FGR Proto-oncogene tyrosine-protein kinase NB25-V FGR IPI00016871 KLDMGGYYITTR pY 208 3.64 0.551 7.57 202 213 FGR Proto-oncogene tyrosine-protein kinase NB61 FGR IPI00016871 KLDMGGYYITTR pY 209 3.55 0.546 0 202 213 FGR Proto-oncogene tyrosine-protein kinase NB25-V FGR IPI00016871 KLDMGGYYITTR pY 208 3.41 0.708 2.12 202 213 FNDC3B Isoform 1 of Fibronectin type III NB12-2 domain-containing protein 3B IPI00217490 DLLPAAQYCCR 1.44 0.553 0 826 836 G3BP1 Ras GTPase-activating protein- NB88 binding protein 1 IPI00012442 NSSYVHGGLDSNGKPADAVYGQK 3.82 0.503 6.55 37 59 G3BP1 Ras GTPase-activating protein- NB12-2 binding protein 1 IPI00012442 NSSYVHGGLDSNGKPADAVYGQK 3.22 0.43 4.16 37 59 G6PD Isoform Long of Glucose-6-phosphate NB12 1-dehydrogenase IPI00216008 VQPNEAVYTK 1.8 0.409 1.36 440 449 G6PD Isoform Long of Glucose-6-phosphate NB25 1-dehydrogenase IPI00216008 VQPNEAVYTK 1.71 0.543 0 440 449
125 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
G6PD Isoform Long of Glucose-6-phosphate NB25-V 1-dehydrogenase IPI00216008 VQPNEAVYTK 1.58 0.484 1.04 440 449 G6PD Isoform Long of Glucose-6-phosphate NB88 1-dehydrogenase IPI00216008 NSYVAGQYDDAASYQR 4.45 0.814 10.1 105 120 G6PD Isoform Long of Glucose-6-phosphate NB12 1-dehydrogenase IPI00216008 NSYVAGQYDDAASYQR 3.68 0.813 7.41 105 120 GAB1 Isoform 1 of GRB2-associated-binding NB88-V protein 1 IPI00031068 TASDTDSSYCIPTAGMSPSR 3.7 0.718 7.77 365 384 GAB1 Isoform 1 of GRB2-associated-binding NB25-V protein 1 IPI00031068 TASDTDSSYCIPTAGMSPSR 3.05 0.786 5 365 384 GAB1 Isoform 1 of GRB2-associated-binding NB25-V protein 1 IPI00031068 KDASSQDCYDIPR 4.15 0.619 3.85 398 410 GAB1 Isoform 1 of GRB2-associated-binding NB88-V protein 1 IPI00031068 KDASSQDCYDIPR 3.95 0.588 3.1 398 410 GAB1 Isoform 1 of GRB2-associated-binding NB25-V protein 1 IPI00031068 HVSISYDIPPTPGNTYQIPR 1.68 0.477 0 302 321 GAB1 Isoform 1 of GRB2-associated-binding NB25-V protein 1 IPI00031068 HGMNGFFQQQMIYDSPPSR 3.47 0.545 4.47 230 248 GAB1 Isoform 1 of GRB2-associated-binding NB88-V protein 1 IPI00031068 DASSQDCYDIPR 2.42 0.662 3.29 399 410 GAB1 Isoform 1 of GRB2-associated-binding NB25-V protein 1 IPI00031068 DASSQDCYDIPR 2.01 0.598 2.16 399 410 GAB1 Isoform 1 of GRB2-associated-binding NB25-V protein 1 IPI00031068 DASSQDCYDIPR 1.66 0.592 0 399 410 GAB1 Isoform 1 of GRB2-associated-binding NB122 protein 1 IPI00031068 DASSQDCYDIPR 1.62 0.535 2.44 399 410 GAB1 Isoform 1 of GRB2-associated-binding NB25-V protein 1 IPI00031068 APSASVDSSLYNLPR 4.58 0.727 5.89 249 263 GAB1 Isoform 1 of GRB2-associated-binding NB88-V protein 1 IPI00031068 APSASVDSSLYNLPR 4.3 0.738 9.42 249 263 GAB2 Isoform 1 of GRB2-associated-binding NB88-V protein 2 IPI00186990 DSTYDLPR 1.8 0.351 0 263 270 GALNT4 Polypeptide N- NB12-2 acetylgalactosaminyltransferase 4 IPI00339297 VYLKTQLETYISNLDR 3.05 0.365 0 180 195 GART Isoform Long of Trifunctional purine NB88 biosynthetic protein adenosine-3 IPI00025273 GYPGDYTK 0.826 0.636 0 343 350 GCET2 Germinal center B-cell-expressed IPI00167003,IPI00549359,IPI00 NB25-V transcript 2 protein 746673 SPEDEYELLMPHR 3.13 0.536 1.38 77 89 GCET2 Germinal center B-cell-expressed NB12-2 transcript 2 protein IPI00167003 SPEDEYELLMPHR 3.02 0.561 0 143 155 IPI00549359,IPI00746673 GCET2 Germinal center B-cell-expressed IPI00167003,IPI00549359,IPI00 NB12 transcript 2 protein 746673 SPEDEYELLMPHR 2.79 0.755 2.6 143 155 GCET2 Germinal center B-cell-expressed IPI00167003,IPI00549359,IPI00 NB61 transcript 2 protein 746673 SPEDEYELLMPHR 1.58 0.38 1.89 143 155 GDI2 cDNA FLJ60299, highly similar to Rab NB88 GDP dissociation inhibitor beta IPI00031461 TDDYLDQPCYETINR 4.29 0.753 6.77 198 212 GDI2 cDNA FLJ60299, highly similar to Rab NB12-2 GDP dissociation inhibitor beta IPI00031461 TDDYLDQPCYETINR 3.64 0.607 7.19 198 212 GDI2 cDNA FLJ60299, highly similar to Rab NB88-2 GDP dissociation inhibitor beta IPI00031461 TDDYLDQPCYETINR 3.62 0.568 7.03 198 212 GDI2 cDNA FLJ60299, highly similar to Rab NB12 GDP dissociation inhibitor beta IPI00031461 TDDYLDQPCYETINR 3.39 0.747 7.43 198 212 GDI2 cDNA FLJ60299, highly similar to Rab NB122-2 GDP dissociation inhibitor beta IPI00031461 TDDYLDQPCYETINR 2.85 0.44 4.09 198 212 GDI2 cDNA FLJ60299, highly similar to Rab NB61 GDP dissociation inhibitor beta IPI00031461 TDDYLDQPCYETINR 2.41 0.714 2 198 212 GDI2 cDNA FLJ60299, highly similar to Rab NB88-V GDP dissociation inhibitor beta IPI00031461 KKNDIYGED 2.26 0.537 2.05 437 445 GDI2 cDNA FLJ60299, highly similar to Rab NB88 GDP dissociation inhibitor beta IPI00031461 KKNDIYGED 1.58 0.426 0 441 449 NB12-2 GEMIN8 40 kDa protein IPI00301879 QQQLDAERLDSYVNADHDLYCNTRR 2.96 0.554 0 261 285 GIT1 Isoform 1 of ARF GTPase-activating NB25-V protein GIT1 IPI00384861 LQPFHSTELEDDAIYSVHVPAGLYR 4.78 0.701 0 531 555 GMPPA Isoform 1 of Mannose-1-phosphate IPI00101782,IPI00383767,IPI00 NB88 guanyltransferase alpha 657888,IPI00893044 SAGSALYASR 3.38 0.586 3.48 250 259 GMPPA Isoform 1 of Mannose-1-phosphate IPI00101782,IPI00383767,IPI00 NB88-2 guanyltransferase alpha 657888,IPI00893044 SAGSALYASR 3.13 0.533 2.77 250 259 GOT1 Aspartate aminotransferase, NB88 cytoplasmic IPI00219029 NFGLYNER 1.89 0.351 0 260 267 GPM6A Neuronal membrane glycoprotein M6- NB88-2 a IPI00019952 MQKYEDIK 1.68 0.301 2.12 248 255
126 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
GPRC5C CDNA FLJ20242 fis, clone NB12-2 COLF6369 IPI00004901 AEDMYSAQSHQAATPPKDGK 3.03 0.626 0 121 140 GRLF1 Isoform 1 of Glucocorticoid receptor NB25-V DNA-binding factor 1 IPI00334715,IPI00718985 SVSSSPWLPQDGFDPSDYAEPMDAVVKPR 4.01 0.622 3.24 1070 1098 GRLF1 Isoform 1 of Glucocorticoid receptor NB88-V DNA-binding factor 1 IPI00334715,IPI00718985 SVSSSPWLPQDGFDPSDYAEPMDAVVKPR 3.68 0.644 3.38 1070 1098 GRLF1 Isoform 1 of Glucocorticoid receptor NB88-2 DNA-binding factor 1 IPI00334715,IPI00718985 SVSSSPWLPQDGFDPSDYAEPMDAVVKPR 3.29 0.488 6.51 1070 1098 GRLF1 Isoform 1 of Glucocorticoid receptor NB122-2 DNA-binding factor 1 IPI00334715,IPI00718985 SVSSSPWLPQDGFDPSDYAEPMDAVVKPR 3.11 0.53 5.48 1070 1098 GRLF1 Isoform 1 of Glucocorticoid receptor NB12 DNA-binding factor 1 IPI00334715,IPI00718985 SVSSSPWLPQDGFDPSDYAEPMDAVVKPR 2.72 0.543 3.82 1070 1098 GRLF1 Isoform 1 of Glucocorticoid receptor NB12-2 DNA-binding factor 1 IPI00334715,IPI00718985 SVSSSPWLPQDGFDPSDYAEPMDAVVKPR 2.47 0.407 3.33 1070 1098 GRLF1 Isoform 1 of Glucocorticoid receptor NB25-V DNA-binding factor 1 IPI00334715,IPI00718985 SVSSSPWLPQDGFDPSDYAEPMDAVVKPR 2.14 0.428 6 1070 1098 GRLF1 Isoform 1 of Glucocorticoid receptor NB12 DNA-binding factor 1 IPI00334715,IPI00718985 NEEENIYSVPHDSTQGK 5.66 0.8 9.06 1099 1115 GRLF1 Isoform 1 of Glucocorticoid receptor NB88-V DNA-binding factor 1 IPI00334715,IPI00718985 NEEENIYSVPHDSTQGK 5.37 0.791 7.27 1099 1115 GRLF1 Isoform 1 of Glucocorticoid receptor NB25-V DNA-binding factor 1 IPI00334715,IPI00718985 NEEENIYSVPHDSTQGK 4.9 0.773 7.89 1099 1115 GRLF1 Isoform 1 of Glucocorticoid receptor NB88 DNA-binding factor 1 IPI00334715,IPI00718985 NEEENIYSVPHDSTQGK 4.39 0.757 6.77 1099 1115 GRLF1 Isoform 1 of Glucocorticoid receptor NB25 DNA-binding factor 1 IPI00334715,IPI00718985 NEEENIYSVPHDSTQGK 4.22 0.722 3.17 1099 1115 GRLF1 Isoform 1 of Glucocorticoid receptor NB25-V DNA-binding factor 1 IPI00334715,IPI00718985 NEEENIYSVPHDSTQGK 3.58 0.683 1.8 1099 1115 GRLF1 Isoform 1 of Glucocorticoid receptor NB122 DNA-binding factor 1 IPI00334715,IPI00718985 NEEENIYSVPHDSTQGK 2.78 0.653 5.12 1099 1115 GRLF1 Isoform 1 of Glucocorticoid receptor NB88-2 DNA-binding factor 1 IPI00334715,IPI00718985 NEEENIYSVPHDSTQGK 2.75 0.642 1.85 1099 1115 GRLF1 Isoform 1 of Glucocorticoid receptor NB122-2 DNA-binding factor 1 IPI00334715,IPI00718985 NEEENIYSVPHDSTQGK 2.69 0.636 1.11 1099 1115 GRLF1 Isoform 1 of Glucocorticoid receptor NB12-2 DNA-binding factor 1 IPI00334715,IPI00718985 NEEENIYSVPHDSTQGK 2.69 0.697 0 1099 1115 GRLF1 Isoform 1 of Glucocorticoid receptor NB61 DNA-binding factor 1 IPI00334715,IPI00718985 NEEENIYSVPHDSTQGK 2.58 0.673 2.3 1099 1115 GRLF1 Isoform 1 of Glucocorticoid receptor NB12-2 DNA-binding factor 1 IPI00334715,IPI00718985 KSVSSSPWLPQDGFDPSDYAEPMDAVVKPR 4.14 0.673 5.03 1069 1098 GRLF1 Isoform 1 of Glucocorticoid receptor NB61 DNA-binding factor 1 IPI00334715,IPI00718985 KSVSSSPWLPQDGFDPSDYAEPMDAVVKPR 3.2 0.54 0 1069 1098 GRLF1 Isoform 1 of Glucocorticoid receptor NB12 DNA-binding factor 1 IPI00334715,IPI00718985 KSVSSSPWLPQDGFDPSDYAEPMDAVVKPR 3.13 0.561 4.96 1069 1098 GSK3B Isoform 1 of Glycogen synthase IPI00028570,IPI00216190,IPI00 NB12-2 kinase-3 beta 292228,IPI00880060 QLVRGEPNVSYICSR 3.62 0.529 4.43 206 220 GSK3B Isoform 1 of Glycogen synthase IPI00028570,IPI00216190,IPI00 NB12 kinase-3 beta 292228,IPI00880060 QLVRGEPNVSYICSR 3.08 0.546 4.44 206 220 GSK3B Isoform 1 of Glycogen synthase IPI00028570,IPI00216190,IPI00 NB88-V kinase-3 beta 292228,IPI00880060 QLVRGEPNVSYICSR 3.04 0.422 1.66 206 220 GSK3B Isoform 1 of Glycogen synthase IPI00028570,IPI00216190,IPI00 NB122-2 kinase-3 beta 292228,IPI00880060 QLVRGEPNVSYICSR 2.89 0.562 2.48 206 220 GSK3B Isoform 1 of Glycogen synthase IPI00028570,IPI00216190,IPI00 NB122 kinase-3 beta 292228,IPI00880060 QLVRGEPNVSYICSR 2.79 0.365 3.12 206 220 GSK3B Isoform 1 of Glycogen synthase IPI00028570,IPI00216190,IPI00 NB88-2 kinase-3 beta 292228,IPI00880060 QLVRGEPNVSYICSR 2.75 0.391 3.85 206 220 GSK3B Isoform 1 of Glycogen synthase IPI00028570,IPI00216190,IPI00 NB88 kinase-3 beta 292228,IPI00880060 QLVRGEPNVSYICSR 1.74 0.656 1.28 206 220 GSK3B Isoform 1 of Glycogen synthase IPI00028570,IPI00216190,IPI00 NB25-V kinase-3 beta 292228,IPI00880060 QLVRGEPNVSYICSR 1.63 0.422 3.32 206 220 GSK3B Isoform 1 of Glycogen synthase IPI00028570,IPI00216190,IPI00 NB61 kinase-3 beta 292228,IPI00880060 QLVRGEPNVSYICSR 1.56 0.453 0 206 220 GSK3B Isoform 1 of Glycogen synthase IPI00028570,IPI00216190,IPI00 NB122 kinase-3 beta 292228,IPI00880060 GEPNVSYICSR 3.12 0.847 0 210 220 GSK3B Isoform 1 of Glycogen synthase IPI00028570,IPI00216190,IPI00 NB61 kinase-3 beta 292228,IPI00880060 GEPNVSYICSR 3.11 0.813 4.49 210 220 GSK3B Isoform 1 of Glycogen synthase IPI00028570,IPI00216190,IPI00 NB12-2 kinase-3 beta 292228,IPI00880060 GEPNVSYICSR 3.09 0.803 2.21 210 220 GSK3B Isoform 1 of Glycogen synthase IPI00028570,IPI00216190,IPI00 NB88 kinase-3 beta 292228,IPI00880060 GEPNVSYICSR 3.06 0.773 1.14 210 220
127 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
GSK3B Isoform 1 of Glycogen synthase IPI00028570,IPI00216190,IPI00 NB122-2 kinase-3 beta 292228,IPI00880060 GEPNVSYICSR 2.94 0.798 3.31 210 220 GSK3B Isoform 1 of Glycogen synthase IPI00028570,IPI00216190,IPI00 NB88-V kinase-3 beta 292228,IPI00880060 GEPNVSYICSR 2.72 0.693 3.66 210 220 GSK3B Isoform 1 of Glycogen synthase IPI00028570,IPI00216190,IPI00 NB12 kinase-3 beta 292228,IPI00880060 GEPNVSYICSR 2.65 0.78 2.11 210 220 GSK3B Isoform 1 of Glycogen synthase IPI00028570,IPI00216190,IPI00 NB88-2 kinase-3 beta 292228,IPI00880060 GEPNVSYICSR 2.65 0.781 2.27 210 220 GSK3B Isoform 1 of Glycogen synthase IPI00028570,IPI00216190,IPI00 NB25-V kinase-3 beta 292228,IPI00880060 GEPNVSYICSR 2.45 0.698 2.47 273 283 GSK3B Isoform 1 of Glycogen synthase IPI00028570,IPI00216190,IPI00 NB25 kinase-3 beta 292228,IPI00880060 GEPNVSYICSR 2.15 0.923 2.74 210 220 GSK3B Isoform 1 of Glycogen synthase IPI00028570,IPI00216190,IPI00 NB25-V kinase-3 beta 292228,IPI00880060 GEPNVSYICSR 2.08 0.769 3.36 210 220 NB88-2 GSS Glutathione synthetase IPI00010706 DGYMPR 1.12 0.753 0 268 273 DKPSVEPVEEYDYEDLKESSNSVSNHQLSGFD NB88-2 HBS1L Isoform 1 of HBS1-like protein IPI00009070,IPI00513811 QAR 3.74 0.333 1.18 46 80 DKPSVEPVEEYDYEDLKESSNSVSNHQLSGFD NB12-2 HBS1L Isoform 1 of HBS1-like protein IPI00009070 QAR 2.65 0.47 0 46 80 IPI00513811 HCK Isoform p59-HCK of Tyrosine-protein NB88-V kinase HCK IPI00029769,IPI00646510 VIEDNEYTAREGAK pY410 4.17 0.57 5.43 384 397 IPI00298625,IPI00432416 HCK Isoform p59-HCK of Tyrosine-protein NB88-2 kinase HCK IPI00029769,IPI00646510 VIEDNEYTAREGAK pY410 3.75 0.535 3.04 384 397 IPI00298625,IPI00432416 HCK Isoform p59-HCK of Tyrosine-protein NB88-2 kinase HCK IPI00029769,IPI00646510 VIEDNEYTAR pY410 3.34 0.639 3.57 384 393 IPI00298625,IPI00432416 HCK Isoform p59-HCK of Tyrosine-protein NB88-V kinase HCK IPI00029769,IPI00646510 VIEDNEYTAR pY410 3.11 0.553 1.34 384 393 IPI00298625,IPI00432416 HCK Isoform p59-HCK of Tyrosine-protein NB88-V kinase HCK IPI00029769,IPI00646510 TLDNGGFYISPR pY 208 3.22 0.413 5.64 181 192 HCK Isoform p59-HCK of Tyrosine-protein NB88-2 kinase HCK IPI00029769,IPI00646510 TLDNGGFYISPR pY 208 3.15 0.52 5.68 181 192 HCK Isoform p59-HCK of Tyrosine-protein TETSASPHCPVYVPDPTSTIKPGPNSHNSNTPG NB88-V kinase HCK IPI00029769,IPI00646510 IR pY 50 3.36 0.428 4.92 19 53 HCLS1 Hematopoietic lineage cell-specific NB25-V protein IPI00026156 GFGGQYGIQK 3.21 0.542 2.92 193 202 HCLS1 Hematopoietic lineage cell-specific NB12-2 protein IPI00026156 GFGGQYGIQK 2.71 0.505 2.34 193 202 HGS Hepatocyte growth factor-regulated NB88-2 tyrosine kinase substrate IPI00006176 VCEPCYEQLNR 3.59 0.651 5.08 211 221 HGS Hepatocyte growth factor-regulated NB88 tyrosine kinase substrate IPI00006176 VCEPCYEQLNR 2.94 0.773 3.64 211 221 HGS Hepatocyte growth factor-regulated NB12-2 tyrosine kinase substrate IPI00006176 VCEPCYEQLNR 2.83 0.711 4.21 211 221 HGS Hepatocyte growth factor-regulated NB122-2 tyrosine kinase substrate IPI00006176 VCEPCYEQLNR 2.16 0.693 3.18 211 221 HGS Hepatocyte growth factor-regulated NB25-V tyrosine kinase substrate IPI00006176 VCEPCYEQLNR 1.71 0.671 0 211 221 HGS Hepatocyte growth factor-regulated NB12 tyrosine kinase substrate IPI00006176 VCEPCYEQLNR 1.47 0.59 0 211 221 IPI00215949,IPI00215950,IPI00 HIPK2 Isoform 2 of Homeodomain- 289892,IPI00302302,IPI006399 NB122-2 interacting protein kinase 2 59,IPI00747261 AVCSTYLQSR 3.05 0.626 2.8 356 365 IPI00215949,IPI00215950,IPI00 HIPK2 Isoform 2 of Homeodomain- 289892,IPI00302302,IPI006399 NB12-2 interacting protein kinase 2 59,IPI00747261 AVCSTYLQSR 3 0.494 2.64 356 365 HIPK2 Isoform 2 of Homeodomain- IPI00639959,IPI00747261,IPI002 NB122 interacting protein kinase 2 IPI00215949 AVCSTYLQSR 2.96 0.612 0 356 365 15950,IPI00289892,IPI00302302 IPI00215949,IPI00215950,IPI00 HIPK2 Isoform 2 of Homeodomain- 289892,IPI00302302,IPI006399 NB88-2 interacting protein kinase 2 59,IPI00747261 AVCSTYLQSR 2.96 0.605 4.21 356 365 IPI00215949,IPI00215950,IPI00 HIPK2 Isoform 2 of Homeodomain- 289892,IPI00302302,IPI006399 NB12 interacting protein kinase 2 59,IPI00747261 AVCSTYLQSR 2.85 0.589 2.31 356 365 HIPK2 Isoform 2 of Homeodomain- IPI00639959,IPI00747261,IPI002 NB25 interacting protein kinase 2 IPI00215949 AVCSTYLQSR 2.83 0.524 0 356 365 15950,IPI00289892,IPI00302302 IPI00215949,IPI00215950,IPI00 HIPK2 Isoform 2 of Homeodomain- 289892,IPI00302302,IPI006399 NB88 interacting protein kinase 2 59,IPI00747261 AVCSTYLQSR 2.8 0.568 4.24 356 365 IPI00215949,IPI00215950,IPI00 HIPK2 Isoform 2 of Homeodomain- 289892,IPI00302302,IPI006399 NB88-V interacting protein kinase 2 59,IPI00747261 AVCSTYLQSR 2.62 0.549 3.12 356 365
128 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
IPI00215949,IPI00215950,IPI00 HIPK2 Isoform 2 of Homeodomain- 289892,IPI00302302,IPI006399 NB61 interacting protein kinase 2 59,IPI00747261 AVCSTYLQSR 2.58 0.476 2.74 356 365 IPI00215949,IPI00215950,IPI00 HIPK2 Isoform 2 of Homeodomain- 289892,IPI00302302,IPI006399 NB25-V interacting protein kinase 2 59,IPI00747261 AVCSTYLQSR 2.55 0.606 1.51 356 365 IPI00215949,IPI00215950,IPI00 HIPK2 Isoform 2 of Homeodomain- 289892,IPI00302302,IPI006399 NB25-V interacting protein kinase 2 59,IPI00747261 AVCSTYLQSR 2.48 0.446 3.66 356 365 HIPK3 Isoform 1 of Homeodomain- NB122-2 interacting protein kinase 3 IPI00099522,IPI00554546 TVCSTYLQSR 3.05 0.456 1.47 354 363 HIPK3 Isoform 1 of Homeodomain- NB25-V interacting protein kinase 3 IPI00099522,IPI00554546 TVCSTYLQSR 3.04 0.629 1.27 354 363 HIPK3 Isoform 1 of Homeodomain- NB12-2 interacting protein kinase 3 IPI00099522,IPI00554546 TVCSTYLQSR 3.02 0.566 3.04 354 363 HIPK3 Isoform 1 of Homeodomain- NB25 interacting protein kinase 3 IPI00099522,IPI00554546 TVCSTYLQSR 2.94 0.609 2.37 354 363 HIPK3 Isoform 1 of Homeodomain- NB88-2 interacting protein kinase 3 IPI00099522,IPI00554546 TVCSTYLQSR 2.86 0.49 1.89 354 363 HIPK3 Isoform 1 of Homeodomain- NB88 interacting protein kinase 3 IPI00099522,IPI00554546 TVCSTYLQSR 2.85 0.533 1.68 354 363 HIPK3 Isoform 1 of Homeodomain- NB12 interacting protein kinase 3 IPI00099522,IPI00554546 TVCSTYLQSR 2.64 0.65 2.77 354 363 HIPK3 Isoform 1 of Homeodomain- NB88-V interacting protein kinase 3 IPI00099522,IPI00554546 TVCSTYLQSR 2.52 0.554 3.64 354 363 HIPK3 Isoform 1 of Homeodomain- NB25-V interacting protein kinase 3 IPI00099522,IPI00554546 TVCSTYLQSR 2.47 0.544 1.72 354 363 HIPK3 Isoform 1 of Homeodomain- NB122 interacting protein kinase 3 IPI00099522 TVCSTYLQSR 2.36 0.586 0 354 363 IPI00554546 HIPK3 Isoform 1 of Homeodomain- NB61 interacting protein kinase 3 IPI00099522 TVCSTYLQSR 2.16 0.45 0 354 363 IPI00554546 HIST1H4F;HIST1H4J;HIST1H4A;HIST1H4C; HIST1H4E;HIST1H4L;HIST1H4K;HIST1H4I;H IST2H4B;HIST4H4;HIST1H4D;HIST1H4B;HI NB88 ST1H4H;HIST2H4A Histone H4 IPI00453473 ISGLIYEETR 2.89 0.621 3.15 47 56 HIST1H4F;HIST1H4J;HIST1H4A;HIST1H4C; HIST1H4E;HIST1H4L;HIST1H4K;HIST1H4I;H IST2H4B;HIST4H4;HIST1H4D;HIST1H4B;HI NB12 ST1H4H;HIST2H4A Histone H4 IPI00453473 ISGLIYEETR 2.88 0.695 1.85 47 56 HLA-C;HLA-B;MICA;XXbac-BPG181B23.1 HLA class I histocompatibility antigen, B-44 ADPPKTHVTHHPISDHEVTLRCWALGFYPAEIT NB88-V alpha chain IPI00743117 LTWQR 1.93 0.406 0 206 243 HNRNPA0 Heterogeneous nuclear NB88-V ribonucleoprotein A0 IPI00011913 KAVPKEDIYSGGGGGGSR 4.08 0.675 4.82 172 189 HNRNPA0 Heterogeneous nuclear NB88 ribonucleoprotein A0 IPI00011913 KAVPKEDIYSGGGGGGSR 3.64 0.592 4.72 172 189 HNRNPA0 Heterogeneous nuclear NB12-2 ribonucleoprotein A0 IPI00011913 KAVPKEDIYSGGGGGGSR 2.79 0.659 4.09 172 189 HNRNPA0 Heterogeneous nuclear NB88-2 ribonucleoprotein A0 IPI00011913 KAVPKEDIYSGGGGGGSR 2.12 0.409 2.36 172 189 HNRNPA0 Heterogeneous nuclear NB88-V ribonucleoprotein A0 IPI00011913 AVPKEDIYSGGGGGGSR 3.11 0.68 6.26 173 189 HNRNPA0 Heterogeneous nuclear NB88 ribonucleoprotein A0 IPI00011913 AVPKEDIYSGGGGGGSR 2.52 0.609 2.54 173 189 HNRNPA1 Isoform A1-B of Heterogeneous NB88-V nuclear ribonucleoprotein A1 IPI00215965 SSGPYGGGGQYFAKPR 3.89 0.609 2.82 337 352 HNRNPA1 Isoform A1-B of Heterogeneous NB88 nuclear ribonucleoprotein A1 IPI00215965 SSGPYGGGGQYFAKPR 3.51 0.586 2.04 337 352 HNRNPA1 Isoform A1-B of Heterogeneous NB25-V nuclear ribonucleoprotein A1 IPI00215965 SSGPYGGGGQYFAKPR 3.17 0.648 2.42 337 352 HNRNPA1 Isoform A1-B of Heterogeneous NB122 nuclear ribonucleoprotein A1 IPI00215965 SSGPYGGGGQYFAKPR 1.81 0.603 0 337 352 HNRNPA1 Isoform A1-B of Heterogeneous NB61 nuclear ribonucleoprotein A1 IPI00215965 SSGPYGGGGQYFAKPR 1.59 0.489 0.921 337 352 HNRNPA1 Isoform A1-B of Heterogeneous NB88-V nuclear ribonucleoprotein A1 IPI00215965 NQGGYGGSSSSSSYGSGR 4.31 0.752 8.28 353 370 HNRNPA2B1 Isoform B1 of Heterogeneous NB88-V nuclear ribonucleoproteins A2/B1 IPI00396378,IPI00414696 NMGGPYGGGNYGPGGSGGSGGYGGR 5.59 0.834 10.9 326 350 HNRNPA3 Isoform 1 of Heterogeneous NB88-V nuclear ribonucleoprotein A3 IPI00419373 SSGSPYGGGYGSGGGSGGYGSR 5.01 0.837 9.66 355 376
129 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
HNRNPC Isoform C1 of Heterogeneous NB88-V nuclear ribonucleoproteins C1/C2 IPI00216592 DYYDRMYSYPAR 2.67 0.564 1.43 118 129 HNRNPH3 Isoform 1 of Heterogeneous NB25-V nuclear ribonucleoprotein H3 IPI00013877 DGMDNQGGYGSVGR 1.98 0.382 3.2 288 301 HNRNPK Isoform 1 of Heterogeneous nuclear NB88-V ribonucleoprotein K IPI00216049 RDYDDMSPR 2.23 0.323 0 278 286 HNRNPK Isoform 1 of Heterogeneous nuclear NB88-V ribonucleoprotein K IPI00216049 GGDLMAYDRR 3.02 0.282 0 317 326 HNRPF Heterogeneous nuclear NB88-V ribonucleoprotein F IPI00003881 FMSVQRPGPYDRPGTAR 1.8 0.244 1.82 201 217 HNRPH1 Heterogeneous nuclear NB88-V ribonucleoprotein H IPI00013881 DLNYCFSGMSDHR 3.46 0.751 7.72 263 275 IPI00386291,IPI00657885,IPI00 NB12 HSH2D HSH2D protein (Fragment) 748347 GSQDHSGDPTSGDRGYTDPCVATSLK 2.47 0.593 1.33 162 187 HSP90AA1 heat shock protein 90kDa alpha NB88 (cytosolic), class A member 1 isoform 1 IPI00382470 HIYYITGETK 3.51 0.661 4.74 612 621 HSP90AA1 heat shock protein 90kDa alpha NB25-V (cytosolic), class A member 1 isoform 1 IPI00382470 HIYYITGETK 3.11 0.599 2.01 612 621 HSP90AA1 heat shock protein 90kDa alpha NB25-V (cytosolic), class A member 1 isoform 1 IPI00382470 HIYYITGETK 3.01 0.558 3.77 612 621 HSP90AA1 heat shock protein 90kDa alpha NB12 (cytosolic), class A member 1 isoform 1 IPI00382470 HIYYITGETK 2.92 0.708 1.8 612 621 HSP90AA1 heat shock protein 90kDa alpha NB25 (cytosolic), class A member 1 isoform 1 IPI00382470 HIYYITGETK 2.67 0.544 2.52 612 621 HSP90AA1 heat shock protein 90kDa alpha NB88-V (cytosolic), class A member 1 isoform 1 IPI00382470 HIYYITGETK 2.49 0.502 4.19 612 621 HSP90AA1 heat shock protein 90kDa alpha NB61 (cytosolic), class A member 1 isoform 1 IPI00382470 HIYYITGETK 1.63 0.456 0 612 621 NB88-V HSP90AB1 Heat shock protein HSP 90-beta IPI00414676 SIYYITGESK 2.89 0.469 2.74 482 491 NB25-V HSP90AB1 Heat shock protein HSP 90-beta IPI00414676 SIYYITGESK 2.8 0.526 0 482 491 NB12 HSP90AB1 Heat shock protein HSP 90-beta IPI00414676 SIYYITGESK 2.74 0.499 2.17 482 491 NB88 HSP90AB1 Heat shock protein HSP 90-beta IPI00414676 SIYYITGESK 2.58 0.58 2.59 482 491 NB25-V HSP90AB1 Heat shock protein HSP 90-beta IPI00414676 SIYYITGESK 2.5 0.421 2.03 482 491 NB122 HSP90AB1 Heat shock protein HSP 90-beta IPI00414676 SIYYITGESK 2.1 0.559 0 482 491 NB88 HSPA4 Heat shock 70 kDa protein 4 IPI00002966 LKKEDIYAVEIVGGATR 3.91 0.53 2.89 330 346 NB12 HSPA4 Heat shock 70 kDa protein 4 IPI00002966 LKKEDIYAVEIVGGATR 3.9 0.645 2.96 330 346 NB25-V HSPA4 Heat shock 70 kDa protein 4 IPI00002966 LKKEDIYAVEIVGGATR 3.47 0.612 4.57 330 346 NB12-2 HSPA4 Heat shock 70 kDa protein 4 IPI00002966 LKKEDIYAVEIVGGATR 3.32 0.502 0 330 346 NB25-V HSPA4 Heat shock 70 kDa protein 4 IPI00002966 LKKEDIYAVEIVGGATR 3.19 0.478 0 330 346 NB88-V HSPA4 Heat shock 70 kDa protein 4 IPI00002966 LKKEDIYAVEIVGGATR 2.59 0.328 0 330 346 NB122 HSPA4 Heat shock 70 kDa protein 4 IPI00002966 LKKEDIYAVEIVGGATR 2.58 0.463 0 330 346 NB88-2 HSPA4 Heat shock 70 kDa protein 4 IPI00002966 LKKEDIYAVEIVGGATR 2.57 0.492 0 330 346 HSPA8 Isoform 1 of Heat shock cognate 71 NB88 kDa protein IPI00003865 VQVEYKGETK 2.21 0.538 0 103 112 HSPA8 Isoform 1 of Heat shock cognate 71 NB12-2 kDa protein IPI00003865 TTPSYVAFTDTER 3.46 0.729 3.89 37 49 NB88 HSPA9 Stress-70 protein, mitochondrial IPI00007765 QAVTNPNNTFYATKR 2.35 0.584 3.89 108 122 NB12-2 HSPA9 Stress-70 protein, mitochondrial IPI00007765 QAVTNPNNTFYATKR 2.15 0.549 0 108 122 NB88-2 HSPA9 Stress-70 protein, mitochondrial IPI00007765 QAVTNPNNTFYATKR 2.05 0.291 0 108 122 INPP5D Isoform 1 of Phosphatidylinositol- NB25-V 3,4,5-trisphosphate 5-phosphatase 1 IPI00329213 TREKLYDFVK 3.92 0.538 1.52 860 869 INPP5D Isoform 1 of Phosphatidylinositol- NB88-V 3,4,5-trisphosphate 5-phosphatase 1 IPI00329213 TREKLYDFVK 3.55 0.402 2.14 860 869 INPP5D Isoform 1 of Phosphatidylinositol- NB88 3,4,5-trisphosphate 5-phosphatase 1 IPI00329213 TREKLYDFVK 3.37 0.539 0 860 869 INPP5D Isoform 1 of Phosphatidylinositol- NB12-2 3,4,5-trisphosphate 5-phosphatase 1 IPI00329213 TREKLYDFVK 2.67 0.443 0 860 869 INPP5D Isoform 1 of Phosphatidylinositol- NB88-2 3,4,5-trisphosphate 5-phosphatase 1 IPI00329213 TREKLYDFVK 2.49 0.508 0 860 869 INPP5D Isoform 1 of Phosphatidylinositol- NB12 3,4,5-trisphosphate 5-phosphatase 1 IPI00329213 TREKLYDFVK 2.25 0.37 0 860 869 INPP5D Isoform 1 of Phosphatidylinositol- NAGDTLPQEDLPLTKPEMFENPLYGSLSSFPKP NB25-V 3,4,5-trisphosphate 5-phosphatase 1 IPI00329213 APR 4 0.665 2.1 999 1034 INPP5D Isoform 1 of Phosphatidylinositol- NAGDTLPQEDLPLTKPEMFENPLYGSLSSFPKP NB25 3,4,5-trisphosphate 5-phosphatase 1 IPI00329213 APR 3.72 0.565 2.02 999 1034 INPP5D Isoform 1 of Phosphatidylinositol- NAGDTLPQEDLPLTKPEMFENPLYGSLSSFPKP NB12-2 3,4,5-trisphosphate 5-phosphatase 1 IPI00329213 APR 3 0.484 2.02 999 1034 INPP5D Isoform 1 of Phosphatidylinositol- NAGDTLPQEDLPLTKPEMFENPLYGSLSSFPKP NB88-2 3,4,5-trisphosphate 5-phosphatase 1 IPI00329213 APR 2.95 0.554 0 999 1034 INPP5D Isoform 1 of Phosphatidylinositol- NAGDTLPQEDLPLTKPEMFENPLYGSLSSFPKP NB12 3,4,5-trisphosphate 5-phosphatase 1 IPI00329213 APR 2.81 0.543 5.52 999 1034
130 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
INPP5D Isoform 1 of Phosphatidylinositol- NAGDTLPQEDLPLTKPEMFENPLYGSLSSFPKP NB88 3,4,5-trisphosphate 5-phosphatase 1 IPI00329213 APR 1.78 0.415 1.82 999 1034 INPP5D Isoform 1 of Phosphatidylinositol- NB12-2 3,4,5-trisphosphate 5-phosphatase 1 IPI00329213 EKLYDFVKTER 2.6 0.531 0 862 872 INPP5D Isoform 1 of Phosphatidylinositol- NB88-V 3,4,5-trisphosphate 5-phosphatase 1 IPI00329213 EKLYDFVKTER 2.46 0.494 0 862 872 INPP5D Isoform 1 of Phosphatidylinositol- NB88 3,4,5-trisphosphate 5-phosphatase 1 IPI00329213 EKLYDFVKTER 2.18 0.428 0 862 872 INPP5D Isoform 1 of Phosphatidylinositol- NB12 3,4,5-trisphosphate 5-phosphatase 1 IPI00329213 EKLYDFVKTER 2.14 0.414 0 862 872 INPP5D Isoform 1 of Phosphatidylinositol- NB25-V 3,4,5-trisphosphate 5-phosphatase 1 IPI00329213 EKLYDFVKTER 1.84 0.427 0 862 872 INPP5D Isoform 1 of Phosphatidylinositol- NB25-V 3,4,5-trisphosphate 5-phosphatase 1 IPI00329213 APPCSGSSITEIINPNYMGVGPFGPPMPLHVK 7.32 0.661 3.57 899 930 INPPL1 Isoform 1 of Phosphatidylinositol- NB25-V 3,4,5-trisphosphate 5-phosphatase 2 IPI00016932,IPI00795687 NSFNNPAYYVLEGVPHQLLPPEPPSPAR 3.56 0.691 4.77 979 1006 INPPL1 Isoform 1 of Phosphatidylinositol- NB25-V 3,4,5-trisphosphate 5-phosphatase 2 IPI00016932,IPI00795687 ERLYEWISIDKDEAGAK 3.53 0.498 0 883 899 INPPL1 Isoform 1 of Phosphatidylinositol- NB88-V 3,4,5-trisphosphate 5-phosphatase 2 IPI00016932,IPI00795687 ERLYEWISIDKDEAGAK 2.7 0.179 2.52 883 899 IPI00025803,IPI00220325,IPI00 NB25-V INSR Insulin receptor precursor 783573,IPI00794155 SYEEHIPYTHMNGGKK pY 1355/ 1361 4.25 0.753 2.8 1374 1389 IPI00025803,IPI00220325,IPI00 NB88-V INSR Insulin receptor precursor 783573,IPI00794155 SYEEHIPYTHMNGGKK pY 1355/ 1361 3.28 0.499 2.07 1374 1389 IPI00025803,IPI00220325,IPI00 NB88-V INSR Insulin receptor precursor 783573,IPI00794155 SYEEHIPYTHMNGGK pY 1355/ 1361 3.58 0.658 2.52 1374 1388 IPI00025803,IPI00220325,IPI00 NB25-V INSR Insulin receptor precursor 783573,IPI00794155 SYEEHIPYTHMNGGK pY 1355/ 1361 3.2 0.66 1.92 1374 1388 IPI00025803,IPI00220325,IPI00 NB88 INSR Insulin receptor precursor 783573 DIYETDYYRKGGK pY 1185/ 1189 3.11 0.754 0 1203 1215 IPI00794155 IPI00025803,IPI00220325,IPI00 NB88-V INSR Insulin receptor precursor 783573,IPI00794155 DIYETDYYRKGGK pY 1185/ 1189 3.02 0.688 1.57 1203 1215 IPI00025803,IPI00220325,IPI00 NB61 INSR Insulin receptor precursor 783573 DIYETDYYRKGGK pY 1185/ 1189 2.3 0.706 0 1203 1215 IPI00794155 IPI00025803,IPI00220325,IPI00 NB25-V INSR Insulin receptor precursor 783573,IPI00794155 DIYETDYYRKGGK pY 1185/ 1189 2.3 0.768 0 1203 1215 IPI00025803,IPI00220325,IPI00 NB88-V INSR Insulin receptor precursor 783573,IPI00794155 DIYETDYYRK pY 1185/ 1189 3.21 0.631 3.89 1203 1212 IPI00025803,IPI00220325,IPI00 NB88 INSR Insulin receptor precursor 783573 DIYETDYYRK pY 1185/ 1189 2.73 0.696 1.62 1203 1212 IPI00794155 IPI00220325,IPI00794155,IPI007 NB122-2 INSR Insulin receptor precursor IPI00025803 DIYETDYYRK pY 1189 1.67 0.626 0 1203 1212 83573 IPI00025803,IPI00220325,IPI00 NB25-V INSR Insulin receptor precursor 783573,IPI00794155 DIYETDYYR pY 1189 2.53 0.609 2.24 1203 1211 IPI00025803,IPI00220325,IPI00 NB61 INSR Insulin receptor precursor 783573 DIYETDYYR pY 1189 2.26 0.425 3.85 1203 1211 IPI00794155 IPI00025803,IPI00220325,IPI00 NB25 INSR Insulin receptor precursor 783573 DIYETDYYR pY 1189 1.93 0.491 2.13 1203 1211 IPI00794155 IPI:IPI00383095.1|TREMBL:Q8TES2 Tax_Id=9606 Gene_Symbol=MYO1G NB12 FLJ00121 protein (Fragment) IPI00383095 DTVIGVLDIYGFEVFPVNSFEQFCINYCNEK 1.75 0.369 0 182 212 IPI:IPI00383095.1|TREMBL:Q8TES2 Tax_Id=9606 Gene_Symbol=MYO1G NB25-V FLJ00121 protein (Fragment) IPI00383095 DTVIGVLDIYGFEVFPVNSFEQFCINYCNEK 1.72 0.42 0 182 212 IPI:IPI00383095.1|TREMBL:Q8TES2 Tax_Id=9606 Gene_Symbol=MYO1G NB25 FLJ00121 protein (Fragment) IPI00383095 DTVIGVLDIYGFEVFPVNSFEQFCINYCNEK 1.28 0.398 0 182 212 IPI:IPI00384562.2|TREMBL:Q86XJ6|ENSEM BL:ENSP00000331602;ENSP00000378217|R EFSEQ:NP_006245;NP_997704|H- INV:HIT000052799|VEGA:OTTHUMP0000016 5024;OTTHUMP00000165025;OTTHUMP0000 NB88-V 0171565 IPI00384562 SDSASSEPVGIYQGFEK 4.22 0.42 4.34 302 318 IPI00329236 IPI:IPI00384562.2|TREMBL:Q86XJ6|ENSEM BL:ENSP00000331602;ENSP00000378217|R EFSEQ:NP_006245;NP_997704|H- INV:HIT000052799|VEGA:OTTHUMP0000016 5024;OTTHUMP00000165025;OTTHUMP0000 NB88-V 0171565 IPI00384562 RSDSASSEPVGIYQGFEKK 5.37 0.712 6.07 301 319 IPI00329236
131 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
IPI:IPI00384562.2|TREMBL:Q86XJ6|ENSEM BL:ENSP00000331602;ENSP00000378217|R EFSEQ:NP_006245;NP_997704|H- INV:HIT000052799|VEGA:OTTHUMP0000016 5024;OTTHUMP00000165025;OTTHUMP0000 NB88-V 0171565 IPI00384562 RSDSASSEPVGIYQGFEK 5.74 0.708 10.2 301 318 IPI00329236 IPI:IPI00384562.2|TREMBL:Q86XJ6|ENSEM BL:ENSP00000331602;ENSP00000378217|R EFSEQ:NP_006245;NP_997704|H- INV:HIT000052799|VEGA:OTTHUMP0000016 5024;OTTHUMP00000165025;OTTHUMP0000 NB25-V 0171565 IPI00384562 RSDSASSEPVGIYQGFEK 4.49 0.735 7.77 301 318 IPI00329236 IPI:IPI00384562.2|TREMBL:Q86XJ6|ENSEM BL:ENSP00000331602;ENSP00000378217|R EFSEQ:NP_006245;NP_997704|H- INV:HIT000052799|VEGA:OTTHUMP0000016 5024;OTTHUMP00000165025;OTTHUMP0000 NB88 0171565 IPI00384562 GRGEYFAIK 2.72 0.361 0 370 378 IPI:IPI00384562.2|TREMBL:Q86XJ6|ENSEM BL:ENSP00000331602;ENSP00000378217|R EFSEQ:NP_006245;NP_997704|H- INV:HIT000052799|VEGA:OTTHUMP0000016 5024;OTTHUMP00000165025;OTTHUMP0000 NB12-2 0171565 IPI00384562 GRGEYFAIK 2.46 0.337 0 370 378 IPI:IPI00384562.2|TREMBL:Q86XJ6|ENSEM BL:ENSP00000331602;ENSP00000378217|R EFSEQ:NP_006245;NP_997704|H- INV:HIT000052799|VEGA:OTTHUMP0000016 5024;OTTHUMP00000165025;OTTHUMP0000 NB12 0171565 IPI00384562 GRGEYFAIK 2.42 0.35 0 370 378 IQGAP1 Ras GTPase-activating-like protein NB88-2 IQGAP1 IPI00009342 LQQTYAALNSK 2.69 0.556 4.96 1506 1516 IQGAP1 Ras GTPase-activating-like protein NB12-2 IQGAP1 IPI00009342 LQQTYAALNSK 2.26 0.495 0 1506 1516 IQGAP1 Ras GTPase-activating-like protein NB88 IQGAP1 IPI00009342 LQQTYAALNSK 1.97 0.448 2.02 1506 1516 RHPQRVDPNGYMMMSPSGGCSPDIGGGPSSS NB88 IRS1 Insulin receptor substrate 1 IPI00019471 SSSSNAVPSGTSYGK 2.56 0.37 0 652 697 RGGHHRPDSSTLHTDDGYMPMSPGVAPVPSG NB88-2 IRS1 Insulin receptor substrate 1 IPI00019471 R 3.21 0.55 1.82 595 626 RGGHHRPDSSTLHTDDGYMPMSPGVAPVPSG NB88 IRS1 Insulin receptor substrate 1 IPI00019471 R 2.7 0.503 0 595 626 RGGHHRPDSSTLHTDDGYMPMSPGVAPVPSG NB88-V IRS1 Insulin receptor substrate 1 IPI00019471 R 1.98 0.412 0 595 626 NB88 IRS1 Insulin receptor substrate 1 IPI00019471 KGSGDYMPMSPK 3.61 0.441 1.66 627 638 NB88-2 IRS1 Insulin receptor substrate 1 IPI00019471 KGSGDYMPMSPK 2.52 0.427 1.03 627 638
NB122-2 IRS1 Insulin receptor substrate 1 IPI00019471 GGHHRPDSSTLHTDDGYMPMSPGVAPVPSGR 2.55 0.387 0 596 626
NB88-V IRS1 Insulin receptor substrate 1 IPI00019471 GGHHRPDSSTLHTDDGYMPMSPGVAPVPSGR 2.44 0.674 0 596 626
NB88-2 IRS1 Insulin receptor substrate 1 IPI00019471 GGHHRPDSSTLHTDDGYMPMSPGVAPVPSGR 2.38 0.616 0 596 626
NB88 IRS1 Insulin receptor substrate 1 IPI00019471 GGHHRPDSSTLHTDDGYMPMSPGVAPVPSGR 2.22 0.503 1.09 596 626
NB25-V IRS1 Insulin receptor substrate 1 IPI00019471 GGHHRPDSSTLHTDDGYMPMSPGVAPVPSGR 1.54 0.538 0 596 626 IRS2 Insulin receptor substrate 2 insertion NB88-V mutant (Fragment) IPI00464978 SYKAPYTCGGDSDQYVLMSSPVGR 4.5 0.713 7 811 834 IRS2 Insulin receptor substrate 2 insertion NB88-2 mutant (Fragment) IPI00464978 SDDYMPMSPASVSAPK 3.19 0.643 3.12 674 689 IRS2 Insulin receptor substrate 2 insertion NB122-2 mutant (Fragment) IPI00464978 SDDYMPMSPASVSAPK 2.81 0.632 5.37 674 689 IRS2 Insulin receptor substrate 2 insertion NB12 mutant (Fragment) IPI00464978 SDDYMPMSPASVSAPK 2.43 0.7 3.82 674 689 IRS2 Insulin receptor substrate 2 insertion NB122 mutant (Fragment) IPI00464978 SDDYMPMSPASVSAPK 1.88 0.578 0 674 689 ITCH Isoform 1 of E3 ubiquitin-protein ligase NB12 Itchy homolog IPI00061780,IPI00176010 FIYGNQDLFATSQSK 4.45 0.539 4 418 432 ITCH Isoform 1 of E3 ubiquitin-protein ligase NB61 Itchy homolog IPI00061780 FIYGNQDLFATSQSK 2.18 0.53 0 418 432 IPI00176010 NB12 ITGB1 Isoform Beta-1A of Integrin beta-1 IPI00217563,IPI00645194 WDTGENPIYK 1.98 0.54 1.34 775 784
132 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
ITGB1 Isoform Beta-1A of Integrin beta-1 NB25-V precursor IPI00217563,IPI00645194 WDTGENPIYK 2.58 0.499 2.82 775 784 IPI00216423,IPI00236575,IPI00 NB88-V ITSN2 Isoform 4 of Intersectin-2 396534,IPI00414027 REEPEALYAAVNK 4.53 0.655 9.7 960 972 IPI00892957 IPI00216423,IPI00236575,IPI00 396534,IPI00414027,IPI008929 NB88-2 ITSN2 Isoform 4 of Intersectin-2 57 REEPEALYAAVNK 4.32 0.598 6.8 960 972 IPI00216423,IPI00396534,IPI00 NB12 ITSN2 Isoform 4 of Intersectin-2 414027,IPI00892957 REEPEALYAAVNK 4.13 0.663 7.77 960 972 IPI00236575 IPI00216423,IPI00396534,IPI00 NB88 ITSN2 Isoform 4 of Intersectin-2 414027,IPI00892957 REEPEALYAAVNK 4.02 0.618 8.22 960 972 IPI00236575 IPI00216423,IPI00236575,IPI00 NB25-V ITSN2 Isoform 4 of Intersectin-2 396534,IPI00414027 REEPEALYAAVNK 4 0.612 4.8 960 972 IPI00892957 IPI00216423,IPI00236575,IPI00 NB25-V ITSN2 Isoform 4 of Intersectin-2 396534,IPI00414027 REEPEALYAAVNK 3.79 0.565 8.32 960 972 IPI00892957 IPI00216423,IPI00396534,IPI00 NB88 ITSN2 Isoform 4 of Intersectin-2 414027,IPI00892957 LIYLVPEK 2.61 0.495 0 550 557 IPI00236575 IPI00216423,IPI00236575,IPI00 NB25-V ITSN2 Isoform 4 of Intersectin-2 396534,IPI00414027 LIYLVPEK 2.28 0.343 0 550 557 IPI00892957 IPI00216423,IPI00396534,IPI00 NB12 ITSN2 Isoform 4 of Intersectin-2 414027,IPI00892957 LIYLVPEK 2.17 0.398 0 550 557 IPI00236575 IPI00892957,IPI00396534,IPI002 NB122-2 ITSN2 Isoform 4 of Intersectin-2 IPI00216423 LIYLVPEK 2.14 0.222 0 550 557 36575,IPI00414027 IPI00216423,IPI00236575,IPI00 NB88-V ITSN2 Isoform 4 of Intersectin-2 396534,IPI00414027 LIYLVPEK 2.09 0.352 0 550 557 IPI00892957 IPI00892957,IPI00236575,IPI004 NB122 ITSN2 Isoform 4 of Intersectin-2 IPI00216423,IPI00396534 LIYLVPEK 1.87 0.356 0 550 557 14027 IPI00216423,IPI00236575,IPI00 396534,IPI00414027,IPI008929 NB88-2 ITSN2 Isoform 4 of Intersectin-2 57 LIYLVPEK 1.8 0.366 1.17 550 557 NB88-V JAK2 Tyrosine-protein kinase JAK2 IPI00031016 REVGDYGQLHETEVLLK 3.69 0.592 3.55 565 581 NB122-2 JAK2 Tyrosine-protein kinase JAK2 IPI00031016 REVGDYGQLHETEVLLK 3.02 0.472 1.6 565 581 NB61 JAK2 Tyrosine-protein kinase JAK2 IPI00031016 REVGDYGQLHETEVLLK 2.72 0.518 0 565 581 NB25-V JAK2 Tyrosine-protein kinase JAK2 IPI00031016 EVGDYGQLHETEVLLK 2.3 0.4 0 566 581 NB88 KIAA1618 Isoform 1 of Protein ALO17 IPI00642126 VHGGTTADMIYSR 2.99 0.779 3 2451 2463 IPI00152853,IPI00445658,IPI00 NB25-V KIAA1949 Isoform 1 of Phostensin 735653 LSPGESAYQK 2.53 0.65 2.46 223 232 NB12-2 KIAA1949 Isoform 1 of Phostensin IPI00152853 LSPGESAYQK 2.11 0.438 0 223 232 IPI00445658,IPI00735653 NB88-2 KIAA1949 Isoform 1 of Phostensin IPI00152853 LSPGESAYQK 1.81 0.4 0 223 232 IPI00445658,IPI00735653 NB12 KIAA1949 Isoform 1 of Phostensin IPI00152853 LSPGESAYQK 1.64 0.692 0 223 232 IPI00445658,IPI00735653 NB88 KIAA1949 Isoform 1 of Phostensin IPI00152853 LSPGESAYQK 1.52 0.623 0 223 232 IPI00445658,IPI00735653 NB25 KIAA1949 Isoform 1 of Phostensin IPI00152853 LSPGESAYQK 1.13 0.573 0 223 232 IPI00445658,IPI00735653 KIF23 Isoform 1 of Kinesin-like protein IPI00291579,IPI00293884,IPI00 NB88 KIF23 873577 KGSQTNLKDPVGVYCR 2.32 0.38 3.41 16 31 LAT2 Isoform 1 of Linker for activation of T- NB88-V cells family member 2 IPI00395993 TYSLVGQAWPGPLADMAPTRK 2.87 0.491 2.72 57 77 IPI00032447 LAT2 Isoform 1 of Linker for activation of T- NB88-2 cells family member 2 IPI00395993 TGPTSGLCPSASPEEDEESEDYQNSASIHQWR 5.88 0.781 4.92 172 203 LAT2 Isoform 1 of Linker for activation of T- NB25-V cells family member 2 IPI00395993 TGPTSGLCPSASPEEDEESEDYQNSASIHQWR 5.35 0.834 7.46 172 203 LAT2 Isoform 1 of Linker for activation of T- NB88-V cells family member 2 IPI00395993 TGPTSGLCPSASPEEDEESEDYQNSASIHQWR 5.23 0.869 11 172 203 LAT2 Isoform 1 of Linker for activation of T- NB12-2 cells family member 2 IPI00395993 TGPTSGLCPSASPEEDEESEDYQNSASIHQWR 4.72 0.645 2.62 172 203 LAT2 Isoform 1 of Linker for activation of T- NB122-2 cells family member 2 IPI00395993 TGPTSGLCPSASPEEDEESEDYQNSASIHQWR 4.11 0.768 4.44 172 203 LAT2 Isoform 1 of Linker for activation of T- NB12 cells family member 2 IPI00395993 TGPTSGLCPSASPEEDEESEDYQNSASIHQWR 3.94 0.694 6.46 172 203 LAT2 Isoform 1 of Linker for activation of T- NB25-V cells family member 2 IPI00395993 TGPTSGLCPSASPEEDEESEDYQNSASIHQWR 3.45 0.843 0 172 203 LAT2 Isoform 1 of Linker for activation of T- NB25 cells family member 2 IPI00395993 TGPTSGLCPSASPEEDEESEDYQNSASIHQWR 3.13 0.821 0 172 203 LAT2 Isoform 1 of Linker for activation of T- NB61 cells family member 2 IPI00395993 TGPTSGLCPSASPEEDEESEDYQNSASIHQWR 2.38 0.68 0 172 203 LAT2 Isoform 1 of Linker for activation of T- NB25-V cells family member 2 IPI00395993 SEKIYQQR 2.69 0.436 0.921 36 43 IPI00032447 LAT2 Isoform 1 of Linker for activation of T- NB88-2 cells family member 2 IPI00395993 FSKPPEDDDANSYENVLICK 4.95 0.66 2.49 124 143 LAT2 Isoform 1 of Linker for activation of T- NB88-V cells family member 2 IPI00395993 FSKPPEDDDANSYENVLICK 4.71 0.686 5.74 124 143
133 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
LAT2 Isoform 1 of Linker for activation of T- NB12 cells family member 2 IPI00395993 FSKPPEDDDANSYENVLICK 4.09 0.538 4.68 124 143 LAT2 Isoform 1 of Linker for activation of T- NB122-2 cells family member 2 IPI00395993 FSKPPEDDDANSYENVLICK 4.07 0.65 2.02 124 143 LAT2 Isoform 1 of Linker for activation of T- NB12-2 cells family member 2 IPI00395993 FSKPPEDDDANSYENVLICK 3.8 0.496 3.74 124 143 LAT2 Isoform 1 of Linker for activation of T- NB25-V cells family member 2 IPI00395993 FSKPPEDDDANSYENVLICK 3.7 0.681 4.89 124 143 LAT2 Isoform 1 of Linker for activation of T- NB25-V cells family member 2 IPI00395993 FSKPPEDDDANSYENVLICK 2.04 0.575 0 124 143 LAT2 Isoform 2 of Linker for activation of T- NB88-V cells family member 2 IPI00032447 TYSLVGQAWPGPLADMAPTRK 2.87 0.491 2.72 57 77 IPI00395993 LAT2 Isoform 2 of Linker for activation of T- NB88-V cells family member 2 IPI00032447 RSEKIYQQR 2.42 0.251 0 35 43 LAT2 Isoform 2 of Linker for activation of T- NB88 cells family member 2 IPI00032447 RSEKIYQQR 2.36 0.34 0 35 43 LAX1 Isoform 1 of Lymphocyte NB88-2 transmembrane adapter 1 IPI00301971 NIYDILPWRQEDLGR 3.71 0.504 2.36 91 105 LAX1 Isoform 1 of Lymphocyte NB88-V transmembrane adapter 1 IPI00301971 NIYDILPWRQEDLGR 3.22 0.511 3.8 91 105 LAX1 Isoform 1 of Lymphocyte NB25-V transmembrane adapter 1 IPI00301971 NIYDILPWRQEDLGR 3.03 0.428 0 91 105 LAX1 Isoform 1 of Lymphocyte NB88 transmembrane adapter 1 IPI00301971 HREEMSNEDSSDYENVLTAK 4.31 0.745 7 361 380 LAX1 Isoform 1 of Lymphocyte NB88-V transmembrane adapter 1 IPI00301971 AKNIYDILPWRQEDLGR 2.88 0.252 0 89 105 LAX1 Isoform 1 of Lymphocyte NB25-V transmembrane adapter 1 IPI00301971 AKNIYDILPWRQEDLGR 2.15 0.462 0 89 105 LCK cDNA FLJ56184, highly similar to Proto- IPI00394952,IPI00515097,IPI00 NB88-2 oncogene tyrosine-protein kinase LCK 555672 NLDNGGFYISPR pY 192 3.86 0.488 4.52 185 196 LCK cDNA FLJ56184, highly similar to Proto- IPI00394952,IPI00515097,IPI00 NB122-2 oncogene tyrosine-protein kinase LCK 555672 NLDNGGFYISPR pY 192 3.59 0.459 4.72 185 196 LCK cDNA FLJ56184, highly similar to Proto- IPI00394952,IPI00515097,IPI00 NB25 oncogene tyrosine-protein kinase LCK 555672 NLDNGGFYISPR pY 192 3.54 0.557 3.29 0 0 LCK cDNA FLJ56184, highly similar to Proto- IPI00394952,IPI00515097,IPI00 NB12-2 oncogene tyrosine-protein kinase LCK 555672 NLDNGGFYISPR pY 192 3.34 0.538 4.59 0 0 LCK cDNA FLJ56184, highly similar to Proto- IPI00394952,IPI00515097,IPI00 NB12 oncogene tyrosine-protein kinase LCK 555672 NLDNGGFYISPR pY 192 3.05 0.59 3.89 0 0 LCK cDNA FLJ56184, highly similar to Proto- NB122 oncogene tyrosine-protein kinase LCK IPI00394952 NLDNGGFYISPR pY 192 2.77 0.541 0 0 0 IPI00555672,IPI00515097 LCK cDNA FLJ56184, highly similar to Proto- IPI00394952,IPI00515097,IPI00 NB88 oncogene tyrosine-protein kinase LCK 555672 NLDNGGFYISPR pY 192 2.64 0.514 4.18 0 0 LCK cDNA FLJ56184, highly similar to Proto- IPI00394952,IPI00515097,IPI00 NB12-2 oncogene tyrosine-protein kinase LCK 555672 LIEDNEYTAREGAK pY 394 5.36 0.535 7.28 432 445 LCK cDNA FLJ56184, highly similar to Proto- IPI00394952,IPI00515097,IPI00 NB12 oncogene tyrosine-protein kinase LCK 555672 LIEDNEYTAREGAK pY 394 4.38 0.474 3.11 432 445 LCK cDNA FLJ56184, highly similar to Proto- IPI00394952,IPI00515097,IPI00 NB122-2 oncogene tyrosine-protein kinase LCK 555672 LIEDNEYTAREGAK pY 394 3.76 0.514 5.28 418 431 LCK cDNA FLJ56184, highly similar to Proto- IPI00394952,IPI00515097,IPI00 NB88 oncogene tyrosine-protein kinase LCK 555672 LIEDNEYTAREGAK pY 394 3.11 0.56 1.24 432 445 LCK cDNA FLJ56184, highly similar to Proto- NB122 oncogene tyrosine-protein kinase LCK IPI00394952 LIEDNEYTAREGAK pY 394 1.53 0.444 0 432 445 IPI00555672,IPI00515097 IPI00166845,IPI00639876,IPI002 LCK cDNA FLJ56184, highly similar to Proto- IPI00394952,IPI00515097,IPI00 19012,IPI00013981,IPI00640091,I NB12 oncogene tyrosine-protein kinase LCK 555672 LIEDNEYTAR pY 394 3.54 0.689 2.96 432 441 PI00641230,IPI00328867 IPI00166845,IPI00639876,IPI002 LCK cDNA FLJ56184, highly similar to Proto- IPI00394952,IPI00515097,IPI00 19012,IPI00013981,IPI00640091,I NB88-2 oncogene tyrosine-protein kinase LCK 555672 LIEDNEYTAR pY 394 3.41 0.673 3.09 418 427 PI00641230,IPI00328867 IPI00166845,IPI00639876,IPI002 LCK cDNA FLJ56184, highly similar to Proto- IPI00394952,IPI00515097,IPI00 19012,IPI00013981,IPI00640091,I NB12-2 oncogene tyrosine-protein kinase LCK 555672 LIEDNEYTAR pY 394 3.05 0.736 4.7 432 441 PI00641230,IPI00328867 IPI00166845,IPI00639876,IPI002 LCK cDNA FLJ56184, highly similar to Proto- IPI00394952,IPI00515097,IPI00 19012,IPI00013981,IPI00640091,I NB61 oncogene tyrosine-protein kinase LCK 555672 LIEDNEYTAR pY 394 3.01 0.666 2.66 432 441 PI00641230,IPI00328867 IPI00166845,IPI00639876,IPI002 LCK cDNA FLJ56184, highly similar to Proto- IPI00394952,IPI00515097,IPI00 19012,IPI00013981,IPI00640091,I NB88 oncogene tyrosine-protein kinase LCK 555672 LIEDNEYTAR pY 394 2.88 0.666 2.34 432 441 PI00641230,IPI00328867 IPI00166845,IPI00639876,IPI002 LCK cDNA FLJ56184, highly similar to Proto- IPI00394952,IPI00515097,IPI00 19012,IPI00013981,IPI00640091,I NB122-2 oncogene tyrosine-protein kinase LCK 555672 LIEDNEYTAR pY 394 2.55 0.637 4.08 418 427 PI00641230,IPI00328867
134 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
LCK cDNA FLJ56184, highly similar to Proto- IPI00394952,IPI00515097,IPI00 NB12-2 oncogene tyrosine-protein kinase LCK 555672 IRNLDNGGFYISPR pY 192 4.95 0.637 6.55 0 0 LCK cDNA FLJ56184, highly similar to Proto- NB122 oncogene tyrosine-protein kinase LCK IPI00394952 IRNLDNGGFYISPR pY 192 3.71 0.713 0 0 0 IPI00555672,IPI00515097 LCK cDNA FLJ56184, highly similar to Proto- IPI00394952,IPI00515097,IPI00 NB12 oncogene tyrosine-protein kinase LCK 555672 IRNLDNGGFYISPR pY 192 3.6 0.681 3.62 0 0 LCK cDNA FLJ56184, highly similar to Proto- IPI00394952,IPI00515097,IPI00 NB122-2 oncogene tyrosine-protein kinase LCK 555672 IRNLDNGGFYISPR pY 192 3.38 0.496 2.52 183 196 LCK Isoform Long of Proto-oncogene IPI00394952,IPI00515097,IPI00 NB25-V tyrosine-protein kinase LCK 555672 NLDNGGFYISPR pY 192 3.29 0.533 3.15 243 254 LCK Isoform Long of Proto-oncogene IPI00394952,IPI00515097,IPI00 NB25-V tyrosine-protein kinase LCK 555672 NLDNGGFYISPR pY 192 2.81 0.447 4.89 243 254 LCK Isoform Long of Proto-oncogene IPI00394952,IPI00515097,IPI00 NB88-V tyrosine-protein kinase LCK 555672 NLDNGGFYISPR pY 192 2.61 0.376 2.55 185 196 LCK Isoform Long of Proto-oncogene IPI00394952,IPI00515097,IPI00 NB25-V tyrosine-protein kinase LCK 555672 LIEDNEYTAREGAK pY 394 3.53 0.395 3.92 395 408 IPI00166845,IPI00639876,IPI002 LCK Isoform Long of Proto-oncogene IPI00394952,IPI00515097,IPI00 19012,IPI00013981,IPI00640091,I NB88-V tyrosine-protein kinase LCK 555672 LIEDNEYTAR pY 394 3.19 0.641 4.24 388 397 PI00641230,IPI00328867 IPI00166845,IPI00639876,IPI002 LCK Isoform Long of Proto-oncogene IPI00394952,IPI00515097,IPI00 19012,IPI00013981,IPI00640091,I NB25-V tyrosine-protein kinase LCK 555672 LIEDNEYTAR pY 394 3.01 0.658 4.12 395 404 PI00641230,IPI00328867 IPI00166845,IPI00639876,IPI002 LCK Isoform Long of Proto-oncogene IPI00394952,IPI00515097,IPI00 19012,IPI00013981,IPI00640091,I NB25-V tyrosine-protein kinase LCK 555672 LIEDNEYTAR pY 394 2.74 0.672 4.44 395 404 PI00641230,IPI00328867 NB88 LCP1 Plastin-2 IPI00010471 WANYHLENAGCNK 5.43 0.694 6.92 273 285 NB12 LCP1 Plastin-2 IPI00010471 WANYHLENAGCNK 4.48 0.711 6.92 273 285 NB122 LCP1 Plastin-2 IPI00010471 WANYHLENAGCNK 2.95 0.69 0 273 285 LDHA Isoform 1 of L-lactate dehydrogenase NB88 A chain IPI00217966 QVVESAYEVIK 3.3 0.589 0 233 243 LDHA Isoform 1 of L-lactate dehydrogenase NB12 A chain IPI00217966 QVVESAYEVIK 2.27 0.61 1.68 233 243 LDHA Isoform 1 of L-lactate dehydrogenase NB88 A chain IPI00217966 EVHKQVVESAYEVIK 2.73 0.395 0 229 243 NB88-2 LDHB L-lactate dehydrogenase B chain IPI00219217 MVVESAYEVIK 3.29 0.505 3.06 234 244 NB25-V LDHB L-lactate dehydrogenase B chain IPI00219217 MVVESAYEVIK 3.16 0.417 1.77 234 244 NB88 LDHB L-lactate dehydrogenase B chain IPI00219217 MVVESAYEVIK 2.98 0.419 1.96 234 244 NB122-2 LDHB L-lactate dehydrogenase B chain IPI00219217 MVVESAYEVIK 2.82 0.241 1.68 234 244 NB12 LDHB L-lactate dehydrogenase B chain IPI00219217 MVVESAYEVIK 2.75 0.368 2.8 234 244 NB88-V LDHB L-lactate dehydrogenase B chain IPI00219217 MVVESAYEVIK 2.64 0.309 3 234 244 NB12-2 LDHB L-lactate dehydrogenase B chain IPI00219217 MVVESAYEVIK 2.24 0.526 1.12 234 244 NB122 LDHB L-lactate dehydrogenase B chain IPI00219217 MVVESAYEVIK 2.09 0.41 1 234 244 LDLR Low-density lipoprotein receptor NB88-V precursor IPI00000070,IPI00792252 TTEDEVHICHNQDGYSYPSR 5.17 0.781 4.3 831 850 IPI00908663 LDLR Low-density lipoprotein receptor NB88 precursor IPI00000070,IPI00908663 TTEDEVHICHNQDGYSYPSR 4.25 0.758 1.49 831 850 IPI00792252 LDLR Low-density lipoprotein receptor NB25-V precursor IPI00000070 TTEDEVHICHNQDGYSYPSR 3.9 0.738 0 831 850 IPI00908663,IPI00792252 NB122 LIFR Leukemia inhibitory factor receptor IPI00444272 YNFFLYGCR 1.04 0.655 0 697 705 LILRB4 leukocyte immunoglobulin-like IPI00289926,IPI00830045,IPI00 NB12-2 receptor, subfamily B, member 4 isoform 2 893119,IPI00893520 SPHDEDPQAVTYAK 2.33 0.681 1.77 348 361 LILRB4 leukocyte immunoglobulin-like IPI00830045,IPI00893520,IPI008 NB25-V receptor, subfamily B, member 4 isoform 2 IPI00289926 SPHDEDPQAVTYAK 1.83 0.703 0 348 361 93119 LIMA1 Isoform Beta of LIM domain and actin- NB12 binding protein 1 IPI00008918 TVYPMER 1.5 0.495 0 396 402 NB25-V LIMD2 LIM domain-containing protein 2 IPI00549972,IPI00789498 SKGNYDEGFGR 3.01 0.597 3.66 98 108 LIME1 Isoform 1 of Lck-interacting NB25-V transmembrane adapter 1 IPI00646802,IPI00658039 TEVTPAAQVDVLYSR 3.37 0.637 2.85 93 107 LIME1 Isoform 1 of Lck-interacting NB88-V transmembrane adapter 1 IPI00646802,IPI00658039 TEVTPAAQVDVLYSR 2.88 0.654 8.57 188 202 LIME1 Isoform 1 of Lck-interacting NB25-V transmembrane adapter 1 IPI00646802,IPI00658039 SPQEPQQGKTEVTPAAQVDVLYSR 4.93 0.671 4.52 84 107 LIME1 Isoform 1 of Lck-interacting NB88-V transmembrane adapter 1 IPI00646802,IPI00658039 SPQEPQQGKTEVTPAAQVDVLYSR 4.21 0.644 6.8 179 202 LMO7 Isoform 3 of LIM domain only protein IPI00291802,IPI00409590,IPI00 NB88 7 409591,IPI00409593 KAQSNPYYNGPHLNLK 4.13 0.661 0 179 194 LMO7 Isoform 3 of LIM domain only protein IPI00291802,IPI00409590,IPI00 NB12-2 7 409591,IPI00409593 KAQSNPYYNGPHLNLK 3.85 0.538 5.06 179 194 LMO7 Isoform 3 of LIM domain only protein IPI00291802,IPI00409590,IPI00 NB88-V 7 409591,IPI00409593 KAQSNPYYNGPHLNLK 3.75 0.522 5.07 179 194
135 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
LMO7 Isoform 3 of LIM domain only protein IPI00291802,IPI00409590,IPI00 NB88-2 7 409591,IPI00409593 KAQSNPYYNGPHLNLK 3.56 0.306 1.89 179 194 LMO7 Isoform 3 of LIM domain only protein IPI00291802,IPI00409590,IPI00 NB61 7 409591,IPI00409593 KAQSNPYYNGPHLNLK 3.01 0.327 0.77 179 194 LMO7 Isoform 3 of LIM domain only protein IPI00291802,IPI00409590,IPI00 NB12-2 7 409591,IPI00409593 AQSNPYYNGPHLNLK 3.67 0.624 8.37 180 194 LMO7 Isoform 3 of LIM domain only protein IPI00291802,IPI00409590,IPI00 NB88 7 409591,IPI00409593 AQSNPYYNGPHLNLK 3.31 0.549 4.05 180 194 LMO7 Isoform 3 of LIM domain only protein IPI00291802,IPI00409590,IPI00 NB88-2 7 409591,IPI00409593 AQSNPYYNGPHLNLK 3.21 0.448 1.25 180 194 LMO7 Isoform 3 of LIM domain only protein IPI00291802,IPI00409590,IPI00 NB12 7 409591,IPI00409593 AQSNPYYNGPHLNLK 2.18 0.436 1.8 180 194 LMO7 Isoform 3 of LIM domain only protein IPI00291802,IPI00409590,IPI00 NB61 7 409591,IPI00409593 AQSNPYYNGPHLNLK 2.05 0.468 0 180 194 LNPEP Isoform 2 of Leucyl-cystinyl IPI00221240,IPI00221241,IPI00 NB88 aminopeptidase 307017,IPI00871815 GLGEHEMEEDEEDYESSAK 3.62 0.696 7.77 43 61 LOC100129063 similar to DKFZP434O047 NB88 protein IPI00888308 VPGQQALSQRDKGIYVPDPR 2.1 0.418 0 222 241 LOC100129063 similar to DKFZP434O047 NB88-V protein IPI00888308 VPGQQALSQRDKGIYVPDPR 1.98 0.313 0 222 241 LOC399491 Isoform 2 of GPS, PLAT and transmembrane domain-containing protein NB61 FLJ00285 IPI00884925 YEDPRSR 1.86 0.403 0 267 273 LOC646048 similar to actin alpha 1 skeletal NB88-2 muscle protein IPI00886911 DSYVGEDAQSKR 3.38 0 3.14 240 251 LOC646048 similar to actin alpha 1 skeletal NB25-V muscle protein IPI00886911 DSYVGEDAQSKR 3.09 0 3.85 240 251 LOC646048 similar to actin alpha 1 skeletal NB25 muscle protein IPI00886911 DSYVGEDAQSKR 2.65 0 2.82 240 251 LOC646048 similar to actin alpha 1 skeletal NB25-V muscle protein IPI00886911 DSYVGEDAQSKR 2.47 0 3.8 240 251 LOC646048 similar to actin alpha 1 skeletal NB61 muscle protein IPI00886911 DSYVGEDAQSKR 1.7 0 2.57 240 251 LOC648998 Putative neutrophil cytosol factor NB12 1C IPI00027007 FTEIYEFHK 2.28 0.648 0 44 52 LOC652708 Uncharacterized protein NB12-2 ENSP00000355193 IPI00514878 QEFGWMLPMQLISYPLSKGK 1.92 0.31 0 53 72 LPXN cDNA FLJ58628, highly similar to NB25-V Leupaxin IPI00299066,IPI00871430 STLQDSDEYSNPAPLPLDQHSR 4.92 0.717 5.47 14 35 LPXN cDNA FLJ58628, highly similar to NB88 Leupaxin IPI00299066,IPI00871430 STLQDSDEYSNPAPLPLDQHSR 4.29 0.708 5.32 19 40 LPXN cDNA FLJ58628, highly similar to NB88-V Leupaxin IPI00299066,IPI00871430 STLQDSDEYSNPAPLPLDQHSR 3.86 0.624 5.28 14 35 LPXN cDNA FLJ58628, highly similar to NB12 Leupaxin IPI00299066,IPI00871430 STLQDSDEYSNPAPLPLDQHSR 3.42 0.566 6.85 19 40 LPXN cDNA FLJ58628, highly similar to NB25-V Leupaxin IPI00299066,IPI00871430 STLQDSDEYSNPAPLPLDQHSR 3.21 0.738 1.34 14 35 LPXN cDNA FLJ58628, highly similar to NB12-2 Leupaxin IPI00299066,IPI00871430 STLQDSDEYSNPAPLPLDQHSR 3.2 0.504 4.02 19 40 LPXN cDNA FLJ58628, highly similar to NB88-2 Leupaxin IPI00299066,IPI00871430 STLQDSDEYSNPAPLPLDQHSR 3.15 0.526 6.04 19 40 LPXN cDNA FLJ58628, highly similar to NB122-2 Leupaxin IPI00299066,IPI00871430 STLQDSDEYSNPAPLPLDQHSR 3.08 0.587 3.37 19 40 LPXN cDNA FLJ58628, highly similar to NB61 Leupaxin IPI00299066 STLQDSDEYSNPAPLPLDQHSR 3.07 0.535 0 19 40 IPI00871430 LSR Isoform 2 of Lipolysis-stimulated NB88 lipoprotein receptor IPI00328218 SRDPHYDDFR 2.78 0.742 1.64 488 497 LSR Isoform 2 of Lipolysis-stimulated NB88-2 lipoprotein receptor IPI00328218 SRDPHYDDFR 1.37 0.39 0 488 497 LYN Isoform LYN A of Tyrosine-protein kinase NB25-V Lyn IPI00298625,IPI00432416 VIEDNEYTAREGAK pY396 4.39 0.653 4.14 370 383 IPI00029769,IPI00646510 LYN Isoform LYN A of Tyrosine-protein kinase NB88 Lyn IPI00298625,IPI00432416 VIEDNEYTAREGAK pY396 4.23 0.617 3 391 404 IPI00029769,IPI00646510 LYN Isoform LYN A of Tyrosine-protein kinase NB88-V Lyn IPI00298625,IPI00432416 VIEDNEYTAREGAK pY396 4.17 0.57 5.43 391 404 IPI00029769,IPI00646510 LYN Isoform LYN A of Tyrosine-protein kinase NB12-2 Lyn IPI00298625,IPI00432416 VIEDNEYTAREGAK pY396 3.95 0.583 4.66 391 404 IPI00029769,IPI00646510 LYN Isoform LYN A of Tyrosine-protein kinase NB88-2 Lyn IPI00298625,IPI00432416 VIEDNEYTAREGAK pY396 3.75 0.535 3.04 391 404 IPI00029769,IPI00646510 LYN Isoform LYN A of Tyrosine-protein kinase NB12 Lyn IPI00298625,IPI00432416 VIEDNEYTAREGAK pY396 3.5 0.548 2.68 391 404 IPI00029769,IPI00646510
136 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
LYN Isoform LYN A of Tyrosine-protein kinase NB122-2 Lyn IPI00298625,IPI00432416 VIEDNEYTAREGAK pY396 3.25 0.448 1.31 391 404 IPI00029769,IPI00646510 LYN Isoform LYN A of Tyrosine-protein kinase NB61 Lyn IPI00298625,IPI00432416 VIEDNEYTAREGAK pY396 1.02 0.487 1.02 391 404 IPI00029769,IPI00646510 LYN Isoform LYN A of Tyrosine-protein kinase NB12 Lyn IPI00298625,IPI00432416 VIEDNEYTAR pY396 3.61 0.708 3.92 391 400 IPI00029769,IPI00646510 LYN Isoform LYN A of Tyrosine-protein kinase NB88-2 Lyn IPI00298625,IPI00432416 VIEDNEYTAR pY396 3.34 0.639 3.57 391 400 IPI00029769,IPI00646510 LYN Isoform LYN A of Tyrosine-protein kinase NB25-V Lyn IPI00298625,IPI00432416 VIEDNEYTAR pY396 3.17 0.726 3.35 370 379 IPI00029769,IPI00646510 LYN Isoform LYN A of Tyrosine-protein kinase NB88-V Lyn IPI00298625,IPI00432416 VIEDNEYTAR pY396 3.11 0.553 1.34 391 400 IPI00029769,IPI00646510 LYN Isoform LYN A of Tyrosine-protein kinase NB12-2 Lyn IPI00298625,IPI00432416 VIEDNEYTAR pY396 2.97 0.571 3.16 391 400 IPI00029769,IPI00646510 LYN Isoform LYN A of Tyrosine-protein kinase NB88 Lyn IPI00298625,IPI00432416 VIEDNEYTAR pY396 2.81 0.523 3.96 391 400 IPI00029769,IPI00646510 LYN Isoform LYN A of Tyrosine-protein kinase NB122-2 Lyn IPI00298625,IPI00432416 VIEDNEYTAR pY396 2.66 0.61 3.1 391 400 IPI00029769,IPI00646510 LYN Isoform LYN A of Tyrosine-protein kinase NB25-V Lyn IPI00298625,IPI00432416 VIEDNEYTAR pY396 2.55 0.519 3.33 391 400 IPI00029769,IPI00646510 LYN Isoform LYN A of Tyrosine-protein kinase NB61 Lyn IPI00298625,IPI00432416 VIEDNEYTAR pY396 1.21 0.673 2 391 400 IPI00029769,IPI00646510 LYN Isoform LYN A of Tyrosine-protein kinase NB12-2 Lyn IPI00298625,IPI00432416 VENCPDELYDIMK pY472 3.27 0.35 4.49 465 477 LYN Isoform LYN A of Tyrosine-protein kinase NB88-2 Lyn IPI00298625,IPI00432416 VENCPDELYDIMK pY472 2.85 0.434 4.77 465 477 LYN Isoform LYN A of Tyrosine-protein kinase NB88-V Lyn IPI00298625,IPI00432416 VENCPDELYDIMK pY472 2.75 0.653 7.11 465 477 LYN Isoform LYN A of Tyrosine-protein kinase NB25 Lyn IPI00298625,IPI00432416 VENCPDELYDIMK pY472 2.47 0.397 3.18 465 477 LYN Isoform LYN A of Tyrosine-protein kinase NB25-V Lyn IPI00298625,IPI00432416 VENCPDELYDIMK pY472 2.23 0.511 3.24 465 477 LYN Isoform LYN A of Tyrosine-protein kinase NB88 Lyn IPI00298625,IPI00432416 VENCPDELYDIMK pY472 2.21 0.584 0 465 477 LYN Isoform LYN A of Tyrosine-protein kinase NB12 Lyn IPI00298625,IPI00432416 VENCPDELYDIMK pY472 2.16 0.604 3.8 465 477 LYN Isoform LYN A of Tyrosine-protein kinase NB12-2 Lyn IPI00298625,IPI00432416 SLDNGGYYISPR pY192 3.59 0.557 5.32 187 198 LYN Isoform LYN A of Tyrosine-protein kinase NB88-2 Lyn IPI00298625,IPI00432416 SLDNGGYYISPR pY192 3.51 0.459 3.16 187 198 LYN Isoform LYN A of Tyrosine-protein kinase NB88 Lyn IPI00298625,IPI00432416 SLDNGGYYISPR pY192 3.13 0.599 1.89 187 198 LYN Isoform LYN A of Tyrosine-protein kinase NB88-V Lyn IPI00298625,IPI00432416 SLDNGGYYISPR pY192 3.02 0.576 4.28 187 198 LYN Isoform LYN A of Tyrosine-protein kinase NB25-V Lyn IPI00298625,IPI00432416 SLDNGGYYISPR pY192 2.79 0.541 1.08 166 177 LYN Isoform LYN A of Tyrosine-protein kinase NB12 Lyn IPI00298625,IPI00432416 SLDNGGYYISPR pY192 2.67 0.521 3.41 187 198 LYN Isoform LYN A of Tyrosine-protein kinase NB12 Lyn IPI00298625,IPI00432416 EKAEERPTFDYLQSVLDDFYTATEGQYQQQP pY 507 3.92 0.54 7.82 482 512 NB88 MAGOHB Protein mago nashi homolog 2 IPI00059292 YANNSNYKNDVMIR 2.51 0.685 0 36 49 NB122-2 MAPK1 Mitogen-activated protein kinase 1 IPI00003479,IPI00874012 VADPDHDHTGFLTEYVATR 5.53 0.708 3.85 173 191 NB88-2 MAPK1 Mitogen-activated protein kinase 1 IPI00003479,IPI00874012 VADPDHDHTGFLTEYVATR 5.43 0.671 2.59 173 191 NB12 MAPK1 Mitogen-activated protein kinase 1 IPI00003479,IPI00874012 VADPDHDHTGFLTEYVATR 4.86 0.69 3.89 173 191 NB88 MAPK1 Mitogen-activated protein kinase 1 IPI00003479,IPI00874012 VADPDHDHTGFLTEYVATR 4.6 0.726 5.64 173 191 NB25-V MAPK1 Mitogen-activated protein kinase 1 IPI00003479,IPI00874012 VADPDHDHTGFLTEYVATR 4.46 0.75 2.25 173 191 NB25-V MAPK1 Mitogen-activated protein kinase 1 IPI00003479,IPI00874012 VADPDHDHTGFLTEYVATR 4.41 0.614 1.43 173 191 NB88-V MAPK1 Mitogen-activated protein kinase 1 IPI00003479,IPI00874012 VADPDHDHTGFLTEYVATR 4.36 0.697 3.96 173 191 NB61 MAPK1 Mitogen-activated protein kinase 1 IPI00003479 VADPDHDHTGFLTEYVATR 3.83 0.628 0 173 191 IPI00874012 NB122 MAPK1 Mitogen-activated protein kinase 1 IPI00003479,IPI00874012 VADPDHDHTGFLTEYVATR 3.58 0.698 2.06 173 191 NB12-2 MAPK1 Mitogen-activated protein kinase 1 IPI00003479,IPI00874012 VADPDHDHTGFLTEYVATR 3.55 0.705 3.77 173 191 NB25 MAPK1 Mitogen-activated protein kinase 1 IPI00003479,IPI00874012 VADPDHDHTGFLTEYVATR 2.8 0.481 2.02 173 191 MAPK14 Isoform CSBP2 of Mitogen-activated IPI00002857,IPI00221141,IPI00 NB88-V protein kinase 14 221142,IPI00221143 HTDDEMTGYVATR 4.55 0.784 13.6 174 186 MAPK14 Isoform CSBP2 of Mitogen-activated IPI00002857,IPI00221141,IPI00 NB122-2 protein kinase 14 221142,IPI00221143 HTDDEMTGYVATR 4.38 0.791 11 174 186 MAPK14 Isoform CSBP2 of Mitogen-activated IPI00002857,IPI00221141,IPI00 NB88 protein kinase 14 221142,IPI00221143 HTDDEMTGYVATR 4.34 0.794 10.6 174 186 MAPK14 Isoform CSBP2 of Mitogen-activated IPI00002857,IPI00221141,IPI00 NB25-V protein kinase 14 221142,IPI00221143 HTDDEMTGYVATR 4.29 0.814 9.13 174 186
137 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
MAPK14 Isoform CSBP2 of Mitogen-activated IPI00002857,IPI00221141,IPI00 NB12 protein kinase 14 221142,IPI00221143 HTDDEMTGYVATR 4.02 0.814 12.3 174 186 MAPK14 Isoform CSBP2 of Mitogen-activated IPI00002857,IPI00221141,IPI00 NB12-2 protein kinase 14 221142,IPI00221143 HTDDEMTGYVATR 3.53 0.7 2.11 174 186 MAPK14 Isoform CSBP2 of Mitogen-activated IPI00002857,IPI00221141,IPI00 NB88-2 protein kinase 14 221142,IPI00221143 HTDDEMTGYVATR 3.52 0.767 5.42 174 186 MAPK14 Isoform CSBP2 of Mitogen-activated IPI00002857,IPI00221141,IPI00 NB122 protein kinase 14 221142,IPI00221143 HTDDEMTGYVATR 3.11 0.647 0 174 186 MAPK14 Isoform CSBP2 of Mitogen-activated IPI00002857,IPI00221141,IPI00 NB25-V protein kinase 14 221142,IPI00221143 HTDDEMTGYVATR 3.06 0.789 6.38 174 186 MAPK14 Isoform CSBP2 of Mitogen-activated IPI00002857,IPI00221141,IPI00 NB61 protein kinase 14 221142,IPI00221143 HTDDEMTGYVATR 2.1 0.593 1.41 174 186 IPI00018195,IPI00304111,IPI00 NB25-V MAPK3 Mitogen-activated protein kinase 3 742900 IADPEHDHTGFLTEYVATR 5.05 0.667 3 190 208 NB25 MAPK3 Mitogen-activated protein kinase 3 IPI00018195 IADPEHDHTGFLTEYVATR 4.99 0.749 0 190 208 IPI00742900,IPI00304111 IPI00018195,IPI00304111,IPI00 NB88 MAPK3 Mitogen-activated protein kinase 3 742900 IADPEHDHTGFLTEYVATR 4.64 0.664 5.82 190 208 IPI00018195,IPI00304111,IPI00 NB88-V MAPK3 Mitogen-activated protein kinase 3 742900 IADPEHDHTGFLTEYVATR 4.52 0.713 2.96 190 208 IPI00018195,IPI00304111,IPI00 NB25-V MAPK3 Mitogen-activated protein kinase 3 742900 IADPEHDHTGFLTEYVATR 4.41 0.645 2.72 190 208 IPI00018195,IPI00304111,IPI00 NB12 MAPK3 Mitogen-activated protein kinase 3 742900 IADPEHDHTGFLTEYVATR 4.21 0.542 1.68 190 208 IPI00018195,IPI00304111,IPI00 NB88-2 MAPK3 Mitogen-activated protein kinase 3 742900 IADPEHDHTGFLTEYVATR 4.18 0.661 0 190 208 IPI00018195,IPI00304111,IPI00 NB122 MAPK3 Mitogen-activated protein kinase 3 742900 IADPEHDHTGFLTEYVATR 4.05 0.594 2.27 190 208 IPI00018195,IPI00304111,IPI00 NB122-2 MAPK3 Mitogen-activated protein kinase 3 742900 IADPEHDHTGFLTEYVATR 3.99 0.625 0 190 208 IPI00018195,IPI00304111,IPI00 NB12-2 MAPK3 Mitogen-activated protein kinase 3 742900 IADPEHDHTGFLTEYVATR 3.9 0.554 4.66 190 208 NB61 MAPK3 Mitogen-activated protein kinase 3 IPI00018195 IADPEHDHTGFLTEYVATR 3.5 0.521 0 190 208 IPI00742900,IPI00304111 IPI00149048,IPI00219601,IPI00 MAPK7 Isoform 3 of Mitogen-activated 376860,IPI00426283,IPI005556 NB88-V protein kinase 7 40 GLCTSPAEHQYFMTEYVATR 3.27 0.653 3.85 67 86 MAPK9 Isoform Alpha-2 of Mitogen-activated IPI00024673,IPI00220382,IPI00 NB88-2 protein kinase 9 220383,IPI00303550 TACTNFMMTPYVVTR 3.48 0.599 3.49 175 189 NB88 MATR3 Matrin-3 IPI00017297 SQESGYYDRMDYEDDRLR 3.36 0.817 0 208 225 NB88-V MATR3 Matrin-3 IPI00017297 SQESGYYDRMDYEDDRLR 2.8 0.71 2.28 208 225 NB88-V MATR3 Matrin-3 IPI00017297 CRDDSFFGETSHNYHKFDSEYER 4.84 0.666 1.15 230 252 MCM7 Isoform 1 of DNA replication licensing NB12-2 factor MCM7 IPI00299904 EAWASKDATYTSAR 2.17 0.473 0 591 604 MERTK Proto-oncogene tyrosine-protein NB25-V kinase MER IPI00029756 KIYSGDYYR 3.4 0.703 2.43 747 755 MERTK Proto-oncogene tyrosine-protein NB88-V kinase MER IPI00029756 KIYSGDYYR 2.81 0.515 3.01 747 755 MERTK Proto-oncogene tyrosine-protein NB25-V kinase MER IPI00029756 KIYSGDYYR 2.57 0.35 1.89 747 755 MERTK Proto-oncogene tyrosine-protein NB25-V kinase MER IPI00029756 IYSGDYYR 1.85 0.441 1.19 748 755 NB122 MGAM Maltase-glucoamylase, intestinal IPI00220143 YEYGTLDNMR 1.72 0.358 0 385 394 NB12 MGAT1 Putative uncharacterized protein IPI00103810 SQGSQIRGTILGYVAGY 1.67 0.483 0 129 145 MPZL1 Isoform 1 of Myelin protein zero-like NB25-V protein 1 IPI00022558 SLPSGSHQGPVIYAQLDHSGGHHSDKINK 2.13 0.628 0 229 257 IPI00760547,IPI00644618 MPZL1 Isoform 1 of Myelin protein zero-like IPI00022558,IPI00644618,IPI00 NB88-V protein 1 760547 SLPSGSHQGPVIYAQLDHSGGHHSDKINK 1.64 0.357 1.32 229 257 NB25-V MUC13 Mucin-13 IPI00011448 DSQMQNPYSR 2.58 0.693 2.19 494 503 NB12 MUC13 Mucin-13 IPI00011448 DSQMQNPYSR 2.16 0.595 1.28 494 503 MX1 Interferon-induced GTP-binding protein NB88-V Mx1 IPI00167949 GKVSYQDYEIEISDASEVEKEINK 8.44 0.7 9.82 122 145 MX1 Interferon-induced GTP-binding protein NB88 Mx1 IPI00167949 GKVSYQDYEIEISDASEVEKEINK 6.82 0.706 9.54 122 145 MX1 Interferon-induced GTP-binding protein NB25-V Mx1 IPI00167949 GKVSYQDYEIEISDASEVEKEINK 5.41 0.478 7.46 122 145 MX1 Interferon-induced GTP-binding protein NB12 Mx1 IPI00167949 GKVSYQDYEIEISDASEVEKEINK 5.27 0.607 4.32 122 145 MX1 Interferon-induced GTP-binding protein NB122 Mx1 IPI00167949 GKVSYQDYEIEISDASEVEKEINK 5.25 0.633 4.3 122 145 MX1 Interferon-induced GTP-binding protein NB88-2 Mx1 IPI00167949 GKVSYQDYEIEISDASEVEKEINK 5.03 0.61 6 122 145
138 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
MX1 Interferon-induced GTP-binding protein NB12-2 Mx1 IPI00167949 GKVSYQDYEIEISDASEVEKEINK 4.64 0.574 4.66 122 145 MX1 Interferon-induced GTP-binding protein NB61 Mx1 IPI00167949 GKVSYQDYEIEISDASEVEKEINK 3.94 0.517 0.854 122 145 MX1 Interferon-induced GTP-binding protein NB122-2 Mx1 IPI00167949 GKVSYQDYEIEISDASEVEKEINK 3.77 0.516 4.06 122 145 NB12 MYH9 Isoform 1 of Myosin-9 IPI00019502 VAAYDKLEK 2.62 0.339 2.68 1405 1413 NB122-2 MYH9 Isoform 1 of Myosin-9 IPI00019502 VAAYDKLEK 2.58 0.457 0 1405 1413 NB88 MYH9 Isoform 1 of Myosin-9 IPI00019502 VAAYDKLEK 2.22 0.351 1.74 1405 1413 NB12 MYH9 Isoform 1 of Myosin-9 IPI00019502 HEMPPHIYAITDTAYR 3.15 0.48 3.77 144 159 NB25-V MYO1E Myosin-Ie IPI00329672 SLYTSMARPPLPR 2.63 0.599 0 988 1000 NARG1 Isoform 2 of NMDA receptor- NB12-2 regulated protein 1 IPI00032158 LKIYEEAWTKYPR 2.33 0.529 1.72 277 289 IPI00028065,IPI00793968,IPI00 RKPSVPDSASPADDSFVDPGERLYDLNMPAYV NB25 NCK1 Cytoplasmic protein NCK1 797459 K 0 0 4.7 82 114 IPI00028065,IPI00793968,IPI00 RKPSVPDSASPADDSFVDPGERLYDLNMPAYV NB88 NCK1 Cytoplasmic protein NCK1 797459 K 0 0 6.02 82 114 IPI00028065,IPI00793968,IPI00 RKPSVPDSASPADDSFVDPGERLYDLNMPAYV NB25-V NCK1 Cytoplasmic protein NCK1 797459 K 0 0 7.27 82 114 IPI00028065,IPI00793968,IPI00 NB25 NCK1 Cytoplasmic protein NCK1 797459 KPSVPDSASPADDSFVDPGERLYDLNMPAYVK 4.55 0.764 9.26 83 114 IPI00028065,IPI00793968,IPI00 NB88-2 NCK1 Cytoplasmic protein NCK1 797459 KPSVPDSASPADDSFVDPGERLYDLNMPAYVK 4.55 0.628 7.24 83 114 NB25-V NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 YQKDVYDIPPSHTTQGVYDIPPSSAK 2.82 0.57 2.49 171 196 NB61 NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 YQKDVYDIPPSHTTQGVYDIPPSSAK 2.43 0.501 0 172 197 NB12 NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 YQKDVYDIPPSHTTQGVYDIPPSSAK 2.05 0.523 0 172 197 NB88-V NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 TGHGYVYEYPSR 3.79 0.669 3.8 160 171 NB88 NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 TGHGYVYEYPSR 3.45 0.631 4.64 160 171 NB25-V NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 TGHGYVYEYPSR 3.43 0.586 2 160 171 NB122-2 NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 TGHGYVYEYPSR 3.34 0.556 4.36 160 171 NB88-2 NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 TGHGYVYEYPSR 3.3 0.623 3.12 160 171 NB25-V NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 TGHGYVYEYPSR 3.29 0.658 4.92 159 170 NB12-2 NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 TGHGYVYEYPSR 3.18 0.694 3.7 160 171 NB12 NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 TGHGYVYEYPSR 3.16 0.699 5.48 160 171 NB25 NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 TGHGYVYEYPSR 3.07 0.718 2.7 160 171 NB122 NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 TGHGYVYEYPSR 2.82 0.632 0 160 171 NB61 NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 TGHGYVYEYPSR 2.2 0.706 2.46 160 171 NB12-2 NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 RHQSLSPNHPPPQLGQSVGSQNDAYDVPR 3.7 0.62 0 293 321 NB25-V NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 RHQSLSPNHPPPQLGQSVGSQNDAYDVPR 3.09 0.517 1.72 292 320 NB122-2 NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 RHQSLSPNHPPPQLGQSVGSQNDAYDVPR 2.62 0.435 1.23 293 321 NB12 NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 LYQVPNPQAAPR 2.98 0.562 6.02 91 102 NB122 NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 LYQVPNPQAAPR 2.33 0.546 3.54 91 102 NB25-V NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 HQSLSPNHPPPQLGQSVGSQNDAYDVPR 6.19 0.622 5.34 294 321 NB122-2 NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 HQSLSPNHPPPQLGQSVGSQNDAYDVPR 6.16 0.7 7.05 294 321 NB61 NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 HQSLSPNHPPPQLGQSVGSQNDAYDVPR 4.59 0.681 0 294 321 NB25-V NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 HQSLSPNHPPPQLGQSVGSQNDAYDVPR 3.96 0.644 7.96 293 320 NB12 NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 HQSLSPNHPPPQLGQSVGSQNDAYDVPR 3.32 0.647 4.66 294 321 NB122-2 NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 GPVFSVPVGEIKPQGVYDIPPTK 2.83 0.477 3.3 198 220 NB25-V NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 GPVFSVPVGEIKPQGVYDIPPTK 2.68 0.588 3.74 198 220 NB25-V NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 GPVFSVPVGEIKPQGVYDIPPTK 2.62 0.65 3.37 197 219 NB12 NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 GPVFSVPVGEIKPQGVYDIPPTK 2.28 0.529 2.77 198 220 NB25-V NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 DEAGLREKDYDFPPPMR 4.04 0.638 4.77 231 247 NB122-2 NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 DEAGLREKDYDFPPPMR 3.57 0.576 0 232 248 NB122 NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 DEAGLREKDYDFPPPMR 3.3 0.474 0 232 248 NB25-V NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 DEAGLREKDYDFPPPMR 2.63 0.701 1.28 232 248 NB25-V NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 ANPQERDGVYDVPLHNPPDAK 3.92 0.593 0 336 356 NB122 NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 ANPQERDGVYDVPLHNPPDAK 3.43 0.479 1.22 336 356 NB25-V NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 ANPQERDGVYDVPLHNPPDAK 3.23 0.459 2.43 335 355 NB12 NEDD9 Enhancer of filamentation 1 IPI00031407,IPI00844476 ANPQERDGVYDVPLHNPPDAK 3.06 0.557 1.37 336 356 NFKB1 Isoform 2 of Nuclear factor NF-kappa- NB88-2 B p105 subunit IPI00292537 RLEPVVSDAIYDSKAPNASNLK 3.97 0.653 7.96 231 252 NHLRC2 Isoform 1 of NHL repeat-containing NB88 protein 2 IPI00301051 FAGSGNEENRNNAYPHK 2.54 0.331 0 390 406 NB88 NME2 Nucleoside diphosphate kinase B IPI00026260 SCAHDWVYE 1.71 0.361 0 144 152 NB88 NPM1 Isoform 1 of Nucleophosmin IPI00549248 ADKDYHFKVDNDENEHQLSLR 4.23 0.689 1.18 25 45 IPI00299594,IPI00643127,IPI00 NB88 NRP1 Neuropilin 1 873301 DKLNTQSTYSEA 2.88 0.544 2.19 142 153 NB88 NUDT5 ADP-sugar pyrophosphatase IPI00296913 TLHYECIVLVK 3.5 0.491 2.92 71 81 NB88 OLA1 Isoform 2 of Obg-like ATPase 1 IPI00216105 FYHDWNDK 1.93 0.672 0 44 51 NB122 OLFML1 Olfactomedin-like protein 1 IPI00394820 GFLFFHNQATSNEIIKYNLQK 2.43 0.333 0 223 243 NB122 OR52A4 Putative olfactory receptor 52A4 IPI00465221 LIYHTYCERVALVK 1.55 0.41 0 178 191
139 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
PABPC1 Isoform 1 of Polyadenylate-binding NB12 protein 1 IPI00008524 IVATKPLYVALAQR 2.24 0.562 4.19 357 370 PABPC1 Isoform 1 of Polyadenylate-binding NB122 protein 1 IPI00008524 IVATKPLYVALAQR 2.12 0.407 1.28 357 370 PAG1 Phosphoprotein associated with NB25-V glycosphingolipid-enriched microdomains 1 IPI00020464 SREEDPTLTEEEISAMYSSVNKPGQLVNK 6.41 0.741 9.82 301 329 PAG1 Phosphoprotein associated with NB12-2 glycosphingolipid-enriched microdomains 1 IPI00020464 SREEDPTLTEEEISAMYSSVNKPGQLVNK 6.07 0.466 3.85 301 329 PAG1 Phosphoprotein associated with NB88-V glycosphingolipid-enriched microdomains 1 IPI00020464 SREEDPTLTEEEISAMYSSVNKPGQLVNK 5.86 0.68 6.6 301 329 PAG1 Phosphoprotein associated with NB12 glycosphingolipid-enriched microdomains 1 IPI00020464 SREEDPTLTEEEISAMYSSVNKPGQLVNK 5.4 0.639 8.66 301 329 PAG1 Phosphoprotein associated with NB88-2 glycosphingolipid-enriched microdomains 1 IPI00020464 SREEDPTLTEEEISAMYSSVNKPGQLVNK 5.35 0.52 3.72 301 329 PAG1 Phosphoprotein associated with NB61 glycosphingolipid-enriched microdomains 1 IPI00020464 SREEDPTLTEEEISAMYSSVNKPGQLVNK 4.26 0.596 1.89 301 329 PAG1 Phosphoprotein associated with NB25-V glycosphingolipid-enriched microdomains 1 IPI00020464 SPSSCNDLYATVKDFEK 3.97 0.67 4.15 351 367 PAG1 Phosphoprotein associated with NB12-2 glycosphingolipid-enriched microdomains 1 IPI00020464 SPSSCNDLYATVKDFEK 2.28 0.302 0 351 367 PAG1 Phosphoprotein associated with NB88-V glycosphingolipid-enriched microdomains 1 IPI00020464 ATLGTNGHHGLVPKENDYESISDLQQGR 4.49 0.538 1.55 400 427 PAG1 Phosphoprotein associated with NB12 glycosphingolipid-enriched microdomains 1 IPI00020464 ATLGTNGHHGLVPKENDYESISDLQQGR 2.82 0.324 0 400 427 PARD3 Isoform 7 of Partitioning-defective 3 NB88-V homolog IPI00045423 ERDYAEIQDFHR 2.98 0.571 0 994 1005 PARD3 Isoform 7 of Partitioning-defective 3 NB88-V homolog IPI00045423 EGHMMDALYAQVK 2.45 0.567 1.26 1036 1048 PARD3 Isoform 7 of Partitioning-defective 3 NB25-V homolog IPI00045423 EGHMMDALYAQVK 1.39 0.592 0 1036 1048 NB88-V PDHA1 Mitochondrial PDHA1 IPI00306301 YHGHSMSDPGVSYR 3.46 0.593 6 327 340 NB88 PDHA1 Mitochondrial PDHA1 IPI00306301 YHGHSMSDPGVSYR 3.35 0.764 4.43 327 340 NB12-2 PDLIM1 PDZ and LIM domain protein 1 IPI00010414 VTPPEGYEVVTVFPK 2.34 0.542 2.96 315 329 NB88-2 PDLIM1 PDZ and LIM domain protein 1 IPI00010414 VTPPEGYEVVTVFPK 2.32 0.537 2.6 315 329 NB88-V PDLIM1 PDZ and LIM domain protein 1 IPI00010414 VITNQYNNPAGLYSSENISNFNNALESK 6.17 0.695 7.85 139 166 NB25-V PDLIM1 PDZ and LIM domain protein 1 IPI00010414 VITNQYNNPAGLYSSENISNFNNALESK 4.19 0.708 0 139 166 NB88-2 PDLIM1 PDZ and LIM domain protein 1 IPI00010414 ERVTPPEGYEVVTVFPK 2.88 0.589 1.03 313 329 NB88-V PDLIM1 PDZ and LIM domain protein 1 IPI00010414 ERVTPPEGYEVVTVFPK 1.79 0.53 4 313 329 NB122 PDXDC1 PDXDC1 protein IPI00329208 LRALPLWLSLQYLGLDGFVERIK 1.51 0.361 0 336 358 IPI00157687,IPI00295618,IPI00 PECAM1 Isoform Long of Platelet endothelial 470719,IPI00470720,IPI008713 NB25-V cell adhesion molecule 52,IPI00872372,IPI00872586 KDTETVYSEVRK 3.34 0.583 3.03 301 312 IPI00157687,IPI00295618,IPI00 PECAM1 Isoform Long of Platelet endothelial 470719,IPI00470720,IPI008713 NB25-V cell adhesion molecule 52,IPI00872372,IPI00872586 DTETVYSEVRK 2.92 0.414 0 302 312 IPI00871352,IPI00470720,IPI008 PECAM1 Isoform Long of Platelet endothelial 72372,IPI00470719,IPI00872586,I NB88 cell adhesion molecule IPI00157687 DTETVYSEVRK 2.16 0.526 0 302 312 PI00295618 NB12-2 PFN1 Profilin-1 IPI00216691 CYEMASHLRR 2.34 0.306 0 128 137 NB88-V PFN1 Profilin-1 IPI00216691 CYEMASHLRR 1.95 0.419 0 128 137 NB88 PFN1 Profilin-1 IPI00216691 CYEMASHLR 2.83 0.675 4.46 128 136 NB88-V PFN1 Profilin-1 IPI00216691 CYEMASHLR 2.6 0.391 5.51 128 136 NB122-2 PFN1 Profilin-1 IPI00216691 CYEMASHLR 2.18 0.55 0 128 136 NB12-2 PFN1 Profilin-1 IPI00216691 CYEMASHLR 1.96 0.313 1.68 128 136 NB88-2 PFN1 Profilin-1 IPI00216691 CYEMASHLR 1.85 0.511 1.08 128 136 NB122 PFN1 Profilin-1 IPI00216691 CYEMASHLR 1.84 0.276 0 128 136 NB61 PFN1 Profilin-1 IPI00216691 CYEMASHLR 1.54 0.663 1.62 128 136 NB12 PGK1 Phosphoglycerate kinase 1 IPI00169383 KELNYFAK 2.37 0.423 0 192 199 NB88 PGK1 Phosphoglycerate kinase 1 IPI00169383 KELNYFAK 2.36 0.371 2 192 199 NB12-2 PGK1 Phosphoglycerate kinase 1 IPI00169383 KELNYFAK 2.18 0.472 2.07 192 199 NB88-2 PGK1 Phosphoglycerate kinase 1 IPI00169383 KELNYFAK 1.84 0.364 0 192 199 NB12 PGK1 Phosphoglycerate kinase 1 IPI00169383 ELNYFAK 1.51 0.443 0 193 199 PIK3AP1 Isoform 3 of Phosphoinositide 3- IPI00180939,IPI00643527,IPI00 NB122-2 kinase adapter protein 1 783519 SQERPGNFYVSSESIR 3.82 0.501 1.89 562 577 PIK3AP1 Isoform 3 of Phosphoinositide 3- IPI00180939,IPI00643527,IPI00 NB88 kinase adapter protein 1 783519 SQERPGNFYVSSESIR 3.64 0.53 1.85 562 577 PIK3AP1 Isoform 3 of Phosphoinositide 3- IPI00180939,IPI00643527,IPI00 NB12-2 kinase adapter protein 1 783519 SQERPGNFYVSSESIR 3.64 0.509 3 562 577 PIK3AP1 Isoform 3 of Phosphoinositide 3- IPI00180939,IPI00643527,IPI00 NB12 kinase adapter protein 1 783519 SQERPGNFYVSSESIR 3.16 0.621 3.92 562 577
140 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
PIK3AP1 Isoform 3 of Phosphoinositide 3- IPI00180939,IPI00643527,IPI00 NB12-2 kinase adapter protein 1 783519 HSQHLPAKVEFGVYESGPR 3.7 0.521 3.33 681 699 PIK3AP1 Isoform 3 of Phosphoinositide 3- IPI00180939,IPI00643527,IPI00 NB88 kinase adapter protein 1 783519 HSQHLPAKVEFGVYESGPR 3.33 0.526 1.49 681 699 PIK3CD Phosphatidylinositol-4,5- bisphosphate 3-kinase catalytic subunit delta NB88-V isoform IPI00298410 RGSGELYEHEKDLVWK 3.71 0.521 3.74 518 533 PIK3CD Phosphatidylinositol-4,5- bisphosphate 3-kinase catalytic subunit delta NB12-2 isoform IPI00298410 RGSGELYEHEKDLVWK 3.65 0.605 1.85 518 533 PIK3CD Phosphatidylinositol-4,5- bisphosphate 3-kinase catalytic subunit delta NB12 isoform IPI00298410 RGSGELYEHEKDLVWK 3.46 0.504 0 518 533 PIK3CD Phosphatidylinositol-4,5- bisphosphate 3-kinase catalytic subunit delta NB88 isoform IPI00298410 RGSGELYEHEKDLVWK 3.33 0.536 2.51 518 533 PIK3CD Phosphatidylinositol-4,5- bisphosphate 3-kinase catalytic subunit delta NB88-2 isoform IPI00298410 RGSGELYEHEKDLVWK 2.81 0.449 0 518 533 PIK3CD Phosphatidylinositol-4,5- bisphosphate 3-kinase catalytic subunit delta NB12-2 isoform IPI00298410 RGSGELYEHEK 2.17 0.32 0 518 528 PIK3CD Phosphatidylinositol-4,5- bisphosphate 3-kinase catalytic subunit delta NB88 isoform IPI00298410 GSGELYEHEKDLVWK 4.9 0.686 3.11 519 533 PIK3CD Phosphatidylinositol-4,5- bisphosphate 3-kinase catalytic subunit delta NB12-2 isoform IPI00298410 GSGELYEHEKDLVWK 4.49 0.61 5.85 519 533 PIK3CD Phosphatidylinositol-4,5- bisphosphate 3-kinase catalytic subunit delta NB25-V isoform IPI00298410 GSGELYEHEKDLVWK 3.79 0.61 3.96 519 533 PIK3CD Phosphatidylinositol-4,5- bisphosphate 3-kinase catalytic subunit delta NB88-2 isoform IPI00298410 GSGELYEHEKDLVWK 3.62 0.519 3.89 519 533 PIK3CD Phosphatidylinositol-4,5- bisphosphate 3-kinase catalytic subunit delta NB88-V isoform IPI00298410 GSGELYEHEKDLVWK 3.38 0.581 3.57 519 533 PIK3CD Phosphatidylinositol-4,5- bisphosphate 3-kinase catalytic subunit delta NB25-V isoform IPI00298410 GSGELYEHEKDLVWK 2.49 0.507 1.31 519 533 PIK3CD Phosphatidylinositol-4,5- bisphosphate 3-kinase catalytic subunit delta NB25 isoform IPI00298410 GSGELYEHEKDLVWK 2.41 0.516 0 519 533 PIK3CD Phosphatidylinositol-4,5- bisphosphate 3-kinase catalytic subunit delta NB122 isoform IPI00298410 GSGELYEHEKDLVWK 1.67 0.295 0 519 533 PIK3CD Phosphatidylinositol-4,5- bisphosphate 3-kinase catalytic subunit delta NB12 isoform IPI00298410 GSGELYEHEKDLVWK 1.48 0.565 0 519 533 PIK3CD Phosphatidylinositol-4,5- bisphosphate 3-kinase catalytic subunit delta NB88-2 isoform IPI00298410 GSGELYEHEK 2.15 0.553 0 519 528 PIK3CD Phosphatidylinositol-4,5- bisphosphate 3-kinase catalytic subunit delta NB12-2 isoform IPI00298410 GSGELYEHEK 1.91 0.472 0 519 528 PIK3CD Phosphatidylinositol-4,5- bisphosphate 3-kinase catalytic subunit delta NB88 isoform IPI00298410 GSGELYEHEK 1.81 0.578 0 519 528 PIK3R1 Isoform 1 of Phosphatidylinositol 3- NB61 kinase regulatory subunit alpha IPI00021448 TRDQYLMWLTQK 2.09 0.1 2.28 576 587 PIK3R1 Isoform 1 of Phosphatidylinositol 3- NB25-V kinase regulatory subunit alpha IPI00021448 SREYDRLYEEYTR 4.07 0.618 1.26 460 472 PIK3R1 Isoform 1 of Phosphatidylinositol 3- NB12-2 kinase regulatory subunit alpha IPI00021448 SREYDRLYEEYTR 4.03 0.593 1.31 460 472 PIK3R1 Isoform 1 of Phosphatidylinositol 3- NB12 kinase regulatory subunit alpha IPI00021448 SREYDRLYEEYTR 3.86 0.582 1.51 460 472 PIK3R1 Isoform 1 of Phosphatidylinositol 3- NB25 kinase regulatory subunit alpha IPI00021448 SREYDRLYEEYTR 3.54 0.615 2.8 460 472 PIK3R1 Isoform 1 of Phosphatidylinositol 3- NB88-2 kinase regulatory subunit alpha IPI00021448 SREYDRLYEEYTR 3.12 0.522 0 460 472
141 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
PIK3R1 Isoform 1 of Phosphatidylinositol 3- NB61 kinase regulatory subunit alpha IPI00021448 SREYDRLYEEYTR 2.15 0.356 0 460 472 PIK3R1 Isoform 1 of Phosphatidylinositol 3- NB88 kinase regulatory subunit alpha IPI00021448 QAAEYREIDKR 2.1 0.321 0 552 562 PIK3R1 Isoform 1 of Phosphatidylinositol 3- NB88 kinase regulatory subunit alpha IPI00021448 KLHEYNTQFQEK 2.72 0.551 0 448 459 PIK3R2 Phosphatidylinositol 3-kinase NB12 regulatory subunit beta IPI00011736,IPI00796770 EYDQLYEEYTR 2.43 0.704 2.17 459 469 PIK3R2 Phosphatidylinositol 3-kinase NB61 regulatory subunit beta IPI00011736 EYDQLYEEYTR 1.83 0.736 0 459 469 IPI00796770 PILRA Isoform 3 of Paired immunoglobulin- NB12 like type 2 receptor alpha IPI00184019 SPQNETLYSVLKA 3.02 0.506 0 218 230 IPI00296897 PILRA Isoform 3 of Paired immunoglobulin- NB88-V like type 2 receptor alpha precursor IPI00184019,IPI00296897 SPQNETLYSVLKA 3.4 0.57 3.13 291 303 PILRA Isoform 3 of Paired immunoglobulin- NB25-V like type 2 receptor alpha precursor IPI00184019,IPI00296897 SPQNETLYSVLKA 3.27 0.537 3.26 218 230 PIN4 protein (peptidyl-prolyl cis/trans NB61 isomerase) NIMA-interacting, 4 IPI00006658 FGYHIIMVEGR 2.92 0.552 0 145 155 PKM2 cDNA FLJ56065, highly similar to NB88-2 Pyruvate kinase isozyme M1 IPI00479186 TATESFASDPILYRPVAVALDTKGPEIR 3.85 0.561 5.12 167 194 PKM2 cDNA FLJ56065, highly similar to NB12 Pyruvate kinase isozyme M1 IPI00479186 TATESFASDPILYRPVAVALDTKGPEIR 2.76 0.305 1.17 167 194 PKM2 cDNA FLJ56065, highly similar to NB88-2 Pyruvate kinase isozyme M1 IPI00479186 TATESFASDPILYRPVAVALDTK 2.32 0.284 1.38 167 189 PKM2 cDNA FLJ56065, highly similar to NB88 Pyruvate kinase isozyme M1 IPI00479186 LNFSHGTHEYHAETIK 2.77 0.528 1.23 148 163 PLCG1 1-phosphatidylinositol-4,5- IPI00016736,IPI00383849,IPI00 NB12 bisphosphate phosphodiesterase gamma-1 745182 ACYRDMSSFPETK 3.62 0.697 4.89 975 987 PLCG1 1-phosphatidylinositol-4,5- NB61 bisphosphate phosphodiesterase gamma-1 IPI00016736 ACYRDMSSFPETK 2.34 0.719 0 975 987 IPI00745182,IPI00383849 PLCG1 1-phosphatidylinositol-4,5- NB122 bisphosphate phosphodiesterase gamma-1 IPI00016736 ACYRDMSSFPETK 2.13 0.802 0 975 987 IPI00745182,IPI00383849 PLCG2 1-phosphatidylinositol-4,5- NB25-V bisphosphate phosphodiesterase gamma-2 IPI00329185 VSNSKFYS pY 1264 2.07 0.595 2.04 1258 1265 PLCG2 1-phosphatidylinositol-4,5- NB12-2 bisphosphate phosphodiesterase gamma-2 IPI00329185 VSNSKFYS pY 1264 1.97 0.638 0 1258 1265 PLCG2 1-phosphatidylinositol-4,5- NB61 bisphosphate phosphodiesterase gamma-2 IPI00329185 RRQEELNNQLFLYDTHQNLR pY 1217 3.83 0.678 0 1205 1224 PLCG2 1-phosphatidylinositol-4,5- NB25-V bisphosphate phosphodiesterase gamma-2 IPI00329185 RRQEELNNQLFLYDTHQNLR pY 1217 3.75 0.531 0 1205 1224 PLCG2 1-phosphatidylinositol-4,5- NB88 bisphosphate phosphodiesterase gamma-2 IPI00329185 RRQEELNNQLFLYDTHQNLR pY 1217 3.27 0.603 1.52 1205 1224 PLCG2 1-phosphatidylinositol-4,5- NB12-2 bisphosphate phosphodiesterase gamma-2 IPI00329185 RRQEELNNQLFLYDTHQNLR pY 1217 2.98 0.411 0 1205 1224 PLCG2 1-phosphatidylinositol-4,5- NB88-V bisphosphate phosphodiesterase gamma-2 IPI00329185 RRQEELNNQLFLYDTHQNLR pY 1217 2.98 0.401 0 1205 1224 PLCG2 1-phosphatidylinositol-4,5- NB12-2 bisphosphate phosphodiesterase gamma-2 IPI00329185 RQEELNNQLFLYDTHQNLR pY 1217 6.97 0.585 5.85 1206 1224 PLCG2 1-phosphatidylinositol-4,5- NB25-V bisphosphate phosphodiesterase gamma-2 IPI00329185 RQEELNNQLFLYDTHQNLR pY 1217 5.95 0.501 5.68 1206 1224 PLCG2 1-phosphatidylinositol-4,5- NB88 bisphosphate phosphodiesterase gamma-2 IPI00329185 RQEELNNQLFLYDTHQNLR pY 1217 5.84 0.592 6.68 1206 1224 PLCG2 1-phosphatidylinositol-4,5- NB88-V bisphosphate phosphodiesterase gamma-2 IPI00329185 RQEELNNQLFLYDTHQNLR pY 1217 5.29 0.644 8.23 1206 1224 PLCG2 1-phosphatidylinositol-4,5- NB61 bisphosphate phosphodiesterase gamma-2 IPI00329185 RQEELNNQLFLYDTHQNLR pY 1217 3.38 0.355 0 1206 1224 PLCG2 1-phosphatidylinositol-4,5- NB12 bisphosphate phosphodiesterase gamma-2 IPI00329185 MYVDPSEINPSMPQR pY 759 4.14 0.63 6.29 758 772 PLCG2 1-phosphatidylinositol-4,5- NB88-V bisphosphate phosphodiesterase gamma-2 IPI00329185 MYVDPSEINPSMPQR pY 759 3.99 0.683 9.43 758 772 PLCG2 1-phosphatidylinositol-4,5- NB88-2 bisphosphate phosphodiesterase gamma-2 IPI00329185 MYVDPSEINPSMPQR pY 759 3.64 0.644 7.89 758 772 PLCG2 1-phosphatidylinositol-4,5- NB12-2 bisphosphate phosphodiesterase gamma-2 IPI00329185 MYVDPSEINPSMPQR pY 759 3.4 0.554 5.92 758 772 PLCG2 1-phosphatidylinositol-4,5- NB61 bisphosphate phosphodiesterase gamma-2 IPI00329185 MYVDPSEINPSMPQR pY 759 1.79 0.486 0 758 772 PLCG2 1-phosphatidylinositol-4,5- NB88-V bisphosphate phosphodiesterase gamma-2 IPI00329185 DINSLYDVSR pY 753 2.84 0.514 4.44 748 757 PLCG2 1-phosphatidylinositol-4,5- NB88 bisphosphate phosphodiesterase gamma-2 IPI00329185 DINSLYDVSR pY 753 2.63 0.614 1.49 748 757
142 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
PLCG2 1-phosphatidylinositol-4,5- NB25-V bisphosphate phosphodiesterase gamma-2 IPI00329185 DINSLYDVSR pY 753 2.46 0.717 2.52 748 757 PLCG2 1-phosphatidylinositol-4,5- NB88-2 bisphosphate phosphodiesterase gamma-2 IPI00329185 CIELDCWDGPDGKPVIYHGWTR pY 371 2.83 0.542 2.46 355 376 NB122 POLQ DNA polymerase theta IPI00794779 VDGYMGSTSPSR 1.19 0.551 0 168 179 PPIL5 Putative uncharacterized protein NB25-V DKFZp686J1525 (Fragment) IPI00465110 FLYTCWRGGMCIVTHLDDSK 1.46 0.522 0 396 415 PPP1R11 Protein phosphatase 1 regulatory NB88-V subunit 11 IPI00030355,IPI00645264 CCCIYEKPR 2.65 0.657 2.06 60 68 PPP1R11 Protein phosphatase 1 regulatory NB88 subunit 11 IPI00030355,IPI00645264 CCCIYEKPR 2.25 0.59 1.4 60 68 PPP1R11 Protein phosphatase 1 regulatory NB12-2 subunit 11 IPI00030355,IPI00645264 CCCIYEKPR 2.23 0.456 3.31 60 68 PPP1R11 Protein phosphatase 1 regulatory NB25-V subunit 11 IPI00030355,IPI00645264 CCCIYEKPR 2.16 0.64 2.92 8 16 NB88-V PRAGMIN Tyrosine-protein kinase SgK223 IPI00739386 CPPAYTMVGLHNLEPR 2.25 0.339 0 155 170 NB88 PRAGMIN Tyrosine-protein kinase SgK223 IPI00739386 CLGLTGEPQPPAHPQEATQPEPIYAESTKR 5.5 0.665 2.74 390 419 NB88-V PRAGMIN Tyrosine-protein kinase SgK223 IPI00739386 CLGLTGEPQPPAHPQEATQPEPIYAESTKR 5.27 0.625 3.74 390 419 NB122-2 PRAGMIN Tyrosine-protein kinase SgK223 IPI00739386 CLGLTGEPQPPAHPQEATQPEPIYAESTKR 4.82 0.615 1.54 390 419 NB122 PRAGMIN Tyrosine-protein kinase SgK223 IPI00739386 CLGLTGEPQPPAHPQEATQPEPIYAESTKR 3.49 0.606 0 390 419 NB88-2 PRAGMIN Tyrosine-protein kinase SgK223 IPI00739386 CLGLTGEPQPPAHPQEATQPEPIYAESTKR 2.91 0.499 0 390 419 NB88-V PRAGMIN Tyrosine-protein kinase SgK223 IPI00739386 CLGLTGEPQPPAHPQEATQPEPIYAESTK 5.42 0.687 4.62 390 418 NB88-2 PRAGMIN Tyrosine-protein kinase SgK223 IPI00739386 CLGLTGEPQPPAHPQEATQPEPIYAESTK 4.99 0.705 6.02 390 418 NB122-2 PRAGMIN Tyrosine-protein kinase SgK223 IPI00739386 CLGLTGEPQPPAHPQEATQPEPIYAESTK 3.84 0.496 3.33 390 418 NB25-V PRDX1 Peroxiredoxin-1 IPI00000874 SKEYFSK 2.75 0.408 0 191 197 NB88-V PRDX1 Peroxiredoxin-1 IPI00000874 SKEYFSK 2.16 0.391 0 191 197 NB88 PREP Prolyl endopeptidase IPI00008164,IPI00747355 MTELYDYPK 2.4 0.544 3.43 67 75 NB88-2 PREP Prolyl endopeptidase IPI00008164,IPI00747355 MTELYDYPK 2.24 0.654 2.85 67 75 NB12 PREP Prolyl endopeptidase IPI00008164,IPI00747355 MTELYDYPK 2.15 0.51 1.92 67 75 NB25-V PREP Prolyl endopeptidase IPI00008164 MTELYDYPK 2.14 0.57 0 67 75 IPI00747355 NB12-2 PREP Prolyl endopeptidase IPI00008164,IPI00747355 MTELYDYPK 2.06 0.63 1.96 67 75 NB122-2 PREP Prolyl endopeptidase IPI00008164,IPI00747355 MTELYDYPK 1.91 0.627 1.74 67 75 NB12-2 PRKCD Protein kinase C delta type IPI00329236 SDSASSEPVGIYQGFEKK 3.49 0.576 0 302 319 NB88 PRKCD Protein kinase C delta type IPI00329236 SDSASSEPVGIYQGFEKK 2.83 0.609 2.57 302 319 NB88-2 PRKCD Protein kinase C delta type IPI00329236 SDSASSEPVGIYQGFEKK 2.72 0.658 0 302 319 NB12-2 PRKCD Protein kinase C delta type IPI00329236 SDSASSEPVGIYQGFEK 3.96 0.512 4.21 302 318 IPI00384562 NB88-2 PRKCD Protein kinase C delta type IPI00329236 SDSASSEPVGIYQGFEK 3.39 0.483 3.36 302 318 IPI00384562 NB88 PRKCD Protein kinase C delta type IPI00329236 SDSASSEPVGIYQGFEK 3.01 0.454 5.2 302 318 IPI00384562 NB88 PRKCD Protein kinase C delta type IPI00329236 RSDSASSEPVGIYQGFEKK 5.37 0.665 4.89 301 319 IPI00384562 NB88-2 PRKCD Protein kinase C delta type IPI00329236 RSDSASSEPVGIYQGFEKK 5.22 0.72 4.31 301 319 IPI00384562 NB12-2 PRKCD Protein kinase C delta type IPI00329236 RSDSASSEPVGIYQGFEKK 5.03 0.665 5.25 301 319 IPI00384562 NB88 PRKCD Protein kinase C delta type IPI00329236 RSDSASSEPVGIYQGFEK 6.12 0.739 7.82 301 318 IPI00384562 NB88-2 PRKCD Protein kinase C delta type IPI00329236 RSDSASSEPVGIYQGFEK 4.39 0.69 4.64 301 318 IPI00384562 NB12-2 PRKCD Protein kinase C delta type IPI00329236 RSDSASSEPVGIYQGFEK 4.33 0.663 4.92 301 318 IPI00384562 PRMT1 HMT1 hnRNP methyltransferase-like NB12 2 isoform 1 IPI00018522 TGFSTSPESPYTHWK 2.5 0.431 0 299 313 PRMT1 HMT1 hnRNP methyltransferase-like NB88 2 isoform 1 IPI00018522 TGFSTSPESPYTHWK 2.03 0.64 0 299 313 PRMT1 HMT1 hnRNP methyltransferase-like NB88-2 2 isoform 1 IPI00018522 TGFSTSPESPYTHWK 1.73 0.458 0 299 313 PRPF4B Serine/threonine-protein kinase NB88-V PRP4 homolog IPI00013721,IPI00552169 LCDFGSASHVADNDITPYLVSR 6.49 0.722 6.28 832 853 PRPF4B Serine/threonine-protein kinase NB12 PRP4 homolog IPI00013721,IPI00552169 LCDFGSASHVADNDITPYLVSR 6.07 0.708 5.34 832 853 PRPF4B Serine/threonine-protein kinase NB88 PRP4 homolog IPI00013721,IPI00552169 LCDFGSASHVADNDITPYLVSR 5.91 0.692 5.59 832 853 PRPF4B Serine/threonine-protein kinase NB122-2 PRP4 homolog IPI00013721,IPI00552169 LCDFGSASHVADNDITPYLVSR 5.8 0.673 2.96 832 853 PRPF4B Serine/threonine-protein kinase NB25-V PRP4 homolog IPI00013721,IPI00552169 LCDFGSASHVADNDITPYLVSR 5.78 0.646 2.49 321 342 PRPF4B Serine/threonine-protein kinase NB122 PRP4 homolog IPI00013721,IPI00552169 LCDFGSASHVADNDITPYLVSR 5.54 0.778 4.24 832 853 PRPF4B Serine/threonine-protein kinase NB61 PRP4 homolog IPI00013721,IPI00552169 LCDFGSASHVADNDITPYLVSR 5.47 0.695 4.16 832 853 PRPF4B Serine/threonine-protein kinase NB25 PRP4 homolog IPI00013721,IPI00552169 LCDFGSASHVADNDITPYLVSR 5.27 0.6 0 832 853 PRPF4B Serine/threonine-protein kinase NB88-2 PRP4 homolog IPI00013721,IPI00552169 LCDFGSASHVADNDITPYLVSR 5.13 0.7 6.09 832 853 PRPF4B Serine/threonine-protein kinase NB25-V PRP4 homolog IPI00013721,IPI00552169 LCDFGSASHVADNDITPYLVSR 4.85 0.696 6.19 832 853
143 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
PRPF4B Serine/threonine-protein kinase NB12-2 PRP4 homolog IPI00013721,IPI00552169 LCDFGSASHVADNDITPYLVSR 4.84 0.646 3.66 832 853 NB12-2 PSMA2 Proteasome subunit alpha type-2 IPI00219622 SILYDER 1.81 0.411 0 54 60 NB88-2 PSMA2 Proteasome subunit alpha type-2 IPI00219622 SILYDER 1.76 0.449 1.62 54 60 NB88-2 PSMA2 Proteasome subunit alpha type-2 IPI00219622 HIGLVYSGMGPDYR 3.62 0.598 5.52 71 84 NB88 PSMA6 Proteasome subunit alpha type-6 IPI00029623 YEAANWKYK 2.08 0.532 0 96 104 PSMA7 Isoform 1 of Proteasome subunit NB88 alpha type-7 IPI00024175 LYQTDPSGTYHAWK 3.62 0.638 4.77 144 157 PSMB8 Isoform 1 of Proteasome subunit IPI00000783,IPI00215824,IPI00 NB88 beta type-8 640155 AIAYATHR 1.51 0.573 0 231 238 PSMD11 Proteasome 26S non-ATPase NB12-2 subunit 11 variant (Fragment) IPI00105598 VVDSLYNK 2.26 0.469 1.36 411 418 PSMD11 Proteasome 26S non-ATPase NB88-2 subunit 11 variant (Fragment) IPI00105598 VVDSLYNK 1.8 0.582 0 411 418 NB12-2 PSME2 Uncharacterized protein PSME2 IPI00384051 GEEKPSMY 1.94 0.704 0 242 249 PTK2B Isoform 1 of Protein tyrosine kinase 2 IPI00029702,IPI00216435,IPI00 NB25-V beta 443767 YIEDEDYYKASVTRLPIK 3.22 0.619 1.32 573 590 PTK2B Isoform 1 of Protein tyrosine kinase 2 IPI00029702,IPI00216435,IPI00 NB12 beta 443767 YIEDEDYYKASVTRLPIK 2.88 0.626 0 573 590 PTK2B Isoform 1 of Protein tyrosine kinase 2 IPI00029702,IPI00216435,IPI00 NB12-2 beta 443767 YIEDEDYYKASVTRLPIK 1.98 0.361 1.24 573 590 PTK2B Isoform 1 of Protein tyrosine kinase 2 NB61 beta IPI00029702 YIEDEDYYKASVTRLPIK 1.82 0.291 0 573 590 IPI00443767,IPI00216435 PTK2B Isoform 1 of Protein tyrosine kinase 2 IPI00029702,IPI00216435,IPI00 NB12 beta 443767 YIEDEDYYKASVTR 4.05 0.545 3.7 573 586 PTPN18 cDNA FLJ51446, highly similar to Tyrosine-protein phosphatase non-receptor NB25-V type 18 IPI00152019,IPI00219132 SAEEAPLYSKVTPR 2.93 0.611 1.77 273 286 PTPN18 cDNA FLJ51446, highly similar to Tyrosine-protein phosphatase non-receptor NB25-V type 18 IPI00152019,IPI00219132 SAEEAPLYSKVTPR 2.76 0.495 6.7 273 286 PTPN18 cDNA FLJ51446, highly similar to Tyrosine-protein phosphatase non-receptor NB88-V type 18 IPI00152019,IPI00219132 SAEEAPLYSKVTPR 2.61 0.642 7.55 273 286 PTPN18 cDNA FLJ51446, highly similar to Tyrosine-protein phosphatase non-receptor NB12 type 18 IPI00152019,IPI00219132 SAEEAPLYSK 2.56 0.605 1.29 275 284 PTPN18 cDNA FLJ51446, highly similar to Tyrosine-protein phosphatase non-receptor NB25-V type 18 IPI00152019,IPI00219132 SAEEAPLYSK 2.56 0.325 2.29 273 282 PTPN18 cDNA FLJ51446, highly similar to Tyrosine-protein phosphatase non-receptor NB12-2 type 18 IPI00152019 SAEEAPLYSK 2.53 0.438 0 275 284 IPI00219132 PTPN18 cDNA FLJ51446, highly similar to Tyrosine-protein phosphatase non-receptor NB25-V type 18 IPI00152019,IPI00219132 SAEEAPLYSK 2.5 0.439 1.43 273 282 PTPN18 cDNA FLJ51446, highly similar to Tyrosine-protein phosphatase non-receptor NB88 type 18 IPI00152019,IPI00219132 SAEEAPLYSK 2.27 0.481 1.48 275 284 PTPN18 cDNA FLJ51446, highly similar to Tyrosine-protein phosphatase non-receptor NB88-V type 18 IPI00152019,IPI00219132 SAEEAPLYSK 2.25 0.368 2.62 273 282 PTPN18 cDNA FLJ51446, highly similar to Tyrosine-protein phosphatase non-receptor NB88-2 type 18 IPI00152019,IPI00219132 SAEEAPLYSK 1.97 0.312 1.08 275 284 PTPN22 Isoform 2 of Tyrosine-protein NB25-V phosphatase non-receptor type 22 IPI00219138 VMHVSSAELNYSLPYDSK 3.79 0.698 1.05 489 506 IPI00183046,IPI00218604,IPI00 PTPN6 Isoform 3 of Tyrosine-protein 396552,IPI00789061,IPI007909 NB25-V phosphatase non-receptor type 6 46 HKEDVYENLHTK 4.96 0.646 3.8 126 137 IPI00183046,IPI00218604,IPI00 PTPN6 Isoform 3 of Tyrosine-protein 396552,IPI00789061,IPI007909 NB25-V phosphatase non-receptor type 6 46 HKEDVYENLHTK 4.77 0.638 3.36 520 531 PTPN6 Isoform 3 of Tyrosine-protein IPI00183046,IPI00218604,IPI00 NB12-2 phosphatase non-receptor type 6 396552 HKEDVYENLHTK 4.29 0.6 1.22 520 531 IPI00790946,IPI00789061 PTPN6 Isoform 3 of Tyrosine-protein IPI00183046,IPI00218604,IPI00 NB88-2 phosphatase non-receptor type 6 396552 HKEDVYENLHTK 3.84 0.737 3.37 520 531 IPI00790946,IPI00789061 IPI00183046,IPI00218604,IPI00 PTPN6 Isoform 3 of Tyrosine-protein 396552,IPI00789061,IPI007909 NB88-V phosphatase non-receptor type 6 46 HKEDVYENLHTK 3.34 0.399 2.66 520 531
144 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
PTPN6 Isoform 3 of Tyrosine-protein IPI00183046,IPI00218604,IPI00 NB88 phosphatase non-receptor type 6 396552 HKEDVYENLHTK 3.13 0.714 4.72 520 531 IPI00790946,IPI00789061 PTPN6 Isoform 3 of Tyrosine-protein IPI00183046,IPI00218604,IPI00 NB122-2 phosphatase non-receptor type 6 396552 HKEDVYENLHTK 2.72 0.502 0 520 531 IPI00790946,IPI00789061 IPI00183046,IPI00218604,IPI00 PTPN6 Isoform 3 of Tyrosine-protein 396552,IPI00789061,IPI007909 NB88-V phosphatase non-receptor type 6 46 GQESEYGNITYPPAMK 4.66 0.734 5.21 492 507 IPI00183046,IPI00218604,IPI00 PTPN6 Isoform 3 of Tyrosine-protein 396552,IPI00789061,IPI007909 NB25-V phosphatase non-receptor type 6 46 GQESEYGNITYPPAMK 4.26 0.731 7.44 492 507 PTPN6 Isoform 3 of Tyrosine-protein IPI00183046,IPI00218604,IPI00 NB12-2 phosphatase non-receptor type 6 396552 GQESEYGNITYPPAMK 4.06 0.705 3.17 492 507 IPI00790946,IPI00789061 PTPN6 Isoform 3 of Tyrosine-protein IPI00183046,IPI00218604,IPI00 NB88 phosphatase non-receptor type 6 396552 GQESEYGNITYPPAMK 3.66 0.694 2.96 492 507 IPI00790946,IPI00789061 PTPN6 Isoform 3 of Tyrosine-protein IPI00183046,IPI00218604,IPI00 NB88-2 phosphatase non-receptor type 6 396552 GQESEYGNITYPPAMK 3.52 0.553 4.28 492 507 IPI00790946,IPI00789061 PTPN6 Isoform 3 of Tyrosine-protein IPI00183046,IPI00218604,IPI00 NB122-2 phosphatase non-receptor type 6 396552 GQESEYGNITYPPAMK 2.5 0.675 4.52 492 507 IPI00790946,IPI00789061 PTPN6 Isoform 3 of Tyrosine-protein IPI00183046,IPI00218604,IPI00 NB61 phosphatase non-receptor type 6 396552 GQESEYGNITYPPAMK 2 0.388 0 492 507 IPI00790946,IPI00789061 PTPRA Isoform 2 of Receptor-type tyrosine- IPI00221067,IPI00304030,IPI00 NB12 protein phosphatase alpha 743698,IPI00843944 VVQEYIDAFSDYANFK 5.48 0.736 11.3 778 793 PTPRA Isoform 2 of Receptor-type tyrosine- IPI00221067,IPI00304030,IPI00 NB25 protein phosphatase alpha 743698,IPI00843944 VVQEYIDAFSDYANFK 4.47 0.626 5.8 778 793 PTPRA Isoform 2 of Receptor-type tyrosine- IPI00221067,IPI00304030,IPI00 NB61 protein phosphatase alpha 743698,IPI00843944 VVQEYIDAFSDYANFK 4.2 0.732 6.92 778 793 PTPRA Isoform 2 of Receptor-type tyrosine- IPI00221067,IPI00304030,IPI00 NB25-V protein phosphatase alpha precursor 743698,IPI00843944 VVQEYIDAFSDYANFK 5.6 0.769 10.1 778 793 PTPRA Isoform 2 of Receptor-type tyrosine- IPI00221067,IPI00304030,IPI00 NB88-V protein phosphatase alpha precursor 743698,IPI00843944 VVQEYIDAFSDYANFK 4.78 0.747 6.77 787 802 PTTG1IP Pituitary tumor-transforming gene 1 IPI00023974,IPI00794805,IPI00 NB12-2 protein-interacting protein 911013 YGLFKEENPYAR 3.18 0.476 2.35 165 176 PTTG1IP Pituitary tumor-transforming gene 1 IPI00023974,IPI00794805,IPI00 NB88 protein-interacting protein 911013 YGLFKEENPYAR 3.12 0.607 3.57 165 176 PTTG1IP Pituitary tumor-transforming gene 1 NB25-V protein-interacting protein IPI00023974,IPI00794805 YGLFKEENPYAR 2.86 0.575 2.12 144 155 IPI00911013 PTTG1IP Pituitary tumor-transforming gene 1 IPI00023974,IPI00794805,IPI00 NB12 protein-interacting protein 911013 YGLFKEENPYAR 2.45 0.557 2.51 165 176 NB12-2 PXN Isoform Alpha of Paxillin IPI00220030 VGEEEHVYSFPNKQK 3.34 0.596 0 111 125 NB122-2 PXN Isoform Alpha of Paxillin IPI00220030 VGEEEHVYSFPNKQK 3.07 0.54 0 111 125 NB122 PXN Isoform Alpha of Paxillin IPI00220030 VGEEEHVYSFPNKQK 2.96 0.605 0 111 125 NB12 PXN Isoform Alpha of Paxillin IPI00220030 VGEEEHVYSFPNKQK 2.44 0.527 0 111 125 NB25-V PXN Isoform Alpha of Paxillin IPI00220030 VGEEEHVYSFPNK 4.08 0.502 3.59 111 123 NB12 PXN Isoform Alpha of Paxillin IPI00220030 VGEEEHVYSFPNK 4.02 0.558 1.74 111 123 NB25-V PXN Isoform Alpha of Paxillin IPI00220030 VGEEEHVYSFPNK 2.85 0.611 2.64 111 123 NB12-2 RAN GTP-binding nuclear protein Ran IPI00643041 NLQYYDISAK 2.77 0.469 2.47 143 152 NB88 RAN GTP-binding nuclear protein Ran IPI00643041 NLQYYDISAK 2.43 0.592 1.77 143 152 NB12 RAN GTP-binding nuclear protein Ran IPI00643041 NLQYYDISAK 2.15 0.332 1.39 143 152 RBMX Heterogeneous nuclear NB25-V ribonucleoprotein G IPI00304692 SDLYSSGRDR 2.53 0.536 1.14 332 341 RBMX Heterogeneous nuclear NB12-2 ribonucleoprotein G IPI00304692 SDLYSSGRDR 2.3 0.234 0 332 341 RBMX Heterogeneous nuclear NB88-V ribonucleoprotein G IPI00304692 SDLYSSGRDR 1.95 0.334 0 332 341 RBMX Heterogeneous nuclear NB88-2 ribonucleoprotein G IPI00304692 SDLYSSGRDR 1.67 0.344 0 332 341 RBMX Heterogeneous nuclear NB88-V ribonucleoprotein G IPI00304692 SDLYSSGR 1.96 0.475 1.82 332 339 RBMX Heterogeneous nuclear NB12-2 ribonucleoprotein G IPI00304692 SDLYSSGR 1.89 0.345 1.66 332 339 RBMX Heterogeneous nuclear NB88-V ribonucleoprotein G IPI00304692 DRDYSDHPSGGSYR 2.82 0.475 2.19 269 282 RIPK2 Isoform 1 of Receptor-interacting NB25-V serine/threonine-protein kinase 2 IPI00021917 KAQDCYFMK 3.38 0.682 0 376 384 RIPK2 Isoform 1 of Receptor-interacting NB88-V serine/threonine-protein kinase 2 IPI00021917 KAQDCYFMK 3.22 0.589 1.47 376 384 RIPK2 Isoform 1 of Receptor-interacting NB25-V serine/threonine-protein kinase 2 IPI00021917 KAQDCYFMK 2.93 0.526 2.92 376 384 RIPK2 Isoform 1 of Receptor-interacting NB88 serine/threonine-protein kinase 2 IPI00021917 KAQDCYFMK 2.86 0.596 0 376 384
145 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
RIPK2 Isoform 1 of Receptor-interacting NB122-2 serine/threonine-protein kinase 2 IPI00021917 KAQDCYFMK 2.85 0.59 0 376 384 RIPK2 Isoform 1 of Receptor-interacting NB88-2 serine/threonine-protein kinase 2 IPI00021917 KAQDCYFMK 2.73 0.551 1.24 376 384 RIPK2 Isoform 1 of Receptor-interacting NB25 serine/threonine-protein kinase 2 IPI00021917 KAQDCYFMK 2.57 0.687 0 376 384 RIPK2 Isoform 1 of Receptor-interacting NB122 serine/threonine-protein kinase 2 IPI00021917 KAQDCYFMK 2.11 0.551 0 376 384 RIPK2 Isoform 1 of Receptor-interacting NB12-2 serine/threonine-protein kinase 2 IPI00021917 KAQDCYFMK 2.06 0.553 0 376 384 RIPK2 Isoform 1 of Receptor-interacting NB12 serine/threonine-protein kinase 2 IPI00021917 KAQDCYFMK 2.04 0.659 0 376 384 RIPK2 Isoform 1 of Receptor-interacting NB88 serine/threonine-protein kinase 2 IPI00021917 AQDCYFMK 1.69 0.435 0 377 384 RIPK2 Isoform 1 of Receptor-interacting NB25-V serine/threonine-protein kinase 2 IPI00021917 AQDCYFMK 1.64 0.668 0 377 384 NB122 RNASE4 Ribonuclease 4 IPI00029699 YCNLMMQRR 1.46 0.369 0 52 60 IPI00219798,IPI00418121,IPI00 418122,IPI00740934,IPI008297 NB12 ROBO1 Isoform 1 of Roundabout homolog 1 39,IPI00896492 NGLTSTYAGIR 2.53 0.244 1.7 926 936 NB61 RP3-402G11.5 Selenoprotein O IPI00031666 VMHANNPKYVLR 1.84 0.324 0 586 597 IPI00008530,IPI00556485,IPI00 NB12 RPLP0 60S acidic ribosomal protein P0 794884 IIQLLDDYPK 3.09 0.638 4.39 17 26 IPI00008530,IPI00556485,IPI00 NB88 RPLP0 60S acidic ribosomal protein P0 794884 IIQLLDDYPK 2.63 0.487 4.28 17 26 IPI00008530,IPI00556485,IPI00 NB122 RPLP0 60S acidic ribosomal protein P0 794884 IIQLLDDYPK 2.17 0.612 1.92 17 26 NB88-2 RPS10 40S ribosomal protein S10 IPI00008438 IAIYELLFKEGVMVAKK 1.75 0.385 0 9 25 NB88-V RPS10 40S ribosomal protein S10 IPI00008438 IAIYELLFK 2.5 0.494 3 9 17 NB25 RPS10 40S ribosomal protein S10 IPI00008438 GEADRDTYRR 1.91 0.524 0 120 129 NB88 RPS10 40S ribosomal protein S10 IPI00008438 GEADRDTYRR 1.35 0.403 1 120 129 NB12 RPS13 40S ribosomal protein S13 IPI00221089 LTSDDVKEQIYK 2.96 0.39 0 28 39 NB12-2 RPS27 40S ribosomal protein S27 IPI00513971 LVQSPNSYFMDVK 4.12 0.639 6.89 24 36 NB88 RPS27 40S ribosomal protein S27 IPI00513971 LVQSPNSYFMDVK 3.43 0.633 6.39 24 36 NB88-V RPS27 40S ribosomal protein S27 IPI00513971 LVQSPNSYFMDVK 3.42 0.506 6.55 24 36 NB122-2 RPS27 40S ribosomal protein S27 IPI00513971 LVQSPNSYFMDVK 3.4 0.617 7.38 24 36 NB12 RPS27 40S ribosomal protein S27 IPI00513971 LVQSPNSYFMDVK 3.13 0.567 8.62 24 36 NB88-2 RPS27 40S ribosomal protein S27 IPI00513971 LVQSPNSYFMDVK 3.02 0.609 7.2 24 36 NB12-2 RPS6 40S ribosomal protein S6 IPI00021840 NKEEAAEYAK 2.1 0.547 2.96 202 211 NB12-2 RPS9 40S ribosomal protein S9 IPI00221088 SPYGGGRPGR 1.89 0.733 1.08 163 172 IPI00413108,IPI00553164,IPI00 NB12 RPSA 33 kDa protein 793137,IPI00847766 ADHQPLTEASYVNLPTIALCNTDSPLR 4.38 0.608 6.1 134 160 SART1 U4/U6.U5 tri-snRNP-associated NB88-V protein 1 IPI00021417 RQDLYSAR 2.56 0.269 0 245 252 SASH3 SAM and SH3 domain-containing NB88 protein 3 IPI00004504 VHTDFTPSPYDHDSLK 2.45 0.38 0 180 195 NB12-2 SDC4 Syndecan-4 IPI00011564 KDEGSYDLGK 2.94 0.551 3.11 175 184 NB12 SDC4 Syndecan-4 IPI00011564 KAPTNEFYA 1.94 0.638 1.3 190 198 NB12-2 SDC4 Syndecan-4 IPI00011564 KAPTNEFYA 1.84 0.533 0 190 198 NB88-2 SDC4 Syndecan-4 IPI00011564 KAPTNEFYA 1.81 0.485 0 190 198 NB25 SDC4 Syndecan-4 IPI00011564 KAPTNEFYA 1.75 0.548 0 190 198 NB122 SDC4 Syndecan-4 IPI00011564 KAPTNEFYA 1.61 0.418 0 190 198 NB122-2 SDC4 Syndecan-4 IPI00011564 KAPTNEFYA 1.59 0.388 0 190 198 NB25-V SDC4 Syndecan-4 IPI00011564 KAPTNEFYA 1.54 0.527 0 190 198 NB88 SDC4 Syndecan-4 IPI00011564 KAPTNEFYA 1.42 0.282 1.19 190 198 SERBP1 Isoform 1 of Plasminogen activator NB88 inhibitor 1 RNA-binding protein IPI00410693 SSFSHYSGLKHEDKR 2.68 0.506 0 202 216 SERBP1 Isoform 1 of Plasminogen activator NB25-V inhibitor 1 RNA-binding protein IPI00410693 SSFSHYSGLKHEDKR 2.46 0.566 2.62 202 216 SERBP1 Isoform 1 of Plasminogen activator NB88-2 inhibitor 1 RNA-binding protein IPI00410693 SSFSHYSGLKHEDKR 2.44 0.498 0 202 216 SERBP1 Isoform 1 of Plasminogen activator NB12-2 inhibitor 1 RNA-binding protein IPI00410693 SSFSHYSGLKHEDKR 2.43 0.527 0 202 216 SERBP1 Isoform 1 of Plasminogen activator NB88-V inhibitor 1 RNA-binding protein IPI00410693 SSFSHYSGLKHEDKR 2.36 0.35 0 202 216 SERBP1 Isoform 1 of Plasminogen activator NB12 inhibitor 1 RNA-binding protein IPI00410693 SSFSHYSGLKHEDKR 2.03 0.584 1.11 202 216 SFRS1 Isoform ASF-1 of Splicing factor, NB122-2 arginine/serine-rich 1 IPI00215884 SHEGETAYIR 2.61 0.703 1.85 182 191 SFRS1 Isoform ASF-1 of Splicing factor, NB12-2 arginine/serine-rich 1 IPI00215884 SHEGETAYIR 2.27 0.576 1.2 182 191
146 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
SFRS1 Isoform ASF-1 of Splicing factor, NB88-2 arginine/serine-rich 1 IPI00215884 SHEGETAYIR 2.16 0.719 2.82 182 191 SFRS1 Isoform ASF-1 of Splicing factor, NB88 arginine/serine-rich 1 IPI00215884 SHEGETAYIR 2.06 0.553 1.96 182 191 SFRS1 Isoform ASF-1 of Splicing factor, NB12 arginine/serine-rich 1 IPI00215884 SHEGETAYIR 1.67 0.72 0 182 191 SFRS1 Isoform ASF-1 of Splicing factor, NB122 arginine/serine-rich 1 IPI00215884 SHEGETAYIR 1.58 0.627 0 182 191 SFRS1 Isoform ASF-1 of Splicing factor, NB88 arginine/serine-rich 1 IPI00215884 KEDMTYAVR 2.67 0.423 0 165 173 SFRS10 Isoform 1 of Splicing factor, NB12-2 arginine/serine-rich 10 IPI00301503 AAQDRDQIYR 2.32 0.255 0 252 261 NB88 SFRS3 Splicing factor, arginine/serine-rich 3 IPI00010204 AFGYYGPLR 2.58 0.541 1.85 29 37 NB88-2 SFRS9 Splicing factor, arginine/serine-rich 9 IPI00012340 GSPHYFSPFRPY 2.52 0.473 0 210 221 NB12-2 SFRS9 Splicing factor, arginine/serine-rich 9 IPI00012340 GSPHYFSPFRPY 2.15 0.497 1.96 210 221 NB122-2 SFRS9 Splicing factor, arginine/serine-rich 9 IPI00012340 GSPHYFSPFRPY 1.91 0.53 0 210 221 SH2D3C Isoform 1 of SH2 domain- IPI00171093,IPI00640927,IPI00 NB122-2 containing protein 3C 641074,IPI00735492 ASPSPSLSSYSDPDSGHYCQLQPPVR 3.42 0.589 4.27 478 503 SH2D3C Isoform 1 of SH2 domain- IPI00171093,IPI00640927,IPI00 NB25-V containing protein 3C 641074,IPI00735492 ASPSPSLSSYSDPDSGHYCQLQPPVR 2.54 0.382 8.7 478 503 SH2D3C Isoform 1 of SH2 domain- IPI00171093,IPI00640927,IPI00 NB88-2 containing protein 3C 641074,IPI00735492 ASPSPSLSSYSDPDSGHYCQLQPPVR 1.82 0.437 2.89 478 503 IPI00021326,IPI00165135,IPI00 SHC1 SHC (Src homology 2 domain 513796,IPI00641285,IPI006431 NB12-2 containing) transforming protein 1 isoform 3 76,IPI00643863,IPI00645103 ELFDDPSYVNVQNLDKAR 2.01 0.405 1.29 421 438 IPI00647867 IPI00021326,IPI00165135,IPI00 513796,IPI00641285,IPI006431 SHC1 SHC (Src homology 2 domain 76,IPI00643863,IPI00645103,IP NB25-V containing) transforming protein 1 isoform 3 I00647867 ELFDDPSYVNVQNLDKAR 1.54 0.602 0 421 438 IPI00021326,IPI00165135,IPI00 SHC1 SHC (Src homology 2 domain 513796,IPI00641285,IPI006431 NB88-2 containing) transforming protein 1 isoform 3 76,IPI00643863,IPI00645103 ELFDDPSYVNVQNLDK 4.04 0.601 5.96 421 436 IPI00647867 IPI00021326,IPI00165135,IPI00 513796,IPI00641285,IPI006431 SHC1 SHC (Src homology 2 domain 76,IPI00643863,IPI00645103,IP NB88-V containing) transforming protein 1 isoform 3 I00647867 ELFDDPSYVNVQNLDK 3.75 0.61 8.74 421 436 IPI00021326,IPI00165135,IPI00 SHC1 SHC (Src homology 2 domain 513796,IPI00641285,IPI006431 NB122-2 containing) transforming protein 1 isoform 3 76,IPI00643863,IPI00645103 ELFDDPSYVNVQNLDK 3.62 0.541 5.74 421 436 IPI00647867 IPI00021326,IPI00165135,IPI00 SHC1 SHC (Src homology 2 domain 513796,IPI00641285,IPI006431 NB12 containing) transforming protein 1 isoform 3 76,IPI00643863,IPI00645103 ELFDDPSYVNVQNLDK 3.42 0.625 7.85 421 436 IPI00647867 IPI00021326,IPI00165135,IPI00 513796,IPI00641285,IPI006431 SHC1 SHC (Src homology 2 domain 76,IPI00643863,IPI00645103,IP NB25-V containing) transforming protein 1 isoform 3 I00647867 ELFDDPSYVNVQNLDK 2.95 0.573 1.14 421 436 IPI00021326,IPI00165135,IPI00 SHC1 SHC (Src homology 2 domain 513796,IPI00641285,IPI006431 NB25 containing) transforming protein 1 isoform 3 76,IPI00643863,IPI00645103 ELFDDPSYVNVQNLDK 2.62 0.693 3.23 421 436 IPI00647867 IPI00021326,IPI00165135,IPI00 SHC1 SHC (Src homology 2 domain 513796,IPI00641285,IPI006431 NB61 containing) transforming protein 1 isoform 3 76,IPI00643863,IPI00645103 ELFDDPSYVNVQNLDK 2.13 0.239 2.49 421 436 IPI00647867 IPI00064982,IPI00219603,IPI00 SIGLEC10 Isoform 1 of Sialic acid-binding Ig- 219605,IPI00219606,IPI004325 NB88-V like lectin 10 precursor 69 SSTQAPESQESQEELHYATLNFPGVRPRPEAR 1.18 0.408 0 651 682 IPI00064982,IPI00219603,IPI00 SIGLEC10 Isoform 1 of Sialic acid-binding Ig- 219605,IPI00219606,IPI004325 NB88-V like lectin 10 precursor 69 GTQADYAEVK 2.55 0.385 1.49 686 695 SIGLEC10 Isoform 1 of Sialic acid-binding Ig- IPI00219606,IPI00432569,IPI002 NB88 like lectin 10 precursor IPI00064982 GTQADYAEVK 2.2 0.595 0 686 695 19605,IPI00219603 SIGLEC10 Isoform 1 of Sialic acid-binding Ig- IPI00219606,IPI00432569,IPI002 NB12-2 like lectin 10 precursor IPI00064982 GTQADYAEVK 2.13 0.425 0 686 695 19605,IPI00219603 SIGLEC10 Isoform 1 of Sialic acid-binding Ig- IPI00219606,IPI00432569,IPI002 NB25-V like lectin 10 precursor IPI00064982 GTQADYAEVK 2.09 0.505 0 686 695 19605,IPI00219603
NB88-2 SLAMF7 Isoform 1 of SLAM family member 7 IPI00099863,IPI00168447 TILKEDPANTVYSTVEIPK 4.6 0.514 4.8 293 311
NB25-V SLAMF7 Isoform 1 of SLAM family member 7 IPI00099863,IPI00168447 TILKEDPANTVYSTVEIPK 4.35 0.668 6.35 293 311
NB25-V SLAMF7 Isoform 1 of SLAM family member 7 IPI00099863,IPI00168447 TILKEDPANTVYSTVEIPK 4.34 0.663 3.48 293 311
147 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
NB122-2 SLAMF7 Isoform 1 of SLAM family member 7 IPI00099863,IPI00168447 TILKEDPANTVYSTVEIPK 3.67 0.526 2.8 293 311
NB12 SLAMF7 Isoform 1 of SLAM family member 7 IPI00099863,IPI00168447 TILKEDPANTVYSTVEIPK 3.64 0.674 5.22 293 311
NB122 SLAMF7 Isoform 1 of SLAM family member 7 IPI00099863,IPI00168447 TILKEDPANTVYSTVEIPK 3.6 0.667 2.89 293 311
NB12-2 SLAMF7 Isoform 1 of SLAM family member 7 IPI00099863,IPI00168447 TILKEDPANTVYSTVEIPK 3.34 0.534 1.39 293 311
NB88-V SLAMF7 Isoform 1 of SLAM family member 7 IPI00099863,IPI00168447 TILKEDPANTVYSTVEIPK 3.21 0.557 4.85 293 311
NB25 SLAMF7 Isoform 1 of SLAM family member 7 IPI00099863,IPI00168447 TILKEDPANTVYSTVEIPK 3.04 0.457 5.74 293 311
NB88-V SLAMF7 Isoform 1 of SLAM family member 7 IPI00099863,IPI00168447 ETPNICPHSGENTEYDTIPHTNR 3.35 0.738 2.55 270 292
NB12-2 SLAMF7 Isoform 1 of SLAM family member 7 IPI00099863,IPI00168447 ETPNICPHSGENTEYDTIPHTNR 3.21 0.826 0 270 292
NB122-2 SLAMF7 Isoform 1 of SLAM family member 7 IPI00099863,IPI00168447 ETPNICPHSGENTEYDTIPHTNR 2.88 0.723 0 270 292
NB12 SLAMF7 Isoform 1 of SLAM family member 7 IPI00099863,IPI00168447 ETPNICPHSGENTEYDTIPHTNR 2.64 0.649 0 270 292
NB25-V SLAMF7 Isoform 1 of SLAM family member 7 IPI00099863,IPI00168447 ETPNICPHSGENTEYDTIPHTNR 2.22 0.653 0 270 292
NB88 SLAMF7 Isoform 1 of SLAM family member 7 IPI00099863 ETPNICPHSGENTEYDTIPHTNR 2.18 0.649 0 270 292 IPI00168447
NB122 SLAMF7 Isoform 1 of SLAM family member 7 IPI00099863,IPI00168447 ETPNICPHSGENTEYDTIPHTNR 2.15 0.413 0 270 292 SNRP70 Isoform 2 of U1 small nuclear NB12-2 ribonucleoprotein 70 kDa IPI00219483 VNYDTTESK 1.59 0.399 2.19 110 118 SNRP70 Isoform 2 of U1 small nuclear NB12-2 ribonucleoprotein 70 kDa IPI00219483 REFEVYGPIKR 3.13 0.571 1.55 121 131 SNRP70 Isoform 2 of U1 small nuclear NB25-V ribonucleoprotein 70 kDa IPI00219483 EFEVYGPIKR 2.06 0.494 0 122 131 SNRP70 Isoform 2 of U1 small nuclear NB12-2 ribonucleoprotein 70 kDa IPI00219483 EFEVYGPIKR 1.76 0.554 0 122 131 SNRP70 Isoform 2 of U1 small nuclear NB122 ribonucleoprotein 70 kDa IPI00219483 EFEVYGPIKR 1.73 0.629 0 122 131 SNRP70 Isoform 2 of U1 small nuclear NB88 ribonucleoprotein 70 kDa IPI00219483 EFEVYGPIKR 1.72 0.49 0 122 131 SNRP70 Isoform 2 of U1 small nuclear NB25-V ribonucleoprotein 70 kDa IPI00219483 EFEVYGPIKR 1.44 0.629 0 122 131 SNRP70 Isoform 2 of U1 small nuclear NB25 ribonucleoprotein 70 kDa IPI00219483 EFEVYGPIKR 1.39 0.69 0 122 131 NB88-V SPRY2 SPRY2 protein IPI00012913 NTNEYTEGPTVVPRPGLKPAPR 2.55 0.542 0 51 72 NB25-V SPRY2 SPRY2 protein IPI00012913 NTNEYTEGPTVVPRPGLKPAPR 2.27 0.493 0 51 72 SPTBN1 Isoform Long of Spectrin beta chain, NB88 brain 1 IPI00005614 IVSSSDVGHDEYSTQSLVKK 2.38 0.561 0 766 785 SPTBN1 Isoform Long of Spectrin beta chain, NB88 brain 1 IPI00005614 IVSSSDVGHDEYSTQSLVK 4.53 0.611 5.47 766 784 SPTBN1 Isoform Long of Spectrin beta chain, NB88-2 brain 1 IPI00005614 IVSSSDVGHDEYSTQSLVK 2.38 0.377 0 766 784 SRC Isoform 2 of Proto-oncogene tyrosine- IPI00166845,IPI00639876,IPI002 NB12-2 protein kinase Src IPI00328867,IPI00641230 LIEDNEYTARQGAK pY 416 3.45 0.485 3.13 419 432 19012,IPI00013981,IPI00640091 IPI00166845,IPI00639876,IPI005 55672,IPI00219012,IPI00013981,I SRC Isoform 2 of Proto-oncogene tyrosine- PI00640091,IPI00515097,IPI0039 NB12 protein kinase Src IPI00328867,IPI00641230 LIEDNEYTAR pY 416 3.54 0.689 2.96 419 428 4952 IPI00166845,IPI00639876,IPI005 55672,IPI00219012,IPI00013981,I SRC Isoform 2 of Proto-oncogene tyrosine- PI00640091,IPI00515097,IPI0039 NB12-2 protein kinase Src IPI00328867,IPI00641230 LIEDNEYTAR pY 416 3.05 0.736 4.7 419 428 4952 SRC Isoform 2 of Proto-oncogene tyrosine- NB12 protein kinase Src IPI00328867,IPI00641230 KEPEERPTFEYLQAFLEDYFTSTEPQYQPGENL pY 529 4.52 0.64 5.21 510 542 SSBP1 Single-stranded DNA-binding protein, NB88 mitochondrial IPI00029744 SGDSEVYQLGDVSQK 4.63 0.597 6.68 67 81 STAT3 Isoform Del-701 of Signal transducer NB12-2 and activator of transcription 3 IPI00306436 YCRPESQEHPEADPGAAPYLKTK 3.66 0.646 1.74 686 708 STAT3 Isoform Del-701 of Signal transducer NB122-2 and activator of transcription 3 IPI00306436 YCRPESQEHPEADPGAAPYLKTK 2.39 0.558 0 686 708 STAT3 Isoform Del-701 of Signal transducer NB88 and activator of transcription 3 IPI00306436 YCRPESQEHPEADPGAAPYLKTK 2.29 0.532 0 686 708
148 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
STAT3 Isoform Del-701 of Signal transducer NB122 and activator of transcription 3 IPI00306436 YCRPESQEHPEADPGAAPYLKTK 2.07 0.4 0 686 708 STAT3 Isoform Del-701 of Signal transducer NB12-2 and activator of transcription 3 IPI00306436 YCRPESQEHPEADPGAAPYLK 3.85 0.622 3.47 686 706 STAT3 Isoform Del-701 of Signal transducer NB25-V and activator of transcription 3 IPI00306436 YCRPESQEHPEADPGAAPYLK 3.81 0.556 4.14 686 706 STAT3 Isoform Del-701 of Signal transducer NB88-V and activator of transcription 3 IPI00306436 YCRPESQEHPEADPGAAPYLK 3.8 0.567 4.92 686 706 STAT3 Isoform Del-701 of Signal transducer NB25 and activator of transcription 3 IPI00306436 YCRPESQEHPEADPGAAPYLK 3.47 0.578 3.2 686 706 STAT3 Isoform Del-701 of Signal transducer NB122-2 and activator of transcription 3 IPI00306436 YCRPESQEHPEADPGAAPYLK 3.3 0.394 4.37 686 706 STAT3 Isoform Del-701 of Signal transducer NB88 and activator of transcription 3 IPI00306436 YCRPESQEHPEADPGAAPYLK 3.22 0.484 2.02 686 706 STAT3 Isoform Del-701 of Signal transducer NB88-2 and activator of transcription 3 IPI00306436 YCRPESQEHPEADPGAAPYLK 3.13 0.453 4.21 686 706 STAT3 Isoform Del-701 of Signal transducer NB12 and activator of transcription 3 IPI00306436 YCRPESQEHPEADPGAAPYLK 3.07 0.33 3.34 686 706 STAT3 Isoform Del-701 of Signal transducer NB61 and activator of transcription 3 IPI00306436 YCRPESQEHPEADPGAAPYLK 2.21 0.348 0 686 706 STAT3 Isoform Del-701 of Signal transducer NB25-V and activator of transcription 3 IPI00306436 YCRPESQEHPEADPGAAPYLK 1.99 0.328 3.48 686 706 STAT3 signal transducer and activator of NB12-2 transcription 3 isoform 3 IPI00412752,IPI00784414 YCRPESQEHPEADPGSAAPYLKTK 3.26 0.66 0 686 709 STAT3 signal transducer and activator of NB25-V transcription 3 isoform 3 IPI00412752,IPI00784414 YCRPESQEHPEADPGSAAPYLKTK 3.04 0.637 0 686 709 STAT3 signal transducer and activator of NB88 transcription 3 isoform 3 IPI00412752,IPI00784414 YCRPESQEHPEADPGSAAPYLKTK 2.82 0.671 2.64 686 709 STAT3 signal transducer and activator of NB122 transcription 3 isoform 3 IPI00412752,IPI00784414 YCRPESQEHPEADPGSAAPYLKTK 2.23 0.585 0 686 709 STAT3 signal transducer and activator of NB12 transcription 3 isoform 3 IPI00412752,IPI00784414 YCRPESQEHPEADPGSAAPYLKTK 1.48 0.461 0 686 709 STAT3 signal transducer and activator of NB25-V transcription 3 isoform 3 IPI00412752,IPI00784414 YCRPESQEHPEADPGSAAPYLK 4.38 0.81 5.09 686 707 STAT3 signal transducer and activator of NB12-2 transcription 3 isoform 3 IPI00412752,IPI00784414 YCRPESQEHPEADPGSAAPYLK 4.22 0.639 4.54 686 707 STAT3 signal transducer and activator of NB25 transcription 3 isoform 3 IPI00412752,IPI00784414 YCRPESQEHPEADPGSAAPYLK 3.87 0.767 7.04 686 707 STAT3 signal transducer and activator of NB88-2 transcription 3 isoform 3 IPI00412752,IPI00784414 YCRPESQEHPEADPGSAAPYLK 3.83 0.551 1.77 686 707 STAT3 signal transducer and activator of NB122-2 transcription 3 isoform 3 IPI00412752,IPI00784414 YCRPESQEHPEADPGSAAPYLK 3.76 0.588 7.85 686 707 STAT3 signal transducer and activator of NB88 transcription 3 isoform 3 IPI00412752,IPI00784414 YCRPESQEHPEADPGSAAPYLK 3.58 0.651 6.59 686 707 STAT3 signal transducer and activator of NB25-V transcription 3 isoform 3 IPI00412752,IPI00784414 YCRPESQEHPEADPGSAAPYLK 3.55 0.646 5.32 686 707 STAT3 signal transducer and activator of NB12 transcription 3 isoform 3 IPI00412752,IPI00784414 YCRPESQEHPEADPGSAAPYLK 3.46 0.483 4.07 686 707 STAT3 signal transducer and activator of NB122 transcription 3 isoform 3 IPI00412752,IPI00784414 YCRPESQEHPEADPGSAAPYLK 3.16 0.734 0 686 707 STAT3 signal transducer and activator of NB88-V transcription 3 isoform 3 IPI00412752,IPI00784414 YCRPESQEHPEADPGSAAPYLK 2.96 0.686 0 686 707 STAT3 signal transducer and activator of NB61 transcription 3 isoform 3 IPI00412752,IPI00784414 YCRPESQEHPEADPGSAAPYLK 2.46 0.43 1.04 686 707 STAT5B Signal transducer and activator of NB88 transcription 5B IPI00103415 AVDGYVKPQIK 1.59 0.314 2.22 695 705 STEAP1 Similar to Six transmembrane NB88-V epithelial antigen of prostate IPI00005846 RNLEEDDYLHKDTGETSMLK 3.7 0.612 0 167 186 STEAP1 Similar to Six transmembrane NB88-V epithelial antigen of prostate IPI00005846 NLEEDDYLHKDTGETSMLK 3.16 0.557 0 168 186 SYK Isoform Long of Tyrosine-protein kinase NB88 SYK IPI00018597 SYSFPKPGHR py 295 2.52 0.398 1.82 295 304 SYK Isoform Long of Tyrosine-protein kinase NB88-2 SYK IPI00018597 QESTVSFNPYEPELAPWAADKGPQR pY 323 3.59 0.589 1.05 314 338 SYK Isoform Long of Tyrosine-protein kinase NB25-V SYK IPI00018597 QESTVSFNPYEPELAPWAADKGPQR pY 323 3.46 0.531 2.31 314 338 SYK Isoform Long of Tyrosine-protein kinase NB25 SYK IPI00018597 QESTVSFNPYEPELAPWAADKGPQR pY 323 3.38 0.524 0 314 338 SYK Isoform Long of Tyrosine-protein kinase NB122 SYK IPI00018597 QESTVSFNPYEPELAPWAADKGPQR pY 323 3.17 0.612 3.39 314 338
149 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
SYK Isoform Long of Tyrosine-protein kinase NB12-2 SYK IPI00018597 QESTVSFNPYEPELAPWAADKGPQR pY 323 3.07 0.462 6.42 314 338 SYK Isoform Long of Tyrosine-protein kinase NB122-2 SYK IPI00018597 QESTVSFNPYEPELAPWAADKGPQR pY 323 2.94 0.604 2.49 314 338 SYK Isoform Long of Tyrosine-protein kinase NB12 SYK IPI00018597 QESTVSFNPYEPELAPWAADKGPQR pY 323 2.66 0.479 4.96 314 338 SYK Isoform Long of Tyrosine-protein kinase NB88-V SYK IPI00018597 QESTVSFNPYEPELAPWAADKGPQR pY 323 2.57 0.572 5.31 314 338 SYK Isoform Long of Tyrosine-protein kinase NB25-V SYK IPI00018597 QESTVSFNPYEPELAPWAADKGPQR pY 323 2.38 0.487 8.6 314 338 SYK Isoform Long of Tyrosine-protein kinase NB61 SYK IPI00018597 QESTVSFNPYEPELAPWAADKGPQR pY 323 2.25 0.449 0 314 338 SYK Isoform Long of Tyrosine-protein kinase NB88 SYK IPI00018597 LRNYYYDVVN pY 630 2.41 0.72 0 626 635 SYK Isoform Long of Tyrosine-protein kinase NB88-2 SYK IPI00018597 LRNYYYDVVN pY 629 2.13 0.517 0 626 635 SYK Isoform Long of Tyrosine-protein kinase NB88-V SYK IPI00018597 LRNYYYDVVN pY 630 1.61 0.59 1.39 626 635 SYK Isoform Long of Tyrosine-protein kinase NB25 SYK IPI00018597 KGYYQMK 2.54 0.307 0 387 393 SYK Isoform Long of Tyrosine-protein kinase NB25-V SYK IPI00018597 IKSYSFPKPGHR pY 296 2.83 0.586 1.92 293 304 SYK Isoform Long of Tyrosine-protein kinase NB88-V SYK IPI00018597 IKSYSFPKPGHR pY 296 2.5 0.593 0 293 304 SYK Isoform Long of Tyrosine-protein kinase NB88-2 SYK IPI00018597 IKSYSFPKPGHR pY 296 2.24 0.461 0 293 304 SYK Isoform Long of Tyrosine-protein kinase NB12-2 SYK IPI00018597 IKSYSFPKPGHR pY 296 2.13 0.322 0 293 304 SYK Isoform Long of Tyrosine-protein kinase NB88 SYK IPI00018597 IKSYSFPKPGHR pY 296 1.93 0.418 1.55 293 304 SYK Isoform Long of Tyrosine-protein kinase NB122-2 SYK IPI00018597 EALPMDTEVYESPYADPEEIRPK pY 532 3.29 0.656 8 339 361 SYK Isoform Long of Tyrosine-protein kinase NB12-2 SYK IPI00018597 EALPMDTEVYESPYADPEEIRPK pY 532 2.98 0.518 5.52 339 361 SYK Isoform Long of Tyrosine-protein kinase NB88-V SYK IPI00018597 EALPMDTEVYESPYADPEEIRPK pY 348/ 532 2.85 0.705 6.74 339 361 SYK Isoform Long of Tyrosine-protein kinase NB88 SYK IPI00018597 EALPMDTEVYESPYADPEEIRPK pY 348/ 532 2.58 0.575 5.12 339 361 SYK Isoform Long of Tyrosine-protein kinase NB88-2 SYK IPI00018597 EALPMDTEVYESPYADPEEIRPK pY 348 2.48 0.641 5.42 339 361 SYK Isoform Long of Tyrosine-protein kinase NB25-V SYK IPI00018597 EALPMDTEVYESPYADPEEIRPK pY 348/ 532 2.05 0.422 0 339 361 SYK Isoform Long of Tyrosine-protein kinase NB25 SYK IPI00018597 EALPMDTEVYESPYADPEEIRPK pY 348/ 532 1.92 0.536 0 339 361 SYK Isoform Long of Tyrosine-protein kinase NB61 SYK IPI00018597 EALPMDTEVYESPYADPEEIRPK pY 348/ 532 1.38 0.463 0 339 361 SYK Isoform Long of Tyrosine-protein kinase NB61 SYK IPI00018597 DNNGSYALCLLHEGK pY 203 1.29 0.431 0 198 212 SYK Isoform Long of Tyrosine-protein kinase NB88-2 SYK IPI00018597 ARDNNGSYALCLLHEGK pY 203 3.91 0.522 1.52 196 212 SYK Isoform Long of Tyrosine-protein kinase NB88 SYK IPI00018597 ARDNNGSYALCLLHEGK pY 203 3.89 0.591 1.02 196 212 SYK Isoform Long of Tyrosine-protein kinase NB88-V SYK IPI00018597 ARDNNGSYALCLLHEGK pY 203 3.43 0.53 1.82 196 212 SYK Isoform Long of Tyrosine-protein kinase NB61 SYK IPI00018597 ARDNNGSYALCLLHEGK pY 203 2.41 0.319 0 196 212 SYK Isoform Long of Tyrosine-protein kinase NB88 SYK IPI00018597 ALRADENYYKAQTHGK pY 525/ 526 2.87 0.601 1.77 518 533 SYK Isoform Long of Tyrosine-protein kinase NB88 SYK IPI00018597 ALRADENYYK pY 525/ 526 2.67 0.633 4.17 518 527 SYK Isoform Long of Tyrosine-protein kinase NB12 SYK IPI00018597 ADENYYKAQTHGKWPVK pY 525/ 526 2.87 0.649 0 521 537 SYK Isoform Long of Tyrosine-protein kinase NB88 SYK IPI00018597 ADENYYKAQTHGKWPVK pY 525/ 526 2.58 0.374 0 521 537 SYK Isoform Long of Tyrosine-protein kinase NB88-V SYK IPI00018597 ADENYYKAQTHGKWPVK pY 525/ 526 2.54 0.471 0 521 537 SYK Isoform Long of Tyrosine-protein kinase NB88 SYK IPI00018597 ADENYYKAQTHGK pY 525/ 526 2.45 0.745 0 521 533 SYK Isoform Long of Tyrosine-protein kinase NB12-2 SYK IPI00018597 ADENYYKAQTHGK pY 525 2.34 0.51 0 521 533
150 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
NB88-2 TARS Threonyl-tRNA synthetase, cytoplasmic IPI00329633 IYGISFPDPK 2.9 0.396 1.82 297 306 NB88 TBCB Tubulin folding cofactor B IPI00293126,IPI00798211 LGEYEDVSRVEK 3.6 0.404 2.29 95 106 NB12-2 TBCB Tubulin folding cofactor B IPI00293126,IPI00798211 LGEYEDVSRVEK 3.3 0.325 1.07 95 106 NB122 TBCB Tubulin folding cofactor B IPI00293126 LGEYEDVSRVEK 2.07 0.34 0 95 106 IPI00798211 NB88-V TBCB Tubulin folding cofactor B IPI00293126,IPI00798211 LGEYEDVSR 2.86 0.526 3.7 95 103 NB88 TBCB Tubulin folding cofactor B IPI00293126,IPI00798211 LGEYEDVSR 2.74 0.701 2.68 95 103 NB12-2 TBCB Tubulin folding cofactor B IPI00293126,IPI00798211 LGEYEDVSR 1.86 0.522 0 95 103 NB88-V TCP1 T-complex protein 1 subunit alpha IPI00290566 DDKHGSYEDAVHSGALND 3.42 0.764 3.14 539 556 NB12-2 TCP1 T-complex protein 1 subunit alpha IPI00290566 DDKHGSYEDAVHSGALND 2.85 0.693 2.3 539 556 NB25-V TCP1 T-complex protein 1 subunit alpha IPI00290566 DDKHGSYEDAVHSGALND 2.79 0.79 3.7 539 556 NB122-2 TCP1 T-complex protein 1 subunit alpha IPI00290566 DDKHGSYEDAVHSGALND 2.71 0.63 1.66 539 556 NB88 TCP1 T-complex protein 1 subunit alpha IPI00290566 DDKHGSYEDAVHSGALND 2.39 0.618 3.48 539 556 NB88-2 TCP1 T-complex protein 1 subunit alpha IPI00290566 DDKHGSYEDAVHSGALND 2.22 0.463 1.92 539 556 NB88 TEC Tyrosine-protein kinase Tec IPI00000878 YVLDDQYTSSSGAKFPVK 5.29 0.56 5.8 513 530 NB88-2 TEC Tyrosine-protein kinase Tec IPI00000878 YVLDDQYTSSSGAKFPVK 4.89 0.555 4.72 513 530 NB12-2 TEC Tyrosine-protein kinase Tec IPI00000878 YVLDDQYTSSSGAKFPVK 4.7 0.553 5.62 513 530 NB12 TEC Tyrosine-protein kinase Tec IPI00000878 YVLDDQYTSSSGAKFPVK 4.49 0.526 6.02 513 530 NB88-V TEC Tyrosine-protein kinase Tec IPI00000878 YVLDDQYTSSSGAKFPVK 4.36 0.653 6.82 513 530 NB25-V TEC Tyrosine-protein kinase Tec IPI00000878 YVLDDQYTSSSGAKFPVK 4.04 0.59 6.32 513 530 NB122-2 TEC Tyrosine-protein kinase Tec IPI00000878 YVLDDQYTSSSGAKFPVK 2.37 0.473 0 513 530 NB88-2 TEC Tyrosine-protein kinase Tec IPI00000878 YVLDDQYTSSSGAK 4.07 0.539 6.07 513 526 NB12-2 TEC Tyrosine-protein kinase Tec IPI00000878 YVLDDQYTSSSGAK 3.82 0.506 5.02 513 526 NB88 TEC Tyrosine-protein kinase Tec IPI00000878 YVLDDQYTSSSGAK 3.49 0.577 6.08 513 526 NB12 TEC Tyrosine-protein kinase Tec IPI00000878 YVLDDQYTSSSGAK 3.45 0.563 5.17 513 526 NB25-V TEC Tyrosine-protein kinase Tec IPI00000878 YVLDDQYTSSSGAK 2.26 0.439 0 513 526 TJP2 Isoform A1 of Tight junction protein ZO- NB25-V 2 IPI00003843 IEIAQKHPDIYAVPIK 3.42 0.439 1.66 1108 1123 TJP2 Isoform A1 of Tight junction protein ZO- NB88-V 2 IPI00003843 IEIAQKHPDIYAVPIK 2.94 0.389 1.02 1108 1123 TJP2 Isoform A1 of Tight junction protein ZO- NB12-2 2 IPI00003843 IEIAQKHPDIYAVPIK 2.28 0.385 0 1108 1123 TJP2 Isoform A1 of Tight junction protein ZO- NB122-2 2 IPI00003843 IEIAQKHPDIYAVPIK 2.08 0.43 0 1108 1123 TJP2 Isoform A1 of Tight junction protein ZO- NB88-V 2 IPI00003843 AYDPDYER 1.71 0.541 0 256 263 TJP2 Isoform A1 of Tight junction protein ZO- NB12-2 2 IPI00003843 AYDPDYER 1.69 0.756 1.11 256 263 TJP2 Isoform A1 of Tight junction protein ZO- NB88 2 IPI00003843 AYDPDYER 1.57 0.767 0 256 263 TJP2 Isoform A1 of Tight junction protein ZO- NB122-2 2 IPI00003843 AYDPDYER 1.24 0.634 1.29 256 263 TJP2 Isoform A1 of Tight junction protein ZO- NB88-2 2 IPI00003843 AYDPDYER 1.24 0.431 1.36 256 263 NB12 TLN1 Talin-1 IPI00298994 TMQFEPSTMVYDACR 2.84 0.731 7.77 16 30 NB25 TLN1 Talin-1 IPI00298994 TMQFEPSTMVYDACR 2.26 0.66 3.64 16 30 NB88 TLN1 Talin-1 IPI00298994 ALDYYMLR 2.56 0.521 0 67 74 NB12 TLN1 Talin-1 IPI00298994 ALDYYMLR 2.54 0.548 0 67 74 NB25 TLN1 Talin-1 IPI00298994 ALDYYMLR 2.22 0.504 0 67 74 NB12-2 TLN1 Talin-1 IPI00298994 ALDYYMLR 2.09 0.535 0 67 74 NB88-2 TLN1 Talin-1 IPI00298994 ALDYYMLR 2.05 0.496 0 67 74 SIVGRTQYSFITGPAVIPGYFSVDVNNVVLILN NB122 TMEM5 Transmembrane protein 5 IPI00003392 GR 1.62 0.312 0 135 169 NB12 TNK2 Activated CDC42 kinase 1 IPI00552750 VSSTHYYLLPERPSYLER 2.44 0.312 2.02 908 925 IPI00442025 NB88 TNK2 Activated CDC42 kinase 1 IPI00552750 VSSTHYYLLPERPSYLER 1.7 0.268 1.41 908 925 IPI00442025 NB25 TNK2 Activated CDC42 kinase 1 IPI00552750 KVSSTHYYLLPERPSYLER 3.05 0.553 4.13 907 925 IPI00442025 NB122-2 TNK2 Activated CDC42 kinase 1 IPI00552750 KVSSTHYYLLPERPSYLER 2.86 0.519 4.57 907 925 IPI00442025 NB88-2 TNK2 Activated CDC42 kinase 1 IPI00552750 KPTYDPVSEDQDPLSSDFKR 3.71 0.648 2.74 571 590 NB25-V TNK2 Activated CDC42 kinase 1 IPI00552750 KPTYDPVSEDQDPLSSDFKR 3.61 0.613 4.54 571 590 NB12 TNK2 Activated CDC42 kinase 1 IPI00552750 KPTYDPVSEDQDPLSSDFKR 3.47 0.669 5.28 571 590 NB122 TNK2 Activated CDC42 kinase 1 IPI00552750 KPTYDPVSEDQDPLSSDFKR 2.93 0.519 2.48 571 590 NB61 TNK2 Activated CDC42 kinase 1 IPI00552750 KPTYDPVSEDQDPLSSDFKR 2.77 0.5 2.16 571 590 NB88-V TNK2 Activated CDC42 kinase 1 IPI00552750 KPTYDPVSEDQDPLSSDFKR 2.75 0.581 1.51 571 590 NB122-2 TNK2 Activated CDC42 kinase 1 IPI00552750 KPTYDPVSEDQDPLSSDFKR 2.65 0.492 2.54 571 590 NB25 TNK2 Activated CDC42 kinase 1 IPI00552750 KPTYDPVSEDQDPLSSDFKR 2.64 0.484 2.51 571 590 NB25-V TNK2 Activated CDC42 kinase 1 IPI00552750 KPTYDPVSEDQDPLSSDFKR 2.56 0.552 0 571 590 NB12-2 TNK2 Activated CDC42 kinase 1 IPI00552750 KPTYDPVSEDQDPLSSDFKR 2.41 0.283 3.33 571 590 NB88 TNK2 Activated CDC42 kinase 1 IPI00552750 KPTYDPVSEDQDPLSSDFKR 2.13 0.498 3.77 571 590 NB88 TNK2 Activated CDC42 kinase 1 IPI00552750 ALPQNDDHYVMQEHR 3.8 0.758 2.02 332 346 IPI00442025 NB88-2 TNK2 Activated CDC42 kinase 1 IPI00552750 ALPQNDDHYVMQEHR 3.71 0.624 2.16 332 346 IPI00442025 NB122-2 TNK2 Activated CDC42 kinase 1 IPI00552750 ALPQNDDHYVMQEHR 3.61 0.724 4.62 332 346 IPI00442025
151 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
NB12-2 TNK2 Activated CDC42 kinase 1 IPI00552750 ALPQNDDHYVMQEHR 3.13 0.633 4.09 332 346 IPI00442025 NB122 TNK2 Activated CDC42 kinase 1 IPI00552750 ALPQNDDHYVMQEHR 3.1 0.689 2.34 332 346 IPI00442025 NB12 TNK2 Activated CDC42 kinase 1 IPI00552750 ALPQNDDHYVMQEHR 2.65 0.771 4.41 332 346 IPI00442025 NB25 TNK2 Activated CDC42 kinase 1 IPI00552750 ALPQNDDHYVMQEHR 1.31 0.696 1.7 332 346 IPI00442025 NB88-V TNK2 Activated CDC42 kinase 1 IPI00552750 ALPQNDDHYVMQEHR 0.821 0.487 1.09 332 346 IPI00442025 TNK2 tyrosine kinase, non-receptor, 2 NB12 isoform 2 IPI00442025 VSSTHYYLLPERPSYLER 2.44 0.312 2.02 932 949 IPI00552750 TNK2 tyrosine kinase, non-receptor, 2 NB12-2 isoform 2 IPI00442025 TMPTTQSFASDPKYATPQVIQAPGPR 3.93 0.724 7.82 892 917 TNK2 tyrosine kinase, non-receptor, 2 NB88-2 isoform 2 IPI00442025 TMPTTQSFASDPKYATPQVIQAPGPR 3.78 0.573 6.16 892 917 TNK2 tyrosine kinase, non-receptor, 2 NB25-V isoform 2 IPI00442025 TMPTTQSFASDPKYATPQVIQAPGPR 2.69 0.577 0 892 917 TNK2 tyrosine kinase, non-receptor, 2 NB12 isoform 2 IPI00442025 TMPTTQSFASDPKYATPQVIQAPGPR 2.53 0.46 4.15 892 917 TNK2 tyrosine kinase, non-receptor, 2 NB88-V isoform 2 IPI00442025 TMPTTQSFASDPKYATPQVIQAPGPR 0 0 6.51 892 917 TNK2 tyrosine kinase, non-receptor, 2 NB88-V isoform 2 IPI00442025 KVSSTHYYLLPERPSYLER 2.78 0.368 0 931 949 IPI00552750 TNK2 tyrosine kinase, non-receptor, 2 NB88-2 isoform 2 IPI00442025 ALPQNDDHYVMQEHR 3.71 0.624 2.16 339 353 IPI00552750 TNK2 tyrosine kinase, non-receptor, 2 NB12-2 isoform 2 IPI00442025 ALPQNDDHYVMQEHR 3.13 0.633 4.09 339 353 IPI00552750 TNK2 tyrosine kinase, non-receptor, 2 NB12 isoform 2 IPI00442025 ALPQNDDHYVMQEHR 2.65 0.771 4.41 339 353 IPI00552750 TNK2 tyrosine kinase, non-receptor, 2 NB88-V isoform 2 IPI00442025 ALPQNDDHYVMQEHR 0.821 0.487 1.09 339 353 IPI00552750 TPM3 Isoform 2 of Tropomyosin alpha-3 NB88-V chain IPI00218319 HIAEEADRKYEEVAR 2.62 0.541 1.59 117 131 NB88-2 TPP2 Tripeptidyl-peptidase 2 IPI00020416 VNESSHYDLAFTDVHFKPGQIR 3.6 0.551 0 639 660 TRAFD1 TRAF-type zinc finger domain- IPI00009146,IPI00788949,IPI00 NB88 containing protein 1 908631 LKEHEDYCGAR 3.43 0.523 1.92 102 112 IPI00237925,IPI00789154,IPI00 NB25-V TSPAN33 Tetraspanin-33 879795 LQLYNQQHR 2.69 0.458 1.21 270 278 NB12 TSPAN33 Tetraspanin-33 IPI00237925 LQLYNQQHR 2.29 0.408 0 270 278 IPI00789154,IPI00879795 NB25-V TUBA1B Tubulin alpha-1B chain IPI00387144 LDHKFDLMYAKR 4.16 0.62 2.33 391 402 NB88 TUBA1B Tubulin alpha-1B chain IPI00387144 LDHKFDLMYAKR 2.83 0.546 1.35 391 402 NB88-V TUBA1B Tubulin alpha-1B chain IPI00387144 LDHKFDLMYAKR 2.1 0.315 2.57 391 402 NB88-V TUBA1B Tubulin alpha-1B chain IPI00387144 LDHKFDLMYAK 4.52 0.446 2.18 391 401 NB12 TUBB Tubulin beta chain IPI00011654 NSSYFVEWIPNNVK 2.15 0.552 1.32 337 350 TXNRD1 Isoform 5 of Thioredoxin reductase NB88 1, cytoplasmic IPI00554786 KVVYENAYGQFIGPHR 3.53 0.645 0 124 139 TYK2 Non-receptor tyrosine-protein kinase NB88 TYK2 IPI00022353 LLAQAEGEPCYIR 4.07 0.555 3.29 282 294 TYK2 Non-receptor tyrosine-protein kinase NB122-2 TYK2 IPI00022353 LLAQAEGEPCYIR 3.99 0.669 5.01 282 294 TYK2 Non-receptor tyrosine-protein kinase NB12-2 TYK2 IPI00022353 LLAQAEGEPCYIR 3.96 0.583 3 282 294 TYK2 Non-receptor tyrosine-protein kinase NB25-V TYK2 IPI00022353 LLAQAEGEPCYIR 3.78 0.609 4.92 282 294 TYK2 Non-receptor tyrosine-protein kinase NB88-2 TYK2 IPI00022353 LLAQAEGEPCYIR 3.58 0.462 3.51 282 294 TYK2 Non-receptor tyrosine-protein kinase NB12 TYK2 IPI00022353 LLAQAEGEPCYIR 3.38 0.621 3.25 282 294 TYK2 Non-receptor tyrosine-protein kinase NB88-V TYK2 IPI00022353 LLAQAEGEPCYIR 3.3 0.483 1.85 282 294 TYK2 Non-receptor tyrosine-protein kinase NB25-V TYK2 IPI00022353 LLAQAEGEPCYIR 2.75 0.558 0 282 294 TYK2 Non-receptor tyrosine-protein kinase NB61 TYK2 IPI00022353 LLAQAEGEPCYIR 2.6 0.585 1.24 282 294 TYK2 Non-receptor tyrosine-protein kinase NB122 TYK2 IPI00022353 LLAQAEGEPCYIR 1.98 0.597 0 282 294 TYK2 Non-receptor tyrosine-protein kinase NB25-V TYK2 IPI00022353 AVPEGHEYYR 1.69 0.666 0 1047 1056
NB12 UBE2E1 Ubiquitin-conjugating enzyme E2 E1 IPI00021346 GDNIYEWR 1.94 0.417 0 73 80 NB122-2 UMODL1 Isoform 3 of Uromodulin-like 1 IPI00166237 MVYRTQYLVVEVPESR 2.46 0.31 0 1 16 UPF1 Isoform 1 of Regulator of nonsense NB88-V transcripts 1 IPI00034049 RFTAQGLPDLNHSQVYAVK 4.09 0.487 1.96 473 491 UPF1 Isoform 1 of Regulator of nonsense NB88-2 transcripts 1 IPI00034049 RFTAQGLPDLNHSQVYAVK 3.65 0.499 1.68 473 491
152 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
UPF1 Isoform 1 of Regulator of nonsense NB12-2 transcripts 1 IPI00034049 RFTAQGLPDLNHSQVYAVK 3.24 0.443 0 473 491 UTP18 U3 small nucleolar RNA-associated NB122-2 protein 18 homolog IPI00000733 LHHYSDF 0.412 0.924 0 567 573
NB88 VASP Vasodilator-stimulated phosphoprotein IPI00301058 VQIYHNPTANSFR 4.58 0.557 4.48 36 48
NB12 VASP Vasodilator-stimulated phosphoprotein IPI00301058 VQIYHNPTANSFR 4.48 0.605 3.46 36 48
NB61 VASP Vasodilator-stimulated phosphoprotein IPI00301058 VQIYHNPTANSFR 3.82 0.503 3.25 36 48
NB25-V VASP Vasodilator-stimulated phosphoprotein IPI00301058 VQIYHNPTANSFR 3.8 0.684 4.07 36 48
NB25-V VASP Vasodilator-stimulated phosphoprotein IPI00301058 VQIYHNPTANSFR 3.6 0.567 5.74 36 48
NB122 VASP Vasodilator-stimulated phosphoprotein IPI00301058 VQIYHNPTANSFR 3.54 0.589 1.74 36 48
NB12-2 VASP Vasodilator-stimulated phosphoprotein IPI00301058 VQIYHNPTANSFR 3.45 0.539 1.62 36 48
NB88-2 VASP Vasodilator-stimulated phosphoprotein IPI00301058 VQIYHNPTANSFR 1.98 0.328 0 36 48 IPI00291175,IPI00307162,IPI00 NB12 VCL Isoform 1 of Vinculin 910496 NPGNQAAYEHFETMK 3.25 0.72 3.66 685 699 IPI00291175,IPI00307162,IPI00 NB12-2 VCL Isoform 1 of Vinculin 910496 ILLRNPGNQAAYEHFETMK 5.79 0.672 6.8 681 699 IPI00291175,IPI00307162,IPI00 NB122-2 VCL Isoform 1 of Vinculin 910496 ILLRNPGNQAAYEHFETMK 5.68 0.618 3.92 681 699 IPI00291175,IPI00307162,IPI00 NB12 VCL Isoform 1 of Vinculin 910496 ILLRNPGNQAAYEHFETMK 4.16 0.374 5.38 681 699 NB88 VIM Vimentin IPI00418471 SLYASSPGGVYATR 4.37 0.619 6.36 51 64 NB88-2 VIM Vimentin IPI00418471 SLYASSPGGVYATR 4.29 0.621 4.19 51 64 NB88-V VIM Vimentin IPI00418471 SLYASSPGGVYATR 3.93 0.551 9.19 51 64 NB25-V VIM Vimentin IPI00418471 SLYASSPGGVYATR 3.67 0.575 6.66 51 64 NB122 VIM Vimentin IPI00418471 SLYASSPGGVYATR 3.56 0.524 8.92 51 64 NB122-2 VIM Vimentin IPI00418471 SLYASSPGGVYATR 3.43 0.635 5.77 51 64 NB12 VIM Vimentin IPI00418471 SLYASSPGGVYATR 3.37 0.716 4.34 51 64 NB12-2 VIM Vimentin IPI00418471 SLYASSPGGVYATR 3.29 0.636 5.14 51 64 NB25 VIM Vimentin IPI00418471 SLYASSPGGVYATR 2.3 0.568 2.39 51 64 NB25-V VIM Vimentin IPI00418471 SLYASSPGGVYATR 2.07 0.56 1.7 51 64 NB61 VIM Vimentin IPI00418471 SLYASSPGGVYATR 2.05 0.531 3.85 51 64 NB88-V VIM Vimentin IPI00418471 QQYESVAAK 1.43 0.478 1.48 274 282 NB12-2 VIM Vimentin IPI00418471 QQYESVAAK 1.23 0.494 0 274 282 NB12-2 VIM Vimentin IPI00418471 FANYIDKVR 1.83 0.483 0 114 122 NB25-V WAS Wiskott-Aldrich syndrome protein IPI00001545 LIYDFIEDQGGLEAVRQEMR 2.8 0.491 0 289 308 NB25-V WAS Wiskott-Aldrich syndrome protein IPI00001545 LIYDFIEDQGGLEAVR 4.35 0.585 0 289 304 AGISEAQLTDAETSKLIYDFIEDQGGLEAVRQE NB12 WAS Wiskott-Aldrich syndrome protein IPI00001545 MR 4.97 0.6 1.1 274 308 NB88 WAS Wiskott-Aldrich syndrome protein IPI00001545 AGISEAQLTDAETSKLIYDFIEDQGGLEAVR 5.16 0.632 7.68 274 304 NB12 WAS Wiskott-Aldrich syndrome protein IPI00001545 AGISEAQLTDAETSKLIYDFIEDQGGLEAVR 3.79 0.521 7.92 274 304 WASL Neural Wiskott-Aldrich syndrome NB88-V protein IPI00011676 VIYDFIEK 2.29 0.429 0 254 261 WASL Neural Wiskott-Aldrich syndrome NB25-V protein IPI00011676 VIYDFIEK 2.16 0.414 0 254 261 WASL Neural Wiskott-Aldrich syndrome NB88-V protein IPI00011676 ETSKVIYDFIEK 2.27 0.388 0 250 261 WASL Neural Wiskott-Aldrich syndrome NB25-V protein IPI00011676 ETSKVIYDFIEK 2.2 0.477 0 250 261 WDR1 Isoform 2 of WD repeat-containing NB12-2 protein 1 IPI00216256 AHDGGIYAISWSPDSTHLLSASGDKTSK 5.44 0.72 3.96 232 259 WDR1 Isoform 2 of WD repeat-containing NB12 protein 1 IPI00216256 AHDGGIYAISWSPDSTHLLSASGDKTSK 4.32 0.685 8.19 232 259 WDR1 Isoform 2 of WD repeat-containing NB25 protein 1 IPI00216256 AHDGGIYAISWSPDSTHLLSASGDKTSK 4.14 0.556 0 232 259 WDR1 Isoform 2 of WD repeat-containing NB88-2 protein 1 IPI00216256 AHDGGIYAISWSPDSTHLLSASGDKTSK 3.46 0.343 0 232 259 WDR1 Isoform 2 of WD repeat-containing NB25-V protein 1 IPI00216256 AHDGGIYAISWSPDSTHLLSASGDKTSK 3.07 0.543 0 232 259 WDR1 Isoform 2 of WD repeat-containing NB88-V protein 1 IPI00216256 AHDGGIYAISWSPDSTHLLSASGDKTSK 3 0.555 1.17 232 259 WDR1 Isoform 2 of WD repeat-containing NB61 protein 1 IPI00216256 AHDGGIYAISWSPDSTHLLSASGDKTSK 2.24 0.428 0 232 259 NB25-V XYLB xylulokinase homolog IPI00021107 RIHAEGLGYR 2.79 0.383 0 420 429
153 Appendix 1: List of identified phospho-tyrosine peptides in NB-TIC pY dataset
IPI00013981,IPI00166845,IPI00 YES1 Proto-oncogene tyrosine-protein kinase 219012,IPI00639876,IPI006400 IPI00555672,IPI00641230,IPI005 NB12 Yes 91 LIEDNEYTAR pY 426 3.54 0.689 2.96 420 429 15097,IPI00394952,IPI00328867 IPI00013981,IPI00166845,IPI00 YES1 Proto-oncogene tyrosine-protein kinase 219012,IPI00639876,IPI006400 IPI00555672,IPI00641230,IPI005 NB61 Yes 91 LIEDNEYTAR pY 426 3.01 0.666 2.66 420 429 15097,IPI00394952,IPI00328867 IPI00013981,IPI00166845,IPI00 YES1 Proto-oncogene tyrosine-protein kinase 219012,IPI00639876,IPI006400 NB12 Yes 91 KLDNGGYYITTR pY 222 3.61 0.488 3.12 216 227 IPI00013981,IPI00166845,IPI00 YES1 Proto-oncogene tyrosine-protein kinase 219012,IPI00639876,IPI006400 NB61 Yes 91 KLDNGGYYITTR pY 223 2.95 0.601 2.68 216 227 NB122 ZNF282 Zinc finger protein 282 IPI00003798 CSECEKTYSR 0.6 0.776 0 576 585 NB12-2 ZNF317 Isoform 1 of Zinc finger protein 317 IPI00216533 STEYAHLFEVFGMDPHLTQPMGR 1.38 0.386 0 133 155 NB61 ZNF804B Zinc finger protein 804B IPI00328308 ANFYCELCDK 1.13 0.466 0 53 62 NB12 ZNF804B Zinc finger protein 804B IPI00328308 ANFYCELCDK 1.12 0.649 0 53 62 NB88 ZNF804B Zinc finger protein 804B IPI00328308 ANFYCELCDK 0.802 0.589 0 53 62
154