A Thesis
Entitled
Identification of Proteins from Lanthionine Ketimine Ethyl Ester (LKE)- treated and
untreated Rat Glioma 2 (RG2) Cells using Proteomic Approaches
by
Siddhita Abhijeet Shirsat
Submitted to the Graduate Faculty as partial fulfillment of the requirements for the
Master of Science Degree in Chemistry
______Dr. Dragan Isailovic, Committee Chair
______Dr. Donald R. Ronning, Committee Member
______Dr. Jon R. Kirchhoff, Committee Member
______Dr. Amanda C. Bryant-Friedrich, Dean College of Graduate Studies
The University of Toledo
August 2016
Copyright 2016, Siddhita Abhijeet Shirsat
This document is copyrighted material.Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of
Identification of Proteins from Lanthionine Ketimine Ethyl Ester (LKE)- treated and
untreated Rat Glioma 2 (RG2) Cells using Proteomic Approaches
by
Siddhita Abhijeet Shirsat
Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Chemistry
The University of Toledo
August 2016
Glioma is a type of tumor which originates in the glial cells that surround and support neurons in the brain. According to the Central Brain Tumor Registry of the
United States (CBTRUS), 29% of all central nervous system tumors diagnosed are gliomas. Rat glioma models and cell lines reflect high-grade brain tumors. Proteomics is commonly used to discover novel glioma biomarkers and study drug-protein interactionsin rat glioma models and cell lines. Proteomic studies are important because they provide a wealth of information about biochemical properties of brain tumors, and assist in discovery and validation of glioma biomarkers and drug targets.
Here, proteomic techniques were used to explore the differences in the proteomes of malignant rat glioma 2 (RG2) cells in the presence and the absence of a sulphur- containing drug, lanthionine ketimine ethyl ester (LKE). In 2010, Hensley et al. synthesized LK from 3-bromopyruvate and L-cysteine hydrochloride as described by
Cavallini et al., and LKE was synthesized using L-cysteine ethyl ester. It was shown that
LKE promotes neurite elongation in neurons at nanomolar concentrations, protects
iii neurons against oxidative stress, and suppresses microglial activation. Recently, Hensley et al. have analyzed the expression of proteins in RG2 cells that were treated with LKE using mostly immunoblotting techniques. A few proteins that are involved in the process of autophagy (cellular recycling) in RG2 cells were influenced by LKE.
The objective of this project was to apply proteomic techniques and mass spectrometry (MS) to further study the differences in the protein expression levels in
LKE-treated and untreated RG2 cells. Electrophoretic methods (e.g., SDS-PAGE), chromatographic techniques (e.g., nano-HPLC), and MS (e.g., MALDI-MS/MS and ESI-
MS/MS) were used to study proteins and phosphoproteins from RG2 cells. Using nano-
HPLC-MALDI-MS/MS, 22 proteins were identified from LKE-treated RG2 cells, and 18 proteins were identified from untreated RG2 cells. The number of identified proteins was significantly improved using nano-HPLC-ESI-MS/MS; 1681 proteins were identified from LKE-treated RG2 cells and 1457 proteins were identified from untreated RG2 cells.
Specifically, the protein vimentin, which was considered as a potential biomarker in glioma cell line U87 was identified in both LKE-treated and untreated RG2 cells. 53 kinases from untreated RG2 cells and 65 kinases from LKE-treated RG2 cells were identified, including serine/threonine protein kinase mTOR and ribosomal protein S6 kinase (p70S6K), which were previously related to the process of autophagy in RG2 cells by Hensley et al. Overall, the present research results provide a long list of glioma-related proteins that can be detected and identified in LKE-treated and untreated RG2 cells using proteomic technologies. Future studies will aim to quantify the effect of LKE on the expression of glioma-related proteins in RG2 cells using HPLC-MS/MS in order to further explore the mechanism of interaction of LKE with proteins in glial cells.
iv
Dedicated to my caring and supportive husband, my loving parents, and to my wonderful
siblings
v
Acknowledgements
First and foremost, I am thankful to my supervisor Dr. Dragan Isailovic for his constant support and guidance from initial to the final level. I want to thank Dr. Kenneth
Hensley for providing me biological samples for my projects and his constructive advice and ideas. I would like to thank my committee members Dr. Ronning and Dr. Kirchhoff for their valuable advice. I would also like to thank Dr. Leif Hanson for giving me training on how to use the MALDI mass spectrometer. I would like to convey my special thanks to Prof. Kippenhan for guiding me to be a good teacher and helping me to become an organized person and, more importantly, a better teaching assistant.
I would like to thank my all current and former group members for their help in the lab. A special note of thanks goes to Rachel Marvin. The completion of this project could not have been possible without her support. I cannot express enough thanks to
Rachel for her continued support and encouragement throughout these 3 years. I appreciate her help and wish her all the best in her future endeavors. Last but not the least; I would like to thank my Pappa, Mummy, my in-laws, and the most important person in my life my husband and my siblings Rani, Siddhu, Priyanka, for everything they have done. Their unconditional love, constant support, and encouragement always made me more confident and gave me strength to keep moving through all challenging situations.
vi
Table of contents:
Abstract……………………………………...………..…………………………………..iii
Acknowledgments………………………………………………………………………..vi
Table of Contents………………………………………………………………………..vii
List of Tables……………………………………………………………………………...x
List of Figures……………………………………………………………………………xi
List of Abbreviations……………………………………………………………………xiii
List of Symbols………………………………………………………………………….xv
1. Introduction
1.1 LKE: synthesis and neurological effects.……...………………………………1
1.2 Proteomics……………………………………………………………………..4
1.3 Mass spectrometry…………………………………………………………….5
1.4 Ion sources…………………………………………………………………….7
1.4.1 Matrix-assisted laser desorption ionization (MALDI)……...……....7
1.4.2 Electrospray ionization (ESI) and nanoelectrospray ionization
(nano-ESI)………………………………….……………………………11
1.5 Mass analyzers……………………………………………………………….13
1.5.1 Time-of-flight mass analyzer………………………………….…...15
1.5.2 Quadrupole mass analyzer….……………………………………...17
vii 1.5.3 Ion trap mass analyzer……….…………………………………….19
1.5.4 Orbitrap mass analyzer……….……………………………………21
1.6. Separation of proteins and peptides………………………………………...23
1.6.1 Gel electrophoresis.………………………………………………..23
1.6.2 Nano-high performance liquid chromatography (nano-HPLC).…..25
1.6.3 Nano-HPLC fraction collector.…………………………………….26
1.7 Protein identification by peptide mass fingerprinting (PMF).……………….27
1.8 Tandem mass spectrometry………………………………………………….28
1.9 Western blotting.…………………………………………………………….29
1.10 Proteomic studies of glial tissues and cells………….…………………..…30
2. Materials and methodology
2.1 Materials and Instruments…………………………………………………....32
2.2 Methodology.………………………………………………………………...33
2.2.1 Sample preparation.………………………………………………..33
2.2.2 Separation of proteins by SDS-PAGE……………….…………….33
2.2.3 In-gel tryptic digestion and peptide mass fingerprinting (PMF)..…35
2.2.4 In-solution tryptic digestion..………………………………………36
2.2.5 Separation and identification of peptides and proteins by
nano-HPLC-MALDI-MS and MS/MS.………………………………….37
2.2.6 Separation and identification of peptides and proteins by
nano-HPLC-ESI-MS and MS/MS.………………………………………38
viii 3. Results and discussion
3.1 The identification and relative quantification of proteins and phosphoproteins
from LKE-treated and untreated RG2 cells using SDS-PAGE, protein staining and
MALDI-MS.……………………………………………………………………..40
3.2 The identification of proteins from LKE-treated and untreated RG2 cells using
nano-HPLC-MALDI-MS/MS……………………………………………………43
3.3 The identification of proteins from LKE-treated and untreated RG2 cells using
nano-HPLC-ESI-MS/MS…………...……………………………………………47
3.4 Glioma related proteins identified using both nano-HPLC-MALDI-MS/MS
and nano-HPLC-ESI-MS/MS…………..………………………………………..50
4. Conclusion and future work..……………………………………………………...55
4.1 Conclusions…………………………………………………………………..55
4.2 Future work…………………………………………………………………..56
References……………………………………………………………………………….58
Appendices………………………………………………………………………………68
ix
List of Tables
1.1 A comparison of common mass analyzers …………………………………………14
3.1 A list of selected proteins related to glioma and cell proliferation identified in untreated and LKE-treated RG2 cells using both nano-HPLC-MALDI and ESI-MS/MS
………...... 51
3.2 An additional list of selected proteins related to glioma and cell proliferation identified in untreated and LKE-treated RG2 cells using nano-LC-ESI-MS/MS……….52
B.1 A list of proteins identified from untreated RG2 cells in two replicateusing nano-
HPLC-MALDI-MS/MS experiments ...…………………………………………………72
B.2 A list of proteins identified from LKE-treated RG2 cells in two replicatenano-HPLC
MALDI-MS/MSexperiments………………………………………………...... 73
B.3 A list of proteins from untreated RG2 cells identified with ≥5 unique peptides in two replicate nano-HPLC-ESI-MS/MS experiments.………………………………………..74
B.4 A list of proteins from LKE-treated RG2 cells identified with ≥5 unique peptides in two replicate nano-HPLC-ESI-MS/MS experiments..…………………………………..90
x
List of Figures
1-1 Structures of lanthionine (Lan), lanthionine ketimine (LK) and lanthionine ketimine ethyl ester (LKE)…………………………………………………………………………..1
1-2 A general schematic of a mass spectrometer………………………………………….6
1-3 Principle of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-
MS) coupled with time-of-flight mass analyzer…………………………………………..9
1-4 Principle of electrospray ionization mass spectrometry (ESI-MS)………………….12
1-5 A schematic of linear time-of-flight (linear TOF) mass analyzer …………………..16
1-6 A schematic of reflectron time-of-flight mass analyzer……………………………..17
1-7 A schematic of quadrupole mass analyzer…………………………………………..18
1-8 A schematic of 3D ion trap………………………………………………………….20
1-9 A schematic of 2D ion trap……………………………………………………….....20
1-10 A schematic of cross section of orbitrap mass analyzer and C-trap...……………..22
1-11 A schematic showing Biemann nomenclature of peptide fragmentation………….28
3-1 Coomassie stained SDS-PAGE gel showing separation of proteins from untreated RG2 cells and LKE-treated RG2 cells………………………………………...41
3-2 In-gel visualization of phosphoproteins from RG2 cells using SDS-PAGE and phosphorylation-specific Pro-Q Diamond stain..………………………………………..42
3-3 Nano-HPLC separation of peptides originating from untreated RG2 cells…………45
xi 3-4 Nano-HPLC separation of peptides originating from rat glioma (RG2) cells treated with 10 µM LKE …………………………………………..……………………………45
3-5 Nano-LC-ESI-MS/MS base peak chromatogram showing separation of peptides obtained by enzymatic digestion of proteins originating from untreated RG2 cells…………..…………………………………………………………………………..48
3-6 Nano-LC-ESI-MS/MS base peakchromatogram showing separation of peptides obtained by enzymatic digestion of proteins originating from RG2 cells treated with
10 µM LKE…………………...………………………………………………………….48
3-7 Venn diagram showingthe number of proteins identified from LKE-treated RG2 cells and untreated RG2 cells………………….…………………………………………49
3-8 Classification of proteins identified from untreated RG2 cells based on subcellular location. Proteins were identified using nano-HPLC-ESI-MS/MS ……...……………...49
3-9 Classification of proteins identified from LKE treated RG2 cells based on subcellular location. Proteins were identified using nano-HPLC-ESI-MS/MS………………..….....50
A-1 Comparison of MALDI mass spectra of peptides eluting out at tR of 24.83 min in untreated RG2 cells (RG2-LKE) and LKE treated RG2 cells (RG2+LKE)…………….69
A-2 Comparison of MALDI mass spectra of peptides eluting out at tR of 73.75 min in untreated RG2 cells (RG2-LKE) and LKE treated RG2 cells (RG2+LKE)……………..70
A-3 Nano-LC-ESI-MS/MS TIC showing separation of peptides obtained by enzymatic digestion of proteins originating from untreated RG2 cells……………………………...71
A-4 Nano-LC-ESI-MS/MS TIC showing separation of peptides obtained by enzymatic digestion of proteins originating from RG2 cells treated with 10 µM LKE……………..71
xii
List of Abbreviations
ACN…………Acetonitrile APCI………..Atmospheric pressure chemical ionization APS…………Ammonium persulfate
Β-ME………..β-mercaptoethanol
CHCA……….α-cyano-4-hydroxycinnamic acid CID…………. Collision-induced dissociation CI……………Chemical ionization CRMP2……...Collapsin response mediator protein-2
DADPA……....Diaminodipropylamine DHB…………2,5-dihydroxy benzoic acid DIT…………..Dithranol DTT……….....Dithiothreitol
EI……………Electron ionization ESI……...... Electrospray ionization
FA…………...Formic acid FAB…………Fast atom bombardment FBS………….Fetal bovine serum FT-ICR……...Fourier transformion cyclotron resonance
GC…………..Gas chromatography GSH………....Glutathione
HPA…………3-hydroxypicolinic acid HPLC……….High-performance liquid chromatography i.d……………Inner diameter IEF………….Isoelectric focusing IR…………....Infrared IT……………Ion trap
LanCL1……..Lanthionine synthetase C-like protein-1 LC-MS……...Liquid chromatography-mass spectrometry
xiii LDI…………Laser desorption ionization LK………….Lanthionine ketimine LKE………...Lanthionine ketimine ethyl ester LIT……….....Linear ion trap
MALDI…...... Matrix-assisted laser desorption/ionization MRI…………Magnetic resonance imaging MS…………..Mass spectrometry MS/MS…….. Tandem mass spectrometry MW…………Molecular weight m/z…………..Mass-to-charge ratio
PMF…………Peptide mass fingerprinting PTM…………Post-translational modification
Q-TOF………Quadrupole-time-of-flight
RF…………...Resonant frequency RG2…………Rat glioma 2
SA………….. Sinapinic acid SDS…………Sodium dodecyl sulfate SDS-PAGE…Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
TEMED……..N,N,N’,N’-tetramethyl-ethylenediamine TFA…………Trifluoroacetic acid THAP……….Trihydroxyacetophenone TOF…………Time-of-flight TOF/TOF…...Time-of-flight/time-of-flight
UV-Vis…….Ultraviolet-visible v/v…………..Volume/volume w/w…………Weight/weight
2D-GE………Two-dimensional gel electrophoresis
xiv
List of Symbols
α ...... alpha
β ...... beta
λ……..lambda, wavelength
µ……..micro n……..nano
Appendices
A. Mass spectra of peptides eluting out at tR 24.83 min and 73.75 min in untreated RG2
cells and LKE-treated RG2 cells
B. Lists of proteins identified from untreated RG2 cells and LKE-treated RG2 cells
using nano-HPLC-MALDI-MS/MS and nano-HPLC-ESI-MS/MS
xv
Chapter 1
Introduction
1.1 LKE: synthesis and neurological effects
The mammalian brain contains a high concentration of non-proteinogenic, sulfurous amino acid lanthionine (Lan, L-cysteine-S-β-L-alanine, Figure 1-1) and its derivative lanthionine ketimine (LK, Figure 1-1).1 Lanthionine is supposed to form in the mammalian brain from cystathionine-β-synthase via a transsulfuration pathway. In the mammalian brain, it undergoes aminotransferase conversion, yielding a cyclic thioether- type metabolite of lanthionine, LK (2H-1,4-thiazine-5,6-dihydro-3,5-dicarboxylic acid).2
Although lanthionine is present in significant concentrations in the mammalian brain, it has no known biological activities. However, its natural metabolite LK possesses neuritigenic, neuroprotective, and anti-inflammatory activities.2
Figure 1-1. Structures of lanthionine (Lan), lanthionine ketimine (LK) and lanthionine ketimine ethyl ester (LKE).1
1 In 2010, Hensley et al. synthesized LK from 3-bromopyruvate and L-cysteine hydrochloride as described by Cavallini et al.,3 and a derivative of LK lanthionine ketimine ester (LKE, Figure 1-1) was synthesized using L-cysteine ethyl ester.1 LK is a dicarboxylic acid and such acids are not permeable through the cell membranes. To increase the cell permeability of LK, the derivatization of the carboxylate group of LK with ethyl groups was performed, and LKE was synthesized. LKE molecule posesses both hydrophobic and hydrophilic functional groups and is capable of penetrating cell membrane.1 It passes through the cell membrane and blood brain barrier by participating in the specific transport mechansism.1 The enantiomer of LKE with esterification of one carboxylic acid group (Figure 1-1) is synthesized using L-cysteine ethyl ester and 3- bromopyruvate, and is pharmaceutically acceptable.1 It was reported that LKE stimulates neurite elongation in neurons at nanomolar concentrations, protects neurons against oxidative stress, suppresses microglial activation, and protects motor neurons from microglial-dependant toxicity.5,6 It is an orally bioavailable, non-toxic, brain cell- penetrating drug metabolite, and has favorable effects in mouse models of neurodegenerative diseases such as Alzheimer's disease5 and cerebral ischemia.6
The mechanism of action of LKE is not well understood. A previous study indicated that LK might bind selectively to brain proteins, but candidate proteins were not identified.7 Hensley et al. showed that LK binds with high affinity to three proteins.4 LK was bound to a solid phase support column comprised of diaminodipropylamine
(DADPA) agarose matrix and bovine brain protein extract was passed through the column. It was found that lanthionine synthetase C-like protein-1 (LanCL1), glutathione
(GSH)-binding protein, and collapsin response mediator protein-2 or
2 dihydropyrimidinase-like protein 2 (CRMP2/DPYL2) bind selectively to LK and not to the uncoupled solid phase support column. These proteins are binding partners of LK and possibly interact with LK through ionic interactions.4 Among the three proteins, CRMP2 was a highly expressed protein that showed interaction with microfilaments and microtubules. CRMP2 also plays a role in axon elongation and neurite extension.8
Floyd et al. performed initial studies to test the effect of LKE in rat C6 glioma model.9 In their research, LKE was injected intraperitoneally into C6 rats glioma models, and their brain tumor volume was compared with control rats without LKE treatment.
The magnetic resonance imaging (MRI) assessment indicated that glioma growth was reduced in the LKE treated C6 rat glioma model. Floyd et al. reported that LKE slows glioma growth either alone or in combination with neuroprotective phenyl-tert- butylnitrones.9
The most common central nervous system tumors or brain tumors observed in humans are gliomas or glioblastomas.10 Since the rat tumor models reflect high-grade brain tumors and have delivered a significant information about biological and biochemical properties of brain tumors, they have been extensively used in neuro- oncology.11 C6 rat glioma and rat glioma 2 (RG2) models or cell lines are commonly used in laboratories for studying these gliomas. An RG2 tumor was produced in Koestner and Wechsler’s laboratory at The Ohio State University in 1971.12 The RG2 glioma has been used in various studies like vascular permeability,13 in vivo tumor growth and its effects on the blood barrier14, and also in in vivo studies of drug transport.15 Since it is malignant glioma and also exhibits invasive growth pattern, the RG2 glioma model is the
3 best model to study human glioblastoma. Additionally, RG2 cells are readily cultured and provide a simple and reproducible glioma model.11
Based on the previous research,16 it is essential to understand the effect of LKE on protein expression in RG2 glioma cells. Thus in this thesis, RG2 [D74] (ATCC CRL-
2433) cell line was used to study the effect of the drug metabolite LKE on protein expression levels. A combination of proteomics and mass spectrometry is used to identify proteins involved in tumorigenesis. The objective of this project is to find the differences in the protein expression levels of the LKE-treated RG2 cells and untreated RG2 cells, and potentially relate these differences to glioma development in cell cultures and animal models. The differences in the protein levels in LKE-treated RG2 cells and untreated
RG2 cells will be examined by electrophoretic methods (i.e., SDS-PAGE), chromatographic methods (i.e., nano-HPLC) and mass spectrometric methods (i.e.,
MALDI-MS/MS and ESI-MS/MS).
1.2 Proteomics
Peptides and proteins have an essential role in almost every biological process.
The study of peptides and proteins in a biological systemis referred to as proteomics.17
Proteins are multifunctional and highly abundant macromolecules; they serve as catalysts, transporters, structural components of cells and tissues, etc.17 The alteration in the composition of proteins can lead to pathological processes, and this has developed a significant interest in applying proteomics in identifying disease markers. Proteomics involves an investigation of the proteins present in cells, tissue, or an organism.
Proteomics is used for protein profiling, for comparing expression levels of two or more protein samples, for the study of protein-protein interaction and also for the identification
4 of post-translational modifications (PTMs).18 Proteomic projects are complex, because of variations in protein count in the cell, and PTMs of these proteins also increase complexity. The exploration of such a complex system is interesting and involves widely used analytical techniques such as mass spectrometry (MS).
The efficacy of mass spectrometry for analyzing peptides and proteins is based on its capability to ionize them under different conditions and provide accurate molecular weight information.19 Mass spectrometry-based (MS-based) proteomics has developed as an indispensable tool to study protein expression. There are two different approaches in
MS-based proteomics: top-down and bottom-up. The traditional bottom-up approach involves a digestion of protein by a proteolytic enzyme into peptides before MS analysis.20 It is well suited for identification of theproteins. Initially, proteins are separated either by gel electrophoresis or by liquid chromatography. Consequently, a sequence-specific endoprotease, such as trypsin, cleaves proteins.21 The masses of digested peptides generated are used, and protein identification is performed by comparing experimentally obtained masses with theoretical peptide masses of proteins stored in a database via mass search programs.21 Several protein databases containing theoretical protein cleavages, along with the enzyme of choice and post-translational modifications, are available to use. A list of probable matches is given and ranked according to a scoring system depending on the best probability of a match. This sequence-specific MS analysis of peptides is known as peptide mass fingerprinting
(PMF).21
Top-down MS is used for analysis of intact proteins without proteolytic digestion.
It first measures the molecular weight of the protein analyte and compares it with the
5 calculated value of the DNA-predicted protein sequence. The difference between these two values will indicate any post-translational modifications present on the protein.
Subsequently, a specifically modified protein can be isolated in the mass spectrometer and fragmented using MS/MS for mapping of the modification site.22
1.3 Mass Spectrometry
Mass spectrometry is an essential analytical technique in chemistry, biochemistry, pharmacy, and medicine. It is used to provide qualitative and quantitative information. In mass spectrometry, analytes are vaporized, ionized, and analyzed according to their mass- to-charge ratios (m/z).23 A typical mass spectrometer is composed of four parts: a sample inlet, an ion source, a mass analyzer, and a detector. The schematic diagram of the typical mass spectrometer is shown in the Figure.1-2.
Sample Inlet
Ion Mass Source Analyzer Detector
Vacuum system
Computer
Figure 1-2. A general schematic of a mass spectrometer. The diagramwas adapted from reference 23.
6 In the mass spectrometer, the sample to be analyzed is introduced through a sample inlet in the form of liquid or solid or in the form of gas.23 Sometimes, an analyte sample is injected into the mass spectrometer by coupling with different separation techniques such as gas chromatography or liquid chromatography.24 The analyte sample then undergoes ionization and formation of gas phase ions in an ion source. The gas phase ions formed in the ion source are transferred into a mass analyzer, where they are separated based on their m/z values. The magnetic sector, time-of-flight (TOF), quadrupole mass filter, quadrupole ion trap, Fourier transform ion cyclotron resonance
(FT-ICR), and Orbitrap are different types of mass analyzers used in mass spectrometry.
Once the ions are separated using amass analyzer, a detector converts ions into electrical signals. These signals are then graphically displayed as a mass spectrum with the relative abundance of the signals according to their m/z ratios. Electron multiplier and microchannel plates are common detectors that are coupled to the mass spectrometer. The key components of the mass spectrometer are under high vacuum conditions (Figure 1.2).
Vacuum prevents unintentional collisions of the ions with other molecules, which could interfere with spectral analysis.25
1.4 Ion sources
The function of an ion source is to ionize the samples to be analyzed. There are two types of ionization sources: hard ionization and soft ionization. Electron ionization
(EI) is an example of hard ionization source that commonly results in the fragmentation of analytes because of high energy.23 Thus, it is typically used with small molecules. The soft ionization sources include matrix-assisted laser desorption ionization (MALDI), electrospray ionization (ESI), and atmospheric pressure chemical ionization (APCI). Soft
7 ionization sources are less energetic, and likely produce molecular ions.23 Commonly, soft ionization sources are used for biomolecules and other large compounds. Among these, MALDI and ESI are frequently used ionization methods to analyze peptides and proteins.26,27
1.4.1 Matrix-Assisted Laser Desorption/Ionization (MALDI)
MALDI-MS is an effective methodology to analyze proteins, synthetic polymers, oligonucleotides, and other large biomolecules.26 The successful use of MALDI for biochemical analysis was first published by Tanaka et al.28 and Hillenkamp and Karas et al.29 in the late 1980’s. It is a soft ionization sourceand offers non-destructive vaporization and ionization of both small and large biomolecules.26 Proteins with masses up to 300,000 Da have been detected by MALDI-MS, often in the femtomole range.26As compared to other ionization techniques, MALDI is less sensitive to contaminants such as buffers, detergents, and salts. Additionally, it tends to produce singly charged ions predominantly,which makes the spectra less complicated to interpret compared to other ionization sources.23 The fundamental principle of MALDI is summarized as rapid photo- volatilization and ionization of an analyte mixed with UV or IR absorbing matrix followed by time-of-flight mass spectralanalysis as shown in the Figure 1-3.30
The analysis of peptides, proteins, or any other biomolecules using MALDI involves co-crystallization of an analyte with a saturated or nearly saturated solution of matrix typically on a MALDI plate. A variety of matrices assist desorption and ionization of different types of molecules.For example, alpha-cyano-4-hydroxycinnamic acid
(CHCA) is usually used for peptides, sinapinic acid (SA) is used for proteins, 2, 5- dihydroxy benzoic acid (DHB) is used for peptides, proteins, and carbohydrates, while
8 trihydroxyacetophenone (THAP), and 3-hydroxypicolinic acid (HPA) are used for oligonucleotides, and dithranol (DIT) and DHB are used for lipids.23
Figure 1-3. Principle of matrix-assisted laser desorption/ionization mass spectrometry
(MALDI-MS) coupled with time-of-flight mass analyzer.30 Reprinted with permission from reference 30.
MALDI matrix plays a significant role in absorbing photons from UV-laser and causing vaporization of analyte indirectly. Matrix molecules have strong absorption at thelaser wavelength. Matrix also acts as both proton donor and acceptor which allows
MALDI-MS analysis in both positive and negative ion modes, respectively.31 In the absence of matrix, the technique is commonly referred to as laser desorption ionization
(LDI). Most commonly used lasers in MALDI are nitrogen laser (λ= 337 nm), Nd:YAG laser (λ= 266 or 355 nm), Er:YAG laser (λ= 2.94 μm) and carbon dioxide laser (λ= 10.6
μm). The co-crystallized analyte-matrix mixture on MALDI plate undergoes photoexcitation using alaser. This step takes place under high vacuum conditions. The laser irradiation results in volatilization and ionization of analyte-matrix mixture into the gas phase.
9 The mechanism of MALDI has not been fully understood. Two theories are generally used to explain analyte ion formation. The older model is photoionization or
Coupled Physical and Chemical Dynamics (CPCD) model, and the more recent one is the
“lucky survivor” model. According to the CPCD model, the analyte molecules are neutral in the matrix crystals and gain their charge in the gaseous desorption plume by charge transfer from photoionized matrix molecules. The neutral analyte molecules collide with either protonated or deprotonated matrix ions in the gas phase. These collisions cause proton transfer reactions by forming either protonated or deprotonated analyte ions.26 An effective charge transfer results from high proton affinities of basic amino acids as compared to the protonated matrix molecules. Thus, the formation of positively charged analyte ion is favorable. The production of both radical matrix ions and the even-electron ions for DHB and CHCA in the form of M+, [M+H]+, and [M-H]- where M is the molecular mass of the analytecan be evidence of this model.26
The “lucky survivor” model assumes the analytes have a solution charge that they retain when incorporated into the matrix, and these charged ions happen to survive neutralization by retaining their charge. It is also likely that the crystals containing matrix and analyte have precharged analyte with a corresponding amount of counter ions generating no net charge.26 After desorption, the plume expands, and the matrix-analyte cluster loses neutral matrix molecules and solvent. Also, the counter ions undergo proton- transfer neutralization via interaction with analyte sites. Consequently, many of the charges have been neutralized, except for a remaining charge. Thus, the charged analyte is a “lucky survivor” of neutralization.26 This model accounts for the low-intensity negative ion analytes that can be seen using acidic matrices. The low intensity of these
10 ions is explained by the fact that many matrix anions have carboxylates, whose acidity is roughly the same as the analyte carboxylic acid groups. Hence, deprotonation of the analyte would not be favored.26 Also, this model explains the process for both UV and
IR-MALDI.
1.4.2 Electrosprayionization (ESI) and nano-ESI
In 1989, a novel method of ionization that ionizes large biomolecules to produce multiply charged ions was introduced by Fennet al.32 The method is known as electrospray ionization. However, smaller molecules with mass less than 1000 Da may be present as singly charged ions. Like MALDI, ESI is also a soft ionization source in mass spectrometry in which the analyte molecule being ionized does not disintegrate or disrupt during the process.26 As the name implies, ESI utilizes electrical energy pneumatically assistedin producing the droplets. The major advantage of ESI is its ability to be directly
(online) coupled to separation techniques, such as high-performance liquid chromatography (HPLC).
In ESI, a liquid analyte is injected into a stainless steel capillary for ionization. An electric energy of high voltage of 2-6 kV is applied to the steel capillary which results in the formation of the “Taylor cone” and the dispersion of sample analyte into an aerosol of electrospray droplets.33 The stainless steel capillary is surrounded by a sheath gas (dry
N2) flow which helps in improved nebulization and also aids in directing aspray of ions towards the mass spectrometer. The elevated temperature of the source causes evaporation of solvent and reduction in the size of charged droplets by the assistance of nitrogen gas flow.33 As the size of the droplet (radius) decreases, it experiences an increase in the surface charge density. A critical point is reached, which is referred as
11 “Rayleigh limit”, and the droplets are transforming into the gas phase by generating ions.
At this point, the columbic repulsion overcomes the surface tension resulting from large surface charge density.34 The schematic of the basic principle of electrospray ionization is shown below in Figure 1-4.
Various other sprayer modifications like pneumatically assisted electrospray35, ultrasonic nebulizer electrospray,36 electrosonicspray,37 and nanoelectrospray34 have been developed in the last 20 years. The most popular among them is nanoelectrospray.
Figure 1-4. Principle of ESI-MS38 Reprinted with permission from reference 38.
A key advantage of nanoelectrospray ionization (nano-ESI) over standard ESI is small sample volume and flow rate. Standard ESI mass spectrometers use flow rates of 1-
300 μL/min while nano-ESI mass spectrometers usea low flow rate of 20-1000 nL/min which reduces waste generation.34 Also, it has ahigher sensitivity which allows samples down to attomol amounts to be analyzed. The low sample concentration and a small sample volume in nano-ESI are obtained by replacing the electrospray needle in standard
12 ESI with a glass capillary of the inner diameter (i.d.) of 10-75 μm. Moreover, the distance between the capillary and the entrance of the mass analyzer is decreased to 0.5-2 mm.34
Although nano-ESI appear to be a minimized version of standard ESI, it uses an altered mechanism that leads to important differences in its use. Standard ESI produces droplets with 1-2 μm in diameter; however, nano-ESI generate droplets with a diameter of ~150 nm. Nano-ESI is easily coupled with nano-HPLC.39 It is advantageous over standard ESI, in thecase of limited sample availability. For example, when 1 µL of a 10-6
M sample analyte solution is injected, this 1 pmol of sample is typically sufficient for 0.5 h measurement time, which enables many experiments to be performed.34 The nano-ESI source with a gold-coated pulled glass capillary emitter with an orifice i.d. of 1-2 µm results in an increase in the stability of electrospray and shows an improvement in ESI-
MS performance.39 A significant increase in MS intensities of detected species was observed when a nano-ESI source was used instead of standard ESI source.39 Also, the elution reproducibility level of nano-HPLC/nano-ESI-MS system is high, and it allows the identification of components of complex mixtures in an efficient way.39
1.5 Mass analyzers
The mass analyzer plays a central role in themass spectrometer since it is a place where gas phase ions from the ionization source are separated according to their m/z ratios. Mass spectrometers can have one mass analyzer or multiple mass analyzers coupled together. Additionally, ion fragmentation can be performed in a mass analyzer.23
Typically, mass analyzers use electric and magnetic fields to separate ions based on their m/z ratios. Mass analyzers are generally classified into two types: 1) scanning analyzers that allow only certain masses to traverse through them, and eventually, ions get detected
13 (e.g., magnetic sector and quadrupole), and 2) simultaneous transmission analyzers that allow transmission of all the ions through the mass analyzer toward the detector (e.g., time-of-flight (TOF), ion trap (IT), ion cyclotron resonance (ICR), and Orbitrap).23 To increase resolution and permit multiple experiments to be performed at once, a trend of combining two or more mass analyzers in a sequence has been developed.25 Hybrid mass spectrometers such as quadrupole-time-of-flight (Q-TOF), triple quadrupole, linear ion trap (LIT)-orbitrap, and Orbitrap Fusion (MS which combines quadrupole, orbitrap, and ion trap mass analyzer) are most prominently used because of their high resolution, extended mass range, and high sensitivity.
Five features measure the performance of each mass analyzer: mass range, transmission, analysis speed, resolution and mass accuracy. Based on these characteristics, mass analyzers are selected for analyzing different molecules. A table comparing different mass analyzers used in this thesis is given below.
Table 1.1. A Comparison of Common Mass Analyzers.23,40
TOF Reflectron Quadrupole Ion Trap Orbitrap
TOF
Mass limit >1,000,000 10,000 4000 6000 6,000
Resolution 5000 20,000 2000 4000 450,000
Accuracy 200 ppm 10 ppm 100 ppm 100 ppm <3 ppm
Ion sampling Pulsed Pulsed Continuous Pulsed Pulsed
Pressure 10-6 Torr 10-6 Torr 10-5Torr 10-3 Torr 10-10 Torr
14 1.5.1 Time-of-flight mass analyzer
Time-of-flight mass analyzers use pulsed ion sampling, which enables them to be coupled with a pulsed ionization source, such as MALDI.23 As its name suggests, the mass of analyte ion is determined by the time it passes through the flight tube. Linear and reflectron TOF’s are two types of TOF mass analyzers.
A linear TOF consists of generated ions that are accelerated towards a mass analyzer and acquire the same kinetic energy. These analyte ions with the same kinetic energy enter a field-free region of the flight tube where they are separated based on their velocity; the velocity with which analyzedions move in the flight tube depends on their
23 m/zratios. The kinetic energy of an analyte ion Ek, having amass of m, and total charge of q, which is the number of charges, z, times the charge of an electron, e, and accelerated by a potential of Vs is given by equation 1.
푚푣2 퐸 = = 푞푉 = 푧푒푉 (1) 푘 2 푠 푠
Since all analyte ions have the same kinetic energy, their travel through the flight tube is given by their velocity and eventually by their m/z ratio. Small analyte ions with low m/z move at a faster rate and reach the detector rapidly, while large analyte ions with higher m/z move at a slower rate and reach the detector slowly. Therefore, Equation 1 can be rearranged to Equation 2 to determine an ion’s velocity.
1/2 푣 = (2푧푒푉푠⁄푚) (2)
If the flight tube length is labeled with L, time (t) that takes for an analyte ion to pass through the flight tube and reach the detector can be given by equation 3.
퐿 푡 = (3) 푣 15 By replacing v by its value in Equation 3 it gives Equation 4
푚 퐿2 t 2 = ( ) (4) 푧 2푒푉푠
The Equation 5 shows that m/z can be calculated by rearranging Equation 4
푚 2푒푉 = ( 푠) t 2 (5) 푧 퐿2
A schematic of linear TOF is shown in Figure 1-5.
Figure 1-5. A schematic of linear time-of-flight (linear TOF) mass analyzer. The diagram was adapted from reference 23.
Linear TOF mass analyzers have numerous advantages. One of them is a huge mass range which makes it appropriate for the analysis of large biomolecules by soft ionization techniques. For example, MALDI-TOF has analyzed samples with masses greater than 300,000 Da.23 Also, linear TOFs have very high sensitivity because of their high transmission efficiency. The detection of 100 attmole of a protein by linear TOF-MS has been observed in previous studies.23
However, there are some disadvantages for the linear TOF mass analyzer. Its main disadvantage compared to other mass analyzers is its low resolution.23 This disadvantage was overcome by employing a reflectron TOF. In a reflectron TOF, the analyte ions travel through a field free region similar to linear TOF. A series of electrodes
16 referred to as reflectron is placed at the opposite end of the ion source, and reflects the ions back into the flight tube.23 There are two ways to position the detector, but the most common is an “off-axis” position. In which, the reflectron deflects the ions at a small angle away from the ion source that allows the detector to be placed next to the ion source. A general schematic of reflectronTOF is shown in Figure 1-6.
Figure 1-6. A schematic of reflectron time-of-flight mass analyzer. The spheres represent two ions with the same mass-to-charge ratios. The ion represented with open circle has higher kinetic energy than the ion represented with ashadedcircle. The diagram was adapted from reference 23.
Ions with the same m/zratio and slight kinetic energy dispersion enter TOF together. The purpose of reflectron is to correct this dispersion.23 Ions with more kinetic energy enter deeper into the reflectron than the ions with lower kinetic energy. As a result, the faster ions spend more time in the reflectron and reach the detector at the same time as slower ions with the same m/z ratio. Thus, without increasing the dimensions of the mass spectrometer, the reflectron increases the flight path. Eventually, the reflectron increases the mass resolution in comparison to linear TOF, but its sensitivity and mass range are limited in comparison with linear TOF mass analyzer.23
17 1.5.2 Quadrupole mass analyzer
A quadrupole mass analyzer is a scanning mass analyzer. It is also known as a
‘mass filter’ that filters out gas phase analyte ions based on their m/z ratio and allows transmission of the selected ions.23 It consists of four perfectly parallel circular metal rods to which potential and electrical fields are applied.23 All the metal rods have an applied radio frequency (RF) voltage. The metals rods are oppositely charged; one pair has negative voltage while the other has positive. For an ion to be detected, its trajectory must be stable through the alternating fields of quadrupole as shown in Figure 1-7. If an ion does not have a stable trajectory, it hits the rod, becomes discharged and is not detectable.23
Quadrupole mass analyzers have a low mass range that is often under 4000 m/z.23Also, they are considered as low resolution instruments. However, this makes them useful by serving as ion transmission guides when only an RF voltage is applied.23
Figure 1-7. A schematic of quadrupole mass analyzer.23 Reprinted with the permission from reference 23.
18 This also allows multiple quadrupole mass analyzers to be used in tandem, where one quadrupole serves as a collision cell for fragmentation of the ions. It is also coupledwith other mass analyzers, especially TOF in Q-TOF instruments.
1.5.3 Ion trap mass analyzer
Ion trap (IT) belongs to the type of mass analyzer that permits transmission of all ions simultaneously. It traps the gas phase ions and measures their m/z ratios. Ion trap mass analyzers can be classified into 2D and 3D ion traps. The 3D ion trap was developed before the 2D ion trap.23 The 2D ion trap is commonly known as a linear ion trap (LIT), and the 3D ion trap is known as quadrupole ion trap. The ion trap permits
MSnexperiments to be executed to obtain multiple fragmentations.
A 3D ion trap consistsof one circular electrode and two ellipsoid end cap electrodes (Figure 1-8). A potential is adjusted on the electrodes for the ions to enter the
3D ion trap. Ions with multiple masses are stored in the trap at the same time, and they are ejected from the trap using a resonant frequency to obtain the mass spectrum.23
Precisely, to inject positively charged ions into the ion trap, a negative potential is applied.23 Too many ions in the trap cause a loss ofresolution and too few ions results in the loss of sensitivity; hence, the number of ions entering the trap is controlled. Since the ions repel each other, their trajectories expand in the ion trap. The ion losses by this expansion are avoided by utilizing helium gas which collides with the ions and reduces their energy. Moreover, an RF voltage is applied to the circular electrode with constant frequencyand varying amplitude. Also, RF voltages are applied to the end caps with the selected frequencies and amplitudes. The 3D ion traps experience space charge effect if
19 too many ions are introduced into an ion trap. The ions on the outside act as a shield, which results in modification of the ions in the middle.
Figure 1-8. A schematic of 3D ion trap.23 Reprinted with the permission from reference 23.
A 2D ion trapis an analyzer comprised of a quadrupole ending with lenses that repel the ions inside the rods (Figure 1-9). By the means of the quadrupolar field, the ions are confined in the radial dimension. The ions oscillate in the xy plane of 2D ion trap due to applied RF potential on the rods similar to the quadrupole. Additional application of the DC voltage at the end part of the quadrupole allows the ions to be trapped.23
20
Figure 1-9. A schematic of 2D ion trap (LIT).23 Reprinted with permission from reference 23. A represents front section of the LIT, B represents acenter section of the
LIT, C represents back section of LIT, while D1 and D2 show the positions of the MS detectors.
The 2D ion trap has ten times higher ion trapping capacity and higher trapping efficiency compared to the 3D ion trap. The 2D ion trap is less vulnerable to space charging effect. Ion trapping efficiency of the 2D ion traps is more than 50 % compared to 5 % in the 3D ion trap. These advantages of the 2D ion trap result in increased sensitivity and dynamic range. The trapped ions in LIT can be ejected via two modes: axial ejection or radial ejection. In axial ejection, AC voltage is applied between the rods and the exit lens; however, in radial ejection, AC voltage is applied to the opposite rods in which the slots have been hollowed.23
1.5.4 Orbitrap mass analyzer
Orbitrap is a simultaneous mass analyzer, which is superior interms of mass resolution compared to ion trap mass analyzers.41 It was introduced commercially in 2005 and a compact design with high field trap version was released in 2011. Orbitrap is coupled with other mass analyzers and is part of a hybrid mass spectrometer in various
21 commercial instruments. For example, in Thermo’s LTQ-Orbitrap it is coupled to a linear ion trap. High resolving power and mass accuracy are the two main advantages of the
Orbitrap, which makes it a powerful analyzer in the field of bottom-up proteomics. It helps to reduce false positive peptide identifications significantly. Thermo’s LTQ-
Orbitrap has a resolving power greater than 150,000 and a mass accuracy of a 2-5 ppm. A schematic of an Orbitrap mass analyzer is shown in Figure 1-10.
Figure 1-10. A Schematic of the cross section of an Orbitrap mass analyzer and
C-trap.41 Reprinted with permission from reference 41.
An orbitrap consists of three electrodes. Two outer electrodes are cup shaped and face each other creating a barrel-likeshape, and the third electrode is an axial inner spindle like electrode. The two cup-shaped electrodes are electrically separated by a hair- thin gap and secured by a central ring made of dielectric. The conventional ion traps use
RF voltage to trap the ions in the cavity, whereas the Orbitrap uses an electrostatic field to trap ions. This electrostatic field forces ions to move in a spiral pattern around the
22 spindle electrode. A spindle-like central electrode holds the trap together and aligns it via dielectric end-spacers. Voltage is applied to the outer and central electrodes for analysis, resulting in a linear electric field along the axis of thecentral electrode with purely harmonic oscillations.41
In 2013, a novel tribrid mass spectrometer named Orbitrap Fusion (Thermo
Fisher) that combines three mass analyzers (quadrupole, ion trap, and orbitrap mass analyzers)was introduced.It has very high resolution (up to 450,000), and very high mass accuracy (1-5 ppm) as compared to traditional mass spectrometers.42 Since, Orbitrap
Fusion contains three mass analyzers, an increase in MS/MS acquisition rates and peptide identification rates are observed. The ion losses are minimized in Orbitrap Fusion compared to some previous versions of this mass spectrometer, because of a reduction in the distance between the ion source and mass analyzer.42 Also, an increase in the number of identification of peptides derived from low abundance proteins is observed compared to earlier hybrid mass spectrometers.42
1.6 Separation of proteins and peptides
1.6.1 Gel electrophoresis
Electrophoresis is a powerful analytical tool that describes the process by which sample molecules migrate in thepresence of an electric field. Polyacrylamide gel electrophoresis (PAGE) is used to carry out electrophoresis of proteins.43 Gels used in polyacrylamide gel electrophoresis are made of acrylamide that is mixed with bis- acrylamide to make a cross-linked network by free radical polymerization. The polymerizing agent used is ammonium persulfate (APS), which forms persulfate free radicals when it is dissolved in water and also activates the acrylamide monomer.43 The
23 N,N,N’,N’-tetramethylene diamine (TEMED) is also added, which acts as a catalyst for accelerating the free radical polymerization reaction because of its ability to carry electrons. The reaction of activated acrylamide monomer with inactivated monomer results in the formation of the long polymer chain. These elongated polymer chains become cross-linked randomly by bisacrylamide (cross-linker) and form a network of acrylamide chains.43 The polyacrylamide gel is made of pores, and the pore size depends on the percentage of bisacrylamide. Proteins loaded on the gel migrate quicker through the gel with large porescompared to gels with smaller pore size, e.g., gel with a concentration of 10% of acrylamide has larger pore size than a gel with aconcentration of
15% of acrylamide.44 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE) is a common form of electrophoresis. Before the electrophoresis, proteins to be separated are heated in sample buffer containing an anionic detergent, sodium dodecylsulfate (SDS), and β-mercaptoethanol (β-ME) or dithiothreitol (DTT) as a reducing agent. SDS denatures proteins before loading on the gel that causes conversion of thesecondary, tertiary and quaternary structure of protein into a linear shape and masks the protein with anegativechargeso that proteins can migrate in an electric field as afunction of their molecular weight (MW), while β-ME reduces the disulfide bonds. The denatured proteinsthen migrate on apolyacrylamide gel and separateelectrophoretically based on their apparent MW and also based on their charge.44
However, in 1975, O’Farrell45 and Klose46 introduced another form of electrophoresis, i.e., a high-resolution method to separate proteins electrophoretically by two independent dimensions: MW and isoelectric point (pI). It is referred as two- dimensional gel electrophoresis (2D-GE). By using two independent parameters, it allows
24 proteins to be separated on the entire rectangular surface of the gel rather than distributing across a diagonal.44 Separation of proteins by the 1st dimension is performed by isoelectric focusing (IEF) in a pH gradient. It is a very sensitive method which separates proteins based on their charges. By applying the electrical field, the proteins carrying positive charge migrate towards the negative electrode (cathode) and negatively charged proteins migrate towards the positive electrode (anode).44 As proteins migrate, their net charges slowly decrease and reach a point at which proteins have zero net charge. This point is known as isoelectric point. After the separation of proteins based on their pI, they become employed on SDS-PAGE for separation based on their MW.
The 2D gel electrophoresis has some shortcomings, such as separation of poorly soluble membrane proteins, very acidic or basic, or very large or small proteins which may be absent on 2D gels. Sometimes, low abundance proteins can be masked by high abundance proteins.44
1.6.2 Nano-high performance liquid chromatography
Nano-high performance liquid chromatography (nano-HPLC) is a miniaturized chromatographic separation technique. It is an environmentally friendly method which minimizes consumption of reagents and generation of waste as compared to conventional
HPLC.47 It becomes easily coupled with ESI-MS to achieve minimal sample consumption and better sensitivity for small amounts of molecules extracted from biological tissues, cells, and any other resources.47 An advantage of nano-HPLC over conventional HPLC involves small sample volume. Generally, samples at microliter to nanoliter range are injected via an autosampler in the nano-HPLC system.47 The analyses using a conventional HPLC system involves the use of columns with an inner diameter
25 (i.d.) of 1.0 - 4.6 mm and flow rates of up to 1 mL/min. However, the nano-HPLC system makes use of columns with i.d. of 10-100 µm and flow rates of 100-500 nL/min.47
Specifically, nano-HPLC columns with 75 µm i.d. are most often used as they offergood detection, loading, and robustness in separations.48 Nano-HPLC systems use similar types of detection methods as conventional HPLC systems. A UV-vis absorption detector is used in nano-HPLC due to its low cost, wide range applications, and online detection.47 A mass spectrometer is commonly used as a nano-HPLC detector in proteomics due to its sensitivity and ability to identify and quantify biomolecules efficiently. In theory, the nano-HPLC system is used to promote analyte enrichment more than conventional
HPLC. In nano-HPLC, the reduced column i.d. decreases the dilution factor, which results in an increase in analyte concentration and, eventually, an increase in analyte detectability compared to conventional HPLC.47
1.6.3 Nano-HPLC fraction collector
The nano-HPLC fraction collector (e.g., Bruker Daltonics Proteineer fc) facilitates automatic liquid handling for MALDI preparation of LC separated molecule fractions. It is an automated fraction collection system which is connected to a nano-HPLC by a fused-silica capillary. Nano-HPLC fractions of isolated molecules are collected continuously and distributed evenly on a 384 spot AnchorChip target plate (Bruker
Daltonics) with a programmed spotting time and movement pattern.49 An online matrix mixing with an analyte is also enabled by coupling an external syringe pump to the spotter. The AnchorChip is a stainless steel MALDI target plate with 600 µm hydrophilic anchor spots surrounded by a hydrophobic coating. The hydrophobic spotting prevents the sample from spreading. About 0.5-2 µL of analyte/matrix mixture is spotted on one
26 AnchorChip spot. The droplets anchor themselves to the target plate after evaporation of a solvent from matrix and analyte.49 In addition to 384 spots, there are 96 calibration spots on one AnchorChip target plate.49 The MALDI plate with spotted fractions is then subjected to mass spectrometric analysis using a MALDI-TOF/TOF mass spectrometer.
1.7 Protein identification by peptide mass fingerprinting (PMF)
PMF is used for the identification of proteins by mass spectrometry. It is an efficient method of identifying proteins separated by gel electrophoresis (SDS-PAGE and
2D-gel electrophoresis). In this technique, protein bands of interest are isolated and enzymatically digested, commonly with trypsin.MALDI-MS analyzes the resulting peptides. After obtaining the mass spectrum, the obtained masses of tryptic peptides are searched against in silico digest of all the proteins in the given database.21
The most common program to perform PMF is MASCOT, which is a probability- based scoring search.50 This probability based scoring is used to calculate the likelihood that observed match of the experimental data is a chance event. The common threshold of p < 0.05 is used. However, for a large database, the probability of a match becomes a very smallnumber, so the probability is converted to a score, where the score is -
50 10Log10(probability). Several factors influence the results of PMF, such as a difference in ionization of the peptides. Specifically, tryptic peptides containing arginine are more intense than peptides containing lysine.21 Another factor is that the digest may contain modified peptides which must be accounted for in the database search. However, as the number of modifications increases in a search, the number of possible random matches increase, which eventually increases the number of false positives.21 Confidence in the identification then depends on the species studied, the mass and pI of the protein
27 observed on a gel, the number of peptides matching a particular protein sequence, the mass accuracy of detection, the protein sequence coverage reached, the number of missed cleavages during the proteolysis process and also the type of modifications observed, which can reflect how the sample is processed.21
1.8 Tandem mass spectrometry
Tandem mass spectrometry (MS/MS) is an essential tool in proteomics, especially in finding the location of post-translational modification, as further fragmentation can help reveal the protein structure. Collision-induced dissociation (CID) is the most common type of fragmentation. Ions are accelerated into the CID cell with a high amount of kinetic energy, and when the ions collide with neutral gas molecules in the cell, their kinetic energy is converted into vibrational energy, which leads to fragmentation of the bonds of the parent ion.23 It can be a high-energy process, which is normally seen in TOF mass analyzers, or it can be a low-energy process which is common in quadrupole mass analyzers.51
Different types of ions are observed in an MS/MS spectrum, and they depend on the composition of the protein/peptide, the amount of internal energy that is transferred, and the ion activation method.23 To denote the types of ions that are formed, Biemann createda widely used nomenclature to describe the fragmentation patterns.52 A schematic representing Biemann nomenclature is shown in Figure 1-11.
28
Figure 1-11. A schematic showing Biemann nomenclature peptide fragmentation. The diagram was adapted from reference 52.
As the peptide fragments internally, a-, b-, and c-ions are observed when the charge is retained on the fragment’s N-terminus, and x-, y-, and z-ions are observed when the charge is retained on the fragment’s C-terminus. In CID, the kinetic energy transfer will fragment the peptide at the most energy-efficient location. In the peptide, this will be the carbon-nitrogen amide bond, which means the predominant fragments will be b- and y-ions.52
1.9 Western blotting
Western blotting (immunoblotting) is a technique used for identification and quantification of target proteins.53 This technique uses antibodies for identification of target proteins among a group of distinct proteins. Specific reactions of antigen-antibody commonly identify the target protein.54 Cell lysates are the common form of samples used for the western blot technique. In this technique, the mixture of proteins is separated by gel electrophoresis and transferred to either a polyvinylidene fluoride (PVDF) or nitrocellulose membrane.53 While transferring the separated proteins onto a membrane, the closure contact between gel and blotting membrane is needed. To avoid nonspecific binding of theantibody to the membrane, blocking is a key step of western blot.53 The membraneis detected by labeling the antibody with an enzyme such as horseradish
29 peroxidase (HRP), which is detected by the signal it produces corresponding the position of the target protein.53 Since, antibody binds to only specific protein or protein of interest, only the band corresponding to that protein isvisible, and thickness of the band provide the quantity of protein present in sample.53
1.10 Proteomic studies of glial tissues and cells
Proteomics has been used to study glioma at different levels including human biopsies and body fluids as well as animal modelsand cell lines.55 To identify potential biomarkersof glioma, differential proteomic profiling techniques have been used, and various markers have been identified in the past.56 However, due to the limited number of identified markers and vagueness in their reproducibility, those markers are not ready for clinical use.56 Recent developments in technological methodologies for high-throughput profiling, including mass spectrometry-based proteomics, are predicted to speed up glioma biomarker discovery.56 Along with amino acid sequences of proteins, proteomics specifically provides information about PTMs such as phosphorylation, glycosylation, acetylation, and ubiquitylation. These PTM’s reveal disease status more effectively.55
Commonly, there are two types of proteomics technologies used to study gliomas: gel-based proteomics and LC-MS/MS-based proteomics. Niclou et al. stated that majority of glioma-related proteomics studies until 2008 involved the use of gel-based proteomics.
It was also mentioned that only medium and high abundance proteins were identified using gel-based proteomics.55 However, LC-MS/MS-based proteomics coupled with either ESI or MALDI can lead to the identification of thousands of proteins.
Phosphorylation of glioma proteins has been studied as well. For example, Shinojima et al. examined the effect of epidermal growth receptor (EGFRvIII) on the expression level
30 of phosphorylated proteins in order to understand phosphotyrosine-mediated cellular signaling networks. The proteins were extracted from the cells and digested into peptides.
Among the four cell lines used in this method, they were able to quantify 99 phosphorylation sites within 69 proteins.57
Recently, Hensley et al. cultured RG2 cells in the presence and absence of LKE and studied the influence of LKE on the expression of proteins in RG2 cells.16 They performed immunoblotting experiments and observed changes in the protein level and phosphorylation states of a few proteins after treatment of RG2 cells with LKE. The proteins affected by LKE include a mammalian target of rapamycin complex-1 (mTOR), ribosomal protein S6 kinase(p70S6), unc-51-like kinase-1, beclin-1, and microtubule- associated protein 1 light chain 3 (LC3). Furthermore, Hensley et al. stated that the lanthionine derivatives are not metabolic waste but important small moleculesinvolved in stimulation of autophagy in RG2 cells.16 Autophagy is a metabolic process where damaged proteins, lipid deposits, and entire organelles get recycled by the cells.16 In the process of autophagy, a double-membrane cytoplasmic vesicle, i.e., autophagosome forms from phagophore by conjugation with protein LC3.58 LC3 controls the expansion of phagophore and assists in completing the formation of autophagosome engulfed with cellular components.58 Next, autolysosome forms by fusing the autophagosome with thelysosomeand lysosomal degradation of componentsoccur. This degradation assists removing damaged cellular organelles and recovering nutrients related to autophagy.58
Since, autophagy is an important process in mammalian physiology, deterioration in autophagy can lead to various human diseases.
31 Encouraged by the previous results,16 the research described in this thesis aims to identify more proteins and further uncoverpotential changes in the proteins expressed in
RG2 cells that were treated with LKE. The application of sensitive and selective proteomics and phosphoproteomics based techniques may lead to the discovery of novel protein targets59 whose expression is affected by LKE.
32
Chapter 2
Materials and methodology
2.1 Materials and instruments
Glacial acetic acid, HPLC-grade acetonitrile (CH3CN), ammonium persulfate
(APS), methanol and sodium acetate were obtained from Fisher Scientific (Pittsburgh,
PA). Ammonium bicarbonate, β-mercaptoethanol, dithiothreitol (DTT), iodoacetamide
(IAM), trypsin from bovine pancreas, trifluoroacetic acid (TFA), formic acid (FA) and
LC-MS-grade water were purchased from Sigma (St. Louis, MO). Bio-safe Coomassie
Brilliant Blue G-250 stain, Laemmli sample buffer and 10% (w/v) SDS were from Bio-
Rad. For polyacrylamide gel casting, 30% acrylamide/bis solution (29:1), 1.5 M Tris-HCl
(pH 8.8), 0.5 M Tris-HCl (pH 6.8) and TEMED were also obtained from Bio-Rad.
Alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix was obtained from Bruker
Daltonics.
Bruker’s MALDI-TOF/TOF UltrafleXtreme (equipped with pulsed 355nm
Nd:YAGlaser) was used to analyze the samples.60 FlexControl (version 3.4, Bruker
Daltonics) software was used to acquire mass spectra in positive ion reflectron mode.
Calibration was done prior to data acquisition using peptide standard obtained from
Bruker Daltonics containing angiotensin II (m/z 1046.5418), angiotensin I (m/z
1296.6848), substance P (m/z 1347.7354), bombesin (m/z 1619.8223), ACTH fragment 1-
17 (m/z 2093.0862), ACTH fragment 18-39 (m/z 2465.1983) and somatostatin (m/z
33 3147.4710). GE Healthcare Life Sciences Typhoon Trio was used to scan gel stained with ProQ Diamond phosphoprotein gel stain. Orbitrap Fusion MS and nano-LC were from Thermo Fisher, while gel electrophoresis equipment was from Bio-Rad.
2.2 Methodology
2.2.1 Sample Preparation
The rat glioma RG2 [D74] (ATCC® CRL-2433™; RG2 cells) cell lysates employed in this project were obtained from our collaborator Dr. Kenneth Hensley from
Department of Pathology (The University of Toledo, College of Medicine). These RG2 cells were cultured in Dr. Hensley’s lab at 37 °C inair containing 5% CO2 using
Dulbecco's Modified Eagle's Medium accompanied by fetal bovine serum (FBS) and penicillin/streptomycin. The cells were plated in tissue culture-treated dishes, and the medium was changed every 2–3 days until the percentage of cell growth became 85–
90%. For cell growth with LKE, LKE was dissolved in saline,neutralized, and diluted into culture medium to get desired final concentrations. The medium in each of the dishes was replaced with medium containing LKE in saline. Cells were left to equilibrate for 15 min, then bafilomycin-A1 (10 nM; LC Laboratories) was added, and cells were incubated for anadditional 4 h. At termination, the culture medium was removed; cells were washed with PBS (Sigma) and lysed on ice in RIPA buffer (Thermo Scientific) containing protease and phosphatase inhibitors. The protein concentrations of these cells lysates were measured by Lowry method, and they were determined to be~ 7-8 µg/µL for each sample.16
34 2.2.2 Separation of proteins by SDS-PAGE
For SDS-PAGE, RG2 cells treated with different concentrations of LKE (1 μM,
10μM, and 100 μM) and untreated RG2 were prepared in a similar manner. Initially, 10
μL of each cell lysate was combined with 9.5 μL of Laemmli sample buffer and 0.5 μL of
β-mercaptoethanol. Hence, the total sample volume of 20 μLwas loaded on the gel.
Before loading, samples were heated at ~90 ºC for five minutes to denature the proteins,after which samples were cooled to room temperature. Denatured proteins in the samples were separated using 12% acrylamide gels ran at 175 V for approximately 50 mins. Two gels were run under identical conditions. For visualization of total proteins, the gel was stained with colloidal Coomassie blue G-250 (Bio-Rad) for 1-2 hours and destained in deionized water overnight. Finally, the stained gel was scanned using a desktop scanner (HP Scanjet 6300C) with maximum resolution (1200 dpi).
For visualization of phosphoproteins in samples, another gel was stained with
Pro-QDiamond phosphoprotein gel stain by following company’s protocol
(Thermo/Invitrogen). Pro-Q Diamond phosphoprotein gel stain is a fluorescent stain, which selectively stains phosphoproteins on SDS-PAGE gel. The staining process initiates with fixation of the gel. For fixation, the gel was immersed in asolution containing 50% methanol and 10% acetic acid and agitated gently for 30 mins. The fixation step was repeated one more time to make sure all SDS was washed out of the gel.
Next, the gel was transferred to the water to remove methanol and acetic acid completely since they could interfere with the staining process. The washing was performed for 10 mins with gentle agitation. Then, staining of the gel was carried out by immersing the gel into Pro-Q Diamond gel stain with shaking it on a rocker in the dark for 90 mins. To
35 reduce background staining, destaining was accomplished by incubating the gel in a solution containing 20% acetonitrile and 50 mM sodium acetate with pH 4.0 for 30 mins in the dark. The destaining process was repeated twice. Finally, the gel was washed twice in water for 5 mins per wash. To visualize the phosphoproteins, the stained gel was scanned using GE Healthcare Life Science’s Typhoon Trio at an excitation wavelength of
532 nm and anemission wavelength of 580 nm.
2.2.3 In-gel tryptic digestion and peptide mass fingerprinting
After separation of proteins by SDS-PAGE, protein bands were subjected to a standard in-gel digestion protocol as described by Shevchenko et al.61 Each Coomassie stained protein band was excised from the gel, and cut into ~1 mm3cubes which were placed into a microcentrifuge tube. The excised gel bands comprising of stained proteins were destained using 100 μL of 100mM NH4HCO3:CH3CN (1:1 v/v) for 30 minutes with occasional vortexing. Subsequent addition of 500 μL of neat CH3CN for 10 minutes was required for dehydration of the pieces. The gel pieces turned white and shrank at that point. Next, all solvent was removed from the microcentrifuge tube, and 50 μL of
13ng/μL trypsin solution in 10 mM NH4HCO3 was added to cover gel pieces completely.
The samples were then kept in the fridge for two hours to saturate them with trypsin.
Later, 15 μL of 100 mM NH4HCO3 was added to gel pieces before transferring the microcentrifuge tubes for incubation to a 37 °C water bath overnight (~15 hours).
The trypsin-digested protein bands were identified using PMF. About 1 μL of the digest was co-spotted with CHCA matrix (10 mg/ml in 60:40 (v:v) CH3CN:H2O containing 0.1% TFA) onto an MTP 384 ground steel target plate (Bruker Daltonics).
Analysis of tryptic peptides was performed using MALDI-MS in positive ion mode from
36 m/z 650 to m/z 4000. The mass spectra were analyzed using FlexAnalysis software
(Bruker Daltonics). Proteins were identified using a MASCOT search (Matrix Science)50 and Swissprot database of the tryptic peptides. The taxonomy was set to Rattus
Norvegicus with peptide mass tolerance of 0.2 Da, and one missed cleavage was allowed with methionine oxidation, carbamidomethylation of cysteine, and phosphorylation of serine, threonine, and tyrosine as variable modifications.
2.2.4 In-solution tryptic digestion
Lysates from LKE-treated RG2 cells and untreated RG2 cells were subjected to digestion with trypsin by following a standard protein digestion protocol. About 50 μL of
RG2 cell lysate wasadded to 25 µL of a 100 mM ammonium bicarbonate stock solution in a microcentrifuge tube. To reduce disulfide bonds in the proteins, 2.5 µL of a 200 mM dithiothreitol (i.e., reducing agent) stock solution was addedto the tube. Briefly, the sample was vortexed thoroughly and transferred to 60°C heating block for incubation for
1 hour. In addition to the reducing agent, 10 µL of a 200 mM iodoacetamide stock solution (i.e., alkylating agent) was also added to the tube, vortexed carefully and kept in a dark environment for 1 hour. About 2.5 µL of a 200 mM DTT stock solution was addedagain,and the sample was kept in the dark for another hour. Finally, 300 µL of water and 100 µL of the ammonium bicarbonate stock solution were added. Next, based on protein: trypsin 100:1 ratio, 20 µL stock solution containing 200 ng/µL trypsin dissolved in 50 mM acetic acid was added to the tube before transferring the tube in a
37°C water bath for incubation overnight (15 hrs.). To quench the enzymatic digestion, 2
µL of trifluoroacetic acid was added to the tube, and the tube was kept in the freezer for further analysis.
37 2.2.5 Separation and identification of peptides by nano-HPLC-MALDI-MS and
MS/MS
The separation of peptides resulting from the in-solution tryptic digested cell lysate was performed using nano-high performance liquid chromatography. Initially, 500 ng/μL of trypsin digested cell lysatewas used for separation. It was found that the intensity of peaks was low (near to the baseline). Therefore, in order to improve the intensity of peaks, an increase in the concentration of the tryptic digest was necessary.
The concentration of trypsin digested cell lysate was increased up to 650 ng/μL, and extracted peptides were separated using an Ultimate3000 nano-HPLC (Dionex) equipped with Acclaim® PepMap RSLC C18 column (75μm x 15cm, 2μm particles, 100Å pore size) with flow rate of 300 nL/min, and the column oven temperature was 35 ºC. Eluting peptides were monitored using a UV-VIS nano-HPLC detector set at 214 nm. Mobile phase A was 0.05% TFA in water and mobile phase B was 0.05% TFA in 80:20 acetonitrile: water (v: v). A multistep gradient system, with concentration of B linearly increasing from 3 % to 60 % over a period of 120 min, and from 60 % to 100 % over 15 min, and then being held constant at 100% for 10 min and dropping down to 3 % in two minand then being constant at 3 % for 13 min, was used. The eluents were mixed online with CHCA matrix using an external syringe pump with aflow rate of 100 µL/h and automatically co-spotted onto an AnchorChip stainless steel MALDI plate using
Proteineer fc (Bruker Daltonics) spotting device. Fractions were collected at 5 second intervals, and the spotted fractions underwent MALDI-MS and MS/MS analysis. Finally, protein identification was performed using MASCOT search of MS/MS data. The
38 taxonomy was set to Rattus Norvegicus with peptide mass tolerance of 0.1 Da and a fragment tolerance of 0.5 Da.
2.2.6 Separation and identification of peptides and proteins by Nano-HPLC-ESI-
MS/MS
To improve the protein identifications, peptides from RG2 cells were separated from anin-solutiontryptic digest of RG2 proteins by nano-HPLC-ESI-MS/MS using an
ESI-Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher, Waltham, MA) equipped with thenanospray source. The peptides were extracted using in-solution tryptic digestion protocol. The extracted peptides were separatedon an Ultimate 3000 HPLC RSLCnano system (Dionex) equipped with an Acclaim® PepMap RSLC C18 column (75μm x 15 cm, 2 μm particles, 100 Å pore size) with a flow rate of 300 nL/min. The column oven temperature was set at 35 ºC. Mobile phase A was 0.1 % formic acid in water and mobile phase B was 0.08 % of formic acid in 80:20 (v:v) acetonitrile: water. The peptides were loaded on a precolumn for five minutes while the C18 column was held at 4 % B. A multistep gradient system was used with concentration of B linearly increasing from 4 % to 55 % over a period of 125 min and from 55 % to 90 % over 15 min, then being held constant at 90 % for 10 min and dropping down to 4 % in 0.1 min and being constant at 4
% for 25 min.
Peptides were analyzed by ESI-Orbitrap Fusion in data-dependent mode after nano-HPLC separation and detection with the UV-Vis detector set at 214 nm. A spray voltage of 1800 V and ion transfer tube temperature of 275 ºC were used. Full MS scans were acquired from m/z 400 to 1600 using quadrupole isolation and orbitrap detection at a resolution of 120,000. MS/MS selection was performed using monoisotopic precursor
39 selection on the charge states of 2+ to 4+ with an intensity threshold of 5.0e3 and a most intense precursor priority. Collision-induced dissociation (CID) MS/MS was performed in the linear ion trap using quadrupole isolation and collision energy of 35%. The spectra were analyzed using Proteome Discoverer (version 1.4, Thermo). A SEQUEST HT search of the tryptic peptides was performed using the SwissProt database. The taxonomy was set to Rattus Norvegicus. Two missed cleavages were allowed with methionine oxidation, carbamidomethylation of cysteine and phosphorylation of serine, threonine, or tyrosine set as variable modifications. The maximum Delta correlation (Cn) was set to
0.05 with precursor mass tolerance of 10 ppm and fragment mass tolerance of 0.6 Da.
Results were run through percolator for validation based on q-value with a false discovery rate (FDR) of 0.05.
40
Chapter 3
Results and discussion
3.1 The identification and relative quantification of proteins and
phosphoproteins from LKE-treated and untreated RG2 cells using SDS-
PAGE, protein staining, and MALDI-MS
SDS-PAGE separations of RG2 cell lysates were performed to investigate if LKE influences protein expression in these cells.Equal volumes of lysates from similar numbersof RG2 cells and RG2 cells treated with different concentrations of LKE (1 μM,
10μM, and 100 μM) were loaded onto 12 % SDS-PAGE gels for protein separation. The
SDS-PAGE analyseswere performedin duplicate, which respectively involved loading of
15 μL and 20 μL of samples on the gels. After separation and staining with Coomassie
(Figure 3-1), protein gel bands were excised and subjected to the in-gel tryptic digestion protocol discussed in Section 2.2.3. The tryptic peptides were analyzed by MALDI-MS and MS/MS, and identified using MASCOT search engine.
Proteins with significant scores (protein score > 52 with p < 0.05), such as coiled- coil domain-containing protein 39, vimentin, glial fibrillary acidic protein, actin cytoplasmic 2, and twinfilin-1 were identified in untreated RG2 cells. Also, elongation factor 2, heat shock cognate 71 kDa protein, vimentin, tubulin beta-5 chain, tubulin alpha-1A chain, alpha-enolase, cytoplasmic actin 1, 40S ribosomal protein S19 and
41 peptidyl-prolyl cis-trans isomerase A were identified in cell lysates of LKE-treated RG2
cells.
RG2+LKE RG2+LKE RG2+LKE Mw 100 µM 10 µM 1 µM RG2-LKE ladder 250 kDa 150 kDa 100 kDa 75 kDa Vimentin
50 kDa
37 kDa
25 kDa 20 kDa
15 kDa
10 kDa
Figure 3-1:Coomassie-stained SDS-PAGE gel showing separation of proteins from
untreated RG2 cells (RG2-LKE lane) and RG2 cells treated with LKE (RG2+LKE lanes)
A Coomassie-stained gel band corresponding to a protein with MW between 50-
70 kDa was the most intense in both LKE-treated and untreated RG2 cells (Figure 3-1).
PMF identified the protein as vimentin (MW 53.7 kDa). Vimentin is a key component of
the intermediate filament family of proteins, and is expressed in mesenchymal cells. It
maintains cellular integrity and provides resistance against stress.62 An increase in
vimentin expression is observed in numerous epithelial cancers including glioma,
prostate cancer, gastrointestinal tumor, central nervous system (CNS) tumors, breast
cancer, malignant melanoma, lung cancer, and other types of cancer.62,63 Proteins that
42 were identified in both cell lines include vimentin and cytoplasmic actin, which are among the most abundant proteins found in RG2 cells.16,64 Glial fibrillary acidic protein, which is an abundant protein involved in cytoskeleton formation in glial cells,64 was identified only in untreated RG2 cells indicating that identifications of proteins using
SDS-PAGE need to be improved. Additional experiments using 2D-GE will be needed to validate assigned proteins and identify additional proteins in RG2 cells. Based on the intensities of Coomassie-stained protein bands in SDS-PAGE gels (Figure 3-1) obtained by separating approximately the same amounts of proteins, similar quantities of vimentin were present in both LKE-treated and untreated RG2 cells. It appears that the total amount of Coomassie-stained proteins is slightly higher in untreated RG2 cells than in
LKE-treated RG2 cells (Figure 3-1) indicating that LKE may affect protein expression in
RG2 cells. However, further quantitative experiments need to be performed using densitometry of Coomassie-stained SDS-PAGE gels to validate this finding.
It was known that few of the proteins identified in glioma cell lysates, such as vimentin, are phosphorylated. To confirm the presence of phosphorylated proteins and examine their amounts in RG2 cells, the SDS-PAGE gel was stained with Pro-Q
Diamond phosphoprotein stain, which selectively binds to phosphoproteins. Figure 3-2 depicts phosphoproteins on an SDS-PAGE gel stained with Pro-Q Diamond. A few gel bands, including vimentin (MW ~ 50 kDa), were stained intensely with Pro-Q Diamond
(e.g., gel bands with MWs of~ 15 kDa, 30kDa, and 40 kDa). This analysis confirms that phosphoproteins were present in cell lysates of both RG2 cells treated with LKE and untreated RG2 cells. However, the intensity of bands corresponding to phosphorylated proteinswas similar in both untreated RG2 cells and LKE-treated RG2 cells (Figure 3-2).
43 RG2+LKE RG2+LKE RG2+LKE MW Peppermint RG2-LKE 100 µM 10 µM 1µM ladder Stick ladder
50 kDa 45 kDa
37 kDa
25 kDa
23.6 kDa
15 kDa
10 kDa
Figure 3-2: In-gel visualization of phosphoproteins from RG2 cells using SDS-PAGE and phosphorylation-specific Pro-Q Diamond stain.
Therefore, SDS-PAGE analysis followed by specific staining of separated RG2
proteins did not indicate significant changes in phosphorylation of the most abundant
phosphoproteins after the addition of LKE to cells.
3.2 The identification of proteins from LKE-treated and untreated RG2 cells
using nano-HPLC-MALDI-MS/MS
SDS-PAGE analyses followed by Coomassie and Pro-Q Diamond staining
indicated that the amounts of most abundant proteins expressed in LKE-treated and
untreated RG2 cells are similar. Since not many proteins were identified using gel-based
separations and the differences in protein amounts were not clearly observed, RG2 cell
lysates were subjected to in-solution tryptic digestion followed by nano-HPLC-MALDI-
MS/MS, as described in sections 2.2.4 and 2.2.5 of this thesis. It was expected that more
44 low-abundance proteinswould be identified in RG2 cells by nano-HPLC-MALDI-
MS/MS than by using PMF. The peptides produced from an in-solution tryptic digest of untreated RG2 cells and RG2 cells treated with LKE were separated by nano-HPLC using 135 min gradient, detected by a UV-VIS HPLC detector set at 214 nm, and collected for further MALDI-MS/MS analyses. Figures 3-3 and 3-4 show the UV chromatograms of the nano-HPLC-separated peptides from untreated RG2 cells and RG2 cells treated with10 µM LKE, respectively. A visual comparison of chromatograms indicates that the intensities of peptide peaks from RG2 cells treated with LKE (Figure 3-
4) were slightly lower than the intensities of the peaks from LKE-untreated RG2 cells
(Figure 3-3). Additionally, significant intensity suppression of couple peaks was observed in RG2 cells treated with LKE. These peaks were circled in red in Figure 3-3 and have retention times (tR) of 24.83 min and 73.75 min. They were suppressed significantly in
RG2 cells treated with 10 µM LKE (Figure 3-4) as well as in RG2 cells treated with 1
µM and 100 µM LKE (data not shown). To identify proteins corresponding to these peptide peaks, the separated peptides were collected from both samples for further
MALDI-MS/MS analyses. The peptide fractions were mixed online with CHCA matrix using an external syringe pump and co-spotted with the matrix onto an AnchorChip stainless steel MALDI plate using Bruker’s Proteineer fc spotting device. The fractions mixed with the matrix were spotted every 5 seconds, and analyzed by MALDI-MS and
MS/MS after completion of a nano-HPLC separation and spotting procedure. Protein identification was performed using MASCOT (Matrix Science) search engine and
Swissprot database.65 The taxonomy was set to Rattus norvegicus with a peptide mass tolerance of 0.1 Da and a fragment tolerance of 0.5 Da.
45
Figure 3-3. Nano-HPLC separation of peptidesoriginating from untreated RG2 cells. The solvents and the gradient used for the peptide separation are shown in the inset.
Figure 3-4. Nano-HPLC separation of peptides originating from RG2 cells treated with 10 µM LKE. The solvents and the gradient used for the separation are shown in the inset.
46 62 proteins corresponding to peptides separated from untreated RG2 cellsand 78 proteins corresponding to peptides separated from RG2 cells treated with LKE were identified. To avoid false-positive identifications, proteins with ≥2 unique peptides were filtered out.66 18 proteins were identified with ≥ 2 unique peptides from untreated RG2 cells and22 proteins were identified with ≥2 unique peptides from LKE treated RG2 cells
(Appendix section, Tables B.1 and B.2). 12 proteins were identified in both LKE-treated
RG2 cells and untreated RG2 cells. Six unique proteins were identified only in untreated
RG2 cells: desmin, fructose-bisphosphate aldolase A, glyceraldehyde-3-phosphate dehydrogenase, peptidyl-prolyl cis-trans isomerase A, phosphoglycerate kinase 1 and vimentin. As shown later (Table 3.2), ezrin was also identified in LKE-treated RG2 cells using nano-HPLC-ESI-MS. Ezrin is a protein which belongs to ezrin-radixin-moesin
(ERM) family,67 and is overexpressed in glioblastoma.68
MALDI mass spectra of peptides eluting out at tR of 24.83 min and 73.75 min in untreated and LKE-treated RG-2 cells (Figures A-1 and A-2) are shown in Appendix A of this thesis. Unfortunately, it was not possible to identify unique proteins in these fractions using nano-HPLC-MALDI-MS/MS and MASCOT.
The subcellular localization of proteins identified from untreated RG2 cells and
LKE treated RG2 cells using nano-HPLC-MALDI-MS/MS were determined. The list of proteins identified with their subcellular localization is shownin Tables B.1 and B.2 in
Appendix B of this thesis. As expected, the majority of identified proteins were from cytoplasm followed by the proteins found in the cell membrane, nucleus, cytosolic ribosomal unit, and endoplasmic reticulum.
47 3.3 The identification of proteins from LKE-treated and untreated RG2 cells
using nano-HPLC-ESI-MS/MS
To identify a larger number of proteins and phosphoproteins, lysates of untreated and LKE-treated RG2 cells were digested with trypsin and analyzed using nano-HPLC-
ESI-MS/MS. Figures 3-5 and 3-6 represent base peak chromatograms showing the nano-
HPLC-ESI-MS separation of peptides from untreated RG2 cells and RG2 cells treated with 10 µM LKE, respectively, using a 180 min gradient. Corresponding total ion chromatograms (TICs) are shown in Figures A-3 and A-4 in Appendix A. The protein identification was performed using SEQUEST search engine and Swissprot database.
2990 proteins were identified in LKE-treated RG2 cells, and 2751 proteins were identified in untreated RG2 cells. To avoid false-positive identifications, proteins with ≥2 unique peptides were filtered out.66 1681 proteins were identified with ≥2 unique peptides from RG2 cells treated with LKE and 1457 proteins were identified from untreated RG2 cells. The total number of commonly identified proteins was 1132 among LKE-treated
RG2 cells and untreated RG2 cells (Figure 3-7). As expected, more RG2 proteins were identified by nano-LC-ESI-MS/MS than by nano-LC-MALDI-MS/MS. Since the list of all identified proteins is extensive, Tables B.3 and B.4 in Appendix B only show proteins with ≥ 5 unique peptides along with their respective cellular locations.
Proteins identified from RG2 cell lysates using nano-HPLC-ESI-MS/MS were also classified based on their subcellular localization. Figures 3-8 and 3-9 depict pie charts of classification of proteins identified from untreated, and LKE treated RG2 cells, respectively. Most of the proteins identified in RG2 cell lysates are nuclear, cytoplasmic, and membrane proteins.
48
Figure 3-5. Nano-LC-ESI-MS/MS base peak chromatogramshowing separation of peptides obtained by enzymatic digestion of proteins originating from untreated RG2 cells.
Figure 3-6. Nano-LC-ESI-MS/MS base peakchromatograms showing separation of peptides obtained by enzymatic digestion of proteins originating from RG2 cells treated with 10 µM LKE.
49
RG2-LKE 1132 RG2+LKE 325 549
Figure 3-7. Venn diagram showing the number of identified proteins from LKE-treated
(+LKE) cells and untreated (-LKE) RG2 cells.
Figure 3-8. Classification of proteins from RG2 cells based on subcellular location.
Proteins wereidentified using nano-HPLC-ESI-MS/MS (Table B.3).
50
Figure 3-9. Classification of proteins from LKE-treated RG2 cells based on subcellular location. Proteins wereidentified using nano-HPLC-ESI-MS/MS (Table B.4).
3.4 Glioma related proteins identified using nano-HPLC-MALDI-MS/MS and
nano-HPLC-ESI-MS/MS.
A set of proteins related to glioma and involved in the proliferation of tumor cells was identified in untreated and LKE-treated RG2 cells with ≥2 unique peptides using both nano-HPLC-MALDI-MS/MS and ESI-MS/MS. A list of such proteins, which were identified using both MALDI-MS/MS and ESI-MS/MS, is shown in Table 3.1 along with the respective number of unique peptides.
51 Table 3.1 A List of Selected Proteins Related to Glioma and Cell Proliferation Identified in Untreated and LKE-treated RG2 Cells Using Both Nano-HPLC-MALDI and ESI-
MS/MS.*
# Accession Protein ID Mw A B C D
1 G3V8C3 Vimentin 53700 4 1 45 51 2 P63018 Heat shock cognate 71 kDa protein 70800 5 6 29 35 3 P06761 78 kDa-glucose regulated protein 72300 3 2 25 29 4 P34058 Heat shock protein HSP 90-beta 83200 3 4 26 28 5 P16617 Phosphoglycerate kinase 1 44500 2 - 24 25 6 P82995 Heat shock protein HSP 90-alpha 84800 6 3 24 21 7 P62630 Elongation factor 1-alpha-1 50100 3 2 19 20 8 Q66HD0 Endoplasmin 92700 4 2 20 19 9 Q63716 Peroxiredoxin-1 22100 3 4 10 10
*A and B: Number of unique peptides in untreated and LKE-treated cells, respectively,identified by MALDI-MS/MS; C and D: Number of unique peptides in untreated and LKE-treated cells, respectively, identified by ESI-MS/MS.
In addition,a few more proteins that are related to tumors and involved in cell proliferation of tumor cells were identified using nano-HPLC-ESI-MS/MS, but not with nano-HPLC-MALDI-MS/MS (Table 3.2). These proteins include, annexin A1,69 annexin
A2,69 annexin A5,69 and annexin A6,70 as well as adenosine deaminase,71 ezrin,67,68 nestin,72 nucleolin,73 peroxiredoxin-2,74 and translationally controlled tumor protein.75
Furthermore, autophagy-related proteins such as serine/threonine-protein kinase mTOR and ribosomal protein S6 kinase (p70S6 kinase), which were identified by Western blotting,16 were also identified by nano-LC-ESI-MS/MS.
52 Table 3.2. An Additional List of Selected Proteins Related to Glioma and Cell
Proliferation Identified in Untreated and LKE-treated RG2 Cells Using Nano-HPLC-ESI-
MS/MS.
# Accession Protein ID Mw A B
1 G3V8F8 Nestin 208800 37 44 2 P07150 Annexin A1 38800 19 16 3 Q920P6 Adenosine deaminase 39900 18 14 4 P13383 Nucleolin 77100 18 26 5 Q07936 Annexin A2 38700 12 11 6 P14668 Annexin A5 35700 11 13 7 P63029 Translationally controlled tumor protein 19400 7 7 8 P31977 Ezrin 69300 6 9 9 D3Z8E0 Ribosomal protein S6 kinase 83700 5 3 10 P48037 Annexin A6 75700 4 3 11 P35704 Peroxiredoxin-2 21800 3 6 12 P42346 Serine/threonine-protein kinase mTOR 288600 2 2
*A and B: Number of unique peptides in untreated and LKE-treated cells, respectively, identified by ESI-MS/MS.
For example, heat shock cognate 71 kDa protein (HSP7C), heat shock protein
HSP 90-alpha (HS90A), and heat shock protein HSP 90-beta (HS90B) were identified in both untreated RG2 cells, and LKE treated RG2 cells using nano-HPLC-MALDI and ESI with ≥2 unique peptides. Heat shock proteins (HSPs) are a group of proteins produced by cells in response to stress conditions. They are molecular chaperones and involved folding of proteins in the cells. They also prevent the formation of nonspecific protein aggregates. It was reported that HSPs are highly expressed in several types of cancer.
Also, they have anti-apoptotic activities and are involved in cell proliferation, differentiation, and invasion.76 It is reported that HSP90 was found in elevated levels in the most malignant pediatric brain tumors.77 Increased expression levels of HSP90 in
53 various other cancers have also been reported. Researchers have specified that HSP90 is a favorable target for cancer treatment due to its key role in malignant transformation.76
Vimentin was identified using MALDI-MS PMF, MALDI-MS/MS and ESI-
MS/MS with highest protein score and sequence coverage. Reinfenberger et al. reported the expression of vimentin and glial fibrillary acidic protein (GFAP) in ethylnitrosourea- induced rat glioma and glioma cell lines.64 Vimentin was also identified in human cell line U87-MG, which was treated with a chemotherapy drug, i.e., temozolomide.63 In
2006, Trog et al. performed experiments with U87-MG using radiotherapy and chemotreatment using temozolomide. Using 2D-GE and Western blotting, they showed up-regulation in expression of vimentin in temozolomide-treated and untreated U87-MG cell lines.63 Similar results were observed in our research; vimentin was identified in both
LKE-treated and untreated RG2 cells, but potential quantitative changes remain to be determined.
Furthermore, peroxiredoxin 1 (Prdx 1), which is related to glioma and other cancers and involved in cell proliferation, was identified using both MALDI and ESI-
MS/MS.Prdx 1 belongs to peroxiredoxins (Prdxs) family, and it is the most abundant protein in that family. Prdxs are abundant proteins found in eukaryotic cells.78 Gong et al. stated that Prdx 1 is highly expressed in some human cancers but the mechanism of action of Prdx 1 is not known so far. They reported that Prdx 1 stimulated tumorigenesis by regulating the activity of mTOR/p70S6 pathway in esophageal cancer.78
Hensley et al. identified mTOR and p70S6 kinases in RG2 cells by immunoblotting.16 They also stated that treatment of LKE on RG2 cells made changes in phosphorylation state of autophagy-related proteins such as mTOR and p70S6.16 Nano-
54 HPLC separation of untreated and LKE treated RG2 cells and protein identification using
ESI-MS/MS confirmed the presence of mTOR and p70S6 kinase. mTOR is a member of a serine-threonine protein kinase family,and it plays a key role in cell growth and proliferation.79 Duzgun et al. has reported in their recent study that mTOR signaling in cell homeostasis is important in the brain. They mentioned that mTOR signaling pathway is crucial in tumor pathogenesis. Also, they suggested that mTOR can be used as a therapeutic target in glioma since the irregularity in mTOR signaling led to the development of glioma.79 Besides mTOR and ribosomal protein S6 kinase identified previously,16 53 kinases from untreated RG2 cells and 65 kinases from LKE-treated RG2 cells were identified. Since kinases have been targets of various cancer drugs,80 it will be important to understand how LKE affects their expression in RG2 and other glial cells.
55
Chapter4
Conclusions and Future Work
4.1. Conclusions
Proteomic techniques are important for the identification and quantification of proteins in different types of cancer cells.81 In this research, proteins expressed in untreated RG2 cells and RG2 cells cultured in the presence of sulphur-containing drug
LKE were successfully identified using gel electrophoresis, nano-HPLC-MALDI-MS, and nano-HPLC-ESI-MS. While dozens of proteins were identified using nano-HPLC-
MALDI-MS/MS, 1681 proteins from LKE-treated RG2 cells and 1457 proteins from untreated RG2 cells were identified with ≥ 2 unique peptides using nano-HPLC-ESI-
MS/MS. A huge number of proteins identified in lysates of RG2 cells demonstrates the power of a high-resolution nano-HPLC-orbitrap-MS system for the identification of cellular proteins.
Importantly, mass spectrometry led to the identification of many glioma-related proteins with high protein scores and sequence coverages. Specifically, proteins such as vimentin, nestin, heat shock proteins, proteins from annexin family, peroxiredoxin 1 and
2. Furthermore, dozens of protein kinases were identified. For example, mTOR and p70S6 kinase, which were identified in RG2 cells using immunoblotting, were identified by nano-HPLC-ESI-MS/MS. The identities of the proteins are significant for our
56 understanding of the proteome of RG2 cells. Additional experiments should reveal the changes in the protein expression in RG2 cells after the treatment with LKE and the influence of LKE on cellular processes such as protein phosphorylation and autophagy.
4.2. Future work
Future experiments will be aimed on nano-HPLC-ESI-MS/MS quantification of
RG2 proteins whose expression is affected by LKE. A label-free quantification method employing SIEVE (Thermo Scientific) software will be used. This label-free quantification method is a useful and inexpensive alternative to isotope-labelling quantification techniques such as isobaric tags for relative and absolute quantitation
(ITRAQ) and stable isotope labeling with amino acids in cell culture (SILAC).
Using SIEVE, peptide peak intensities will bemeasured to investigate and compare expression of proteins in untreated and LKE-treated RG2 cells. SIEVE provides semi-quantitative analysis of proteins and peptides based on LC-MS/MS data through the following steps.82 First, the experiment definition wizard allows choosing experiment type (e.g., control vs. treated sample), data files, and parameters to execute differential analysis. Next, using full scan data from each data file, SIEVE can align LC-MS/MS chromatograms.82 Further, using MS intensities from raw LC-MS/MS data, the software can generate a frame for each group of peaks for samples based on theirm/z and tR. Later, the significant abundant differences between control and treated samples will be determined by the algorithm and will create the frame report, which includes information about MS and MS/MS data as well as their statistics. Finally, a report will be formed that combines both qualitative and quantitative results.82
57 Quantitative studies will be necessaryin order to discover additional glioma- related proteins that may be targetsof LKEin addition tothe autophagy-related proteins that were previously described by Hensley et al.16,83,84 They will also help elucidating the influence of LKE on the growth of rat glioma, which can be beneficial for potential treatment of glioma using LKE.
58
References
1. Hensley, K. Lanthionine-related compounds for the treatment of inflammatory diseases. US 2007/0197515 A1. https://www.google.com/patents/US7683055Accessed on June 16th, 2016.
2. Hensley, K.; Venkova, K.; Christov, A. Emerging biological importance of central nervous system lanthionines. Molecules 2010, 15 (8), 5581-5594.
3. Cavallini, D.; Ricci, G.; Federici, G. The ketamine derivatives of thialysine, lanthionine, cystathionine, cystine: preparation and properties. Prog. Clin. Biol. Res. 1982, 125, 355-363.
4. Hensley, K.; Christov, A.; Kamat, S.; Zhang, X. C.; Jackson, K. W.; Snow, S.; Post, J. Proteomic identification of binding partners for the brain metabolite lanthionine ketimine (LK) and documentation of LK effects on microglia and motoneuron cell cultures. J. Neurosci. 2010, 30 (8), 2979-2988.
5. Hensley, K.; Gabbita, S. P.; Venkova, K.; Hristov, A.; Johnson, M. F.; Eslami, P.; Harris-White, M. E. A Derivative of the Brain Metabolite Lanthionine Ketimine Improves Cognition and Diminishes Pathology in the 3× Tg-AD Mouse Model of Alzheimer Disease. J. Neuropath. Exp. Neurol . 2013, 72 (10), 955-969.
6. Nada, S. E.; Tulsulkar, J.; Raghavan, A.; Hensley, K.; Shah, Z. A. A derivative of the CRMP2 binding compound lanthionine ketimine provides neuroprotection in a mouse model of cerebral ischemia. Neurochem. Int. 2012, 61 (8), 1357-1363.
7. Fontana, M.; Ricci, G.; Solinas, S.; Antonucci, A.; Serao, I.; Dupre, S.; Cavallini, D. [35 S] Lanthionine ketimine binding to bovine brain membranes. Biochem.Biophys.Res. Commun. 1990, 171 (1), 480-486.
8. Hensley, K.; Venkova, K.; Christov, A.; Gunning, W.; Park, J. Collapsin response mediator protein-2: an emerging pathologic feature and therapeutic target for neurodisease indications. Mol. Neurobiol. 2011, 43 (3), 180-191.
59 9. Floyd, R. A.; Neto, H. C. C. F.; Zimmerman, G. A.; Hensley, K.; Towner, R. A. Nitrone-based therapeutics for neurodegenerative diseases: Their use alone or in combination with lanthionines. Free Radic. Biol. Med. 2013, 62, 145-156.
10. Dolecek, T. A.; Propp, J. M.; Stroup, N. E.; Kruchko, C. CBTRUS statistical report: primary brain and central nervous system tumors diagnosed in the United States in 2005–2009. Neuro-oncol. 2012, 14 (suppl 5), v1-v49.
11. Barth, R. F. Rat brain tumor models in experimental neuro-oncology: the 9L, C6, T9, F98, RG2 (D74), RT-2 and CNS-1 gliomas. J.Neurooncol. 1998, 36 (1), 91-102.
12. Koestner, A.; Swenberg, J. A.; Wechsler, W. Transplacental production with ethylnitrosourea of neoplasms of the nervous system in Sprague-Dawley rats. Am. J. Pathol. 1971, 63 (1), 37.
13. Groothuis, D. R.; Pasternak, J. F.; Fischer, J. M.; Blasberg, R. G.; Bigner, D. D.; Vick, N. A. Regional measurements of blood flow in experimental RG-2 rat gliomas. Cancer Res. 1983, 43 (7), 3362-3367.
14. Aas, A. T.; Brun, A.; Blennow, C.; Strömblad, S.; Salford, L. G. The RG2 rat glioma model. J. Neurooncol. 1995, 23 (3), 175-183.
15. Gallo, J. M.; Varkonyi, P.; Hassan, E. E.; Groothius, D. R. Targeting anticancer drugs to the brain: II. Physiological pharmacokinetic model of oxantrazole following intraarterial administration to rat glioma-2 (RG-2) bearing rats. J. Pharmacokinet. Biopharm. 1993, 21 (5), 575-592.
16. Harris-White, M. E.; Ferbas, K. G.; Johnson, M. F.; Eslami, P.; Poteshkina, A.; Venkova, K.; Christov, A.; Hensley, K. A cell-penetrating ester of the neural metabolite lanthionine ketimine stimulates autophagy through the mTORC1 pathway: evidence for a mechanism of action with pharmacological implications for neurodegenerative pathologies. Neurobiol. Dis. 2015, 84, 60-68.
17. Berg, J. M.; Tymoczko, J. L.; Stryer, L.Biochemistry 5th. Edition, New York: WH Freeman: 2002.
18. Chandramouli, K.; Qian, P.-Y. Proteomics: challenges, techniques, and possibilities to overcome biological sample complexity. Hum. Genomics Proteomics 2009, 1 (1), 1-22.
60 19. Fidalgo, M. J.; Olmedo Casas, E.; Fernández Vidal, I.; Estanyol i Ullate, J. M. Analyzing peptides and proteins by mass spectrometry: principles and applications in proteomics. Capítol del llibre: Handbook of instrumental techniques for materials, chemical and biosciences research, Centres Científics i Tecnològics. Universitat de Barcelona, Barcelona, 2012. Part III. Biosciences technologies (BT), 8, 10 p. 2012.
20. Zhang, Y.; Fonslow, B. R.; Shan, B.; Baek, M.-C.; Yates III, J. R., Protein analysis by shotgun/bottom-up proteomics. Chem. Rev. 2013, 113 (4), 2343-2394.
21. Hjernø, K. Protein identification by peptide mass fingerprinting. Mass Spectrometry Data Analysis in Proteomics 2007, 61-75.
22. Zhang, H.; Ge, Y. Comprehensive analysis of protein modifications by top-down mass spectrometry. Circ.Cardiovasc Genet. 2011, 4 (6), 711-711.
23. Schalley, C. A.Mass Spectrometry-Principles and Applications-Edmond de Hoffmann and Vincent Stroobant (Eds.), Wiley, Chichester, 2001, 407 pp, ISBN 0-471- 48565-9. 2002, 173.
24. Kitson, F. G. Larsen, B. S.; McEwen, C. N. Gas chromatography and mass spectrometry: a practical guide. Academic Press: 1996.
25. Gross, J. H. Mass spectrometry: a textbook. Springer Science & Business Media: 2006.
26. Hillenkamp, F.; Peter-Katalinic, J., MALDI MS: a practical guide to instrumentation, methods, and applications. John Wiley & Sons: 2013.
27. Loo, J. A.; Udseth, H. R.; Smith, R. D. Peptide and protein analysis by electrospray ionization-mass spectrometry and capillary electrophoresis-mass spectrometry. Anal.Biochem. 1989, 179 (2), 404-412.
28. Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T.; Matsuo, T. Protein and polymer analyses up to m/z 100 000 by laser ionization time of flight mass spectrometry. Rapid Commun.Mass Spectrom. 1988, 2 (8), 151-153.
61 29. Karas, M.; Hillenkamp, F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal.Chem. 1988, 60 (20), 2299-2301.
30. Marvin, L. F.; Roberts, M. A.; Fay, L. B. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in clinical chemistry. Clin.Chim.Acta. 2003, 337 (1), 11-21.
31. Hillenkamp, F.; Karas, M.; Holtkamp, D.; Klüsener, P. Energy deposition in ultraviolet laser desorption mass spectrometry of biomolecules.Int. J. Mass Spectrom. Ion Process. 1986, 69 (3), 265-276.
32. Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Electrospray ionization for mass spectrometry of large biomolecules. Science 1989, 246 (4926), 64-71.
33. Banerjee, S.; Mazumdar, S., Electrospray ionization mass spectrometry: a technique to access the information beyond the molecular weight of the analyte. Int.J. Anal.Chem. 2012, 2012, 1-40.
34. Karas, M.; Bahr, U.; Dülcks, T. Nano-electrospray ionization mass spectrometry: addressing analytical problems beyond routine. Fresen. J.Anal.Chem. 2000, 366 (6-7), 669-676.
35. Ikonomou, M. G.; Blades, A. T.; Kebarle, P. Electrospray-ion spray: a comparison of mechanisms and performance. Anal.Chem. 1991, 63 (18), 1989-1998.
36. Banks Jr, J. F.; Quinn, J. P.; Whitehouse, C. M. LC/ESI-MS determination of proteins using conventional liquid chromatography and ultrasonically assisted electrospray. Anal.Chem. 1994, 66 (21), 3688-3695.
37. Takats, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G. Electrosonic spray ionization. A gentle technique for generating folded proteins and protein complexes in the gas phase and for studying ion-molecule reactions at atmospheric pressure. Anal. Chem. 2004, 76 (14), 4050-4058.
38. Konermann, L.; Ahadi, E.; Rodriguez, A. D.; Vahidi, S. Unraveling the mechanism of electrospray ionization. Anal.Chem. 2012, 85 (1), 2-9.
62 39. Shen, Y.; Zhao, R.; Berger, S. J.; Anderson, G. A.; Rodriguez, N.; Smith, R. D. High-efficiency nanoscale liquid chromatography coupled on-line with mass spectrometry using nanoelectrospray ionization for proteomics. Anal.Chem. 2002, 74 (16), 4235-4249.
40. Nie, L.; Zhu, M.; Sun, S.; Zhai, L.; Wu, Z.; Qian, L.; Tan, M. An optimization of the LC-MS/MS workflow for deep proteome profiling on an Orbitrap Fusion. Anal. Methods 2016, 8 (2), 425-434.
41. Zubarev, R. A.; Makarov, A. Orbitrap mass spectrometry. Anal.Chem. 2013, 85 (11), 5288-5296.
42. Senko, M. W.; Remes, P. M.; Canterbury, J. D.; Mathur, R.; Song, Q.; Eliuk, S. M.; Mullen, C.; Earley, L.; Hardman, M.; Blethrow, J. D. Novel parallelized quadrupole/linear ion trap/Orbitrap tribrid mass spectrometer improving proteome coverage and peptide identification rates.Anal.Chem. 2013, 85 (24), 11710-11714.
43. Hames, B. D.Gel Electrophoresis of Proteins: A Practical Approach OUP Oxford: 1998; Vol. 197.
44. Rosenberg, I. M.Protein analysis and purification: benchtop techniques. Springer Science & Business Media:2013.
45. O'Farrell, P. H., High resolution two-dimensional electrophoresis of proteins. J.Biol. Chem. 1975, 250 (10), 4007-4021.
46. Klose, J. Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. Humangenetik 1975, 26 (3), 231-243.
47. Gama, M. R.; Collins, C. H.; Bottoli, C. B. Nano-liquid chromatography in pharmaceutical and biomedical research. J.Chromatogr.Sci. 2013, 51 (7), 694-703.
48. Rieux, L.; Sneekes, E.-J.; Swart, R. Nano LC: principles, evolution, and state-of- the-art of the technique. LC/GC North America 2011, 29 (10), 926-934.
49. Leung, S.-M.; Pitts, R. L. A novel approach using MALDI-TOF/TOF mass spectrometry and prestructured sample supports (AnchorChip Technology) for proteomic profiling and protein identification. Tissue Proteomics, Springer: 2008; pp 57-70.
63 50. Cottrell, J. S.; London, U. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 1999, 20 (18), 3551- 3567.
51. Desiderio, D. M.; Nibbering, N. M.; Ekman, R.; Silberring, J.; Brinkmalm, A. M., Mass Spectrometry-Instrumentation, Interpretation, and Applications. Wiley, 2008. ISBN: 978-0-471-71395-1.
52. Biemann, K. Contributions of mass spectrometry to peptide and protein structure. Biol. Mass Spectrom. 1988, 16 (1‐12), 99-111.
53. Mahmood, T.; Yang, P.-C. Western blot: technique, theory, and trouble shooting. North Am.J.Medical Sci. 2012, 4 (9), 429.
54. Magi, B.; Liberatori, S. Immunoblotting techniques. Immuno. Protoc. Springer, 2005, vol 295, 227-253.
55. Niclou, S. P.; Fack, F.; Rajcevic, U. Glioma proteomics: status and perspectives. J. Proteomics 2010, 73 (10),1823-1838.
56. Kalinina, J.; Peng, J.; Ritchie, J. C.; Van Meir, E. G. Proteomics of gliomas: initial biomarker discovery and evolution of technology. Neuro-oncology 2011, 13 (9), 926-942.
57. Shinojima, N.; Tada, K.; Shiraishi, S.; Kamiryo, T.; Kochi, M.; Nakamura, H.; Makino, K.; Saya, H.; Hirano, H.; Kuratsu, J.-i. Prognostic value of epidermal growth factor receptor in patients with glioblastoma multiforme. Cancer Res. 2003, 63 (20), 6962-6970.
58. Jing, K.; Lim, K. Why is autophagy important in human diseases? Exp. Mol. Med. 2012, 44 (2), 69-72.
59. Guo, S.; Zou, J.; Wang, G., Advances in the proteomic discovery of novel therapeutic targets in cancer. Drug design, development and therapy 2013, 7, 1259.
60. Suckau, D.; Resemann, A.; Schuerenberg, M.; Hufnagel, P.; Franzen, J.; Holle, A. A novel MALDI LIFT-TOF/TOF mass spectrometer for proteomics. Anal.Bioanal. Chem. 2003, 376 (7), 952-965.
64 61. Shevchenko A, T. H., Havlis J, Olsen JV, Mann M.In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nature protocols 2006, 1 (6), 2856-2860.
62. Satelli, A.; Li, S. Vimentin in cancer and its potential as a molecular target for cancer therapy. Cell.Mol. Life Sci. 2011, 68 (18), 3033-3046.
63. Trog, D.; Fountoulakis, M.; Friedlein, A.; Golubnitschaja, O. Is current therapy of malignant gliomas beneficial for patients? Proteomics evidence of shifts in glioma cells expression patterns under clinically relevant treatment conditions. Proteomics 2006, 6 (9), 2924-2930.
64. Reifenberger, G.; Bilzer, T.; Seitz, R.; Wechsler, W. Expression of vimentin and glial fibrillary acidic protein in ethylnitrosourea-induced rat gliomas and glioma cell lines. Acta Neuropathol. 1989, 78 (3), 270-282.
65. Bairoch, A.; Apweiler, R.The SWISS-PROT protein sequence database and its supplement TrEMBL in 2000,.Nucleic Acids Res. 2000, 28 (1), 45-48.
66. Käll, L.; Storey, J. D.; MacCoss, M. J.; Noble, W. S., Assigning significance to peptides identified by tandem mass spectrometry using decoy databases. J. Proteome Res. 2007, 7 (01), 29-34.
67. Li, J.; Wei, K.; Yu, H.; Jin, D.; Wang, G.; Yu, B. Prognostic Value of Ezrin in Various Cancers: A Systematic Review and Updated Meta-analysis. Sci. Rep. 2015, 5, 1- 13.
68. Morales, F. C.; Molina, J. R.; Hayashi, Y.; Georgescu, M.-M. Overexpression of ezrin inactivates NF2 tumor suppressor in glioblastoma. Neuro-oncology 2010, 12 (6), 528-539.
69. Deng, S.; Wang, J.; Hou, L.; Li, J.; Chen, G.; Jing, B.; Zhang, X.; Yang, Z. Annexin A1, A2, A4 and A5 play important roles in breast cancer, pancreatic cancer and laryngeal carcinoma, alone and/or synergistically. Oncol. Lett. 2013, 5 (1), 107-112.
70. Qi, H.; Liu, S.; Guo, C.; Wang, J.; Greenaway, F. T.; Sun, M. Z. Role of annexin A6 in cancer (Review). Oncol.Lett. 2015, 10 (4), 1947-1952.
65 71. Huang, J.; He, Y.; Chen, M.; Du, J.; Li, G.; Li, S.; Liu, W.; Long, X. Adenosine deaminase and adenosine kinase expression in human glioma and their correlation with glioma‑associated epilepsy. Mol. Med. Rep. 2015, 12 (5), 6509-6516.
72. Jin, X.; Jin, X.; Jung, J.-E.; Beck, S.; Kim, H. Cell surface Nestin is a biomarker for glioma stem cells. Biochem. Biophysic. Res. Comm. 2013, 433 (4), 496-501.
73. Xu, Z.; Joshi, N.; Agarwal, A.; Dahiya, S.; Bittner, P.; Smith, E.; Taylor, S.; Piwnica-Worms, D.; Weber, J.; Leonard, J. R. Knocking down nucleolin expression in gliomas inhibits tumor growth and induces cell cycle arrest. J. Neurooncol. 2012, 108 (1), 59-67.
74. Smith-Pearson, P. S.; Kooshki, M.; Spitz, D. R.; Poole, L. B.; Zhao, W.; Robbins, M. E. Decreasing peroxiredoxin II expression decreases glutathione, alters cell cycle distribution, and sensitizes glioma cells to ionizing radiation and H2O2. Free Radic. Biol. Med. 2008, 45 (8), 1178-1189.
75. Gu, X.; Yao, L.; Ma, G.; Cui, L.; Li, Y.; Liang, W.; Zhao, B.; Li, K. TCTP promotes glioma cell proliferation in vitro and in vivo via enhanced β-catenin/TCF-4 transcription. Neuro-oncology 2013, 16 (2), 217-227.
76. Lianos, G. D.; Alexiou, G. A.; Mangano, A.; Mangano, A.; Rausei, S.; Boni, L.; Dionigi, G.; Roukos, D. H. The role of heat shock proteins in cancer. Cancer lett. 2015, 360 (2), 114-118.
77. Alexiou, G. A.; Vartholomatos, G.; Stefanaki, K.; Patereli, A.; Dova, L.; Karamoutsios, A.; Lallas, G.; Sfakianos, G.; Moschovi, M.; Prodromou, N. Expression of heat shock proteins in medulloblastoma: Laboratory investigation. J. Neurosurge. Pediatr. 2013, 12 (5), 452-457.
78. Gong, F.; Hou, G.; Liu, H.; Zhang, M. Peroxiredoxin 1 promotes tumorigenesis through regulating the activity of mTOR/p70S6K pathway in esophageal squamous cell carcinoma. Med.Onc. 2015, 32 (2), 1-9.
79. Duzgun, Z.; Eroglu, Z.; Avci, C. B. Role of mTOR in glioblastoma. Gene 2016, 575 (2), 187-190.
66 80. Cohen, P.; Alessi, D. R., Kinase drug discovery–what’s next in the field? ACS Chem. Biol. 2012, 8 (1), 96-104.
81. Veenstra, T. D. Proteomic Applications in Cancer Detection and Discovery. John Wiley & Sons: 2013.
82. Thermo Scientific SIEVE Software for Differential Expression Analysis. planetorbitrap.com/download.php?filename=ZFS1334684932913_SIEVE.pdf. Accessed on 16th June 2016
83. Autelitano, F.; Loyaux, D.; Roudières, S.; Déon, C.; Guette, F.; Fabre, P.; Ping, Q.; Wang, S.; Auvergne, R.; Badarinarayana, V. Identification of novel tumor-associated cell surface sialoglycoproteins in human glioblastoma tumors using quantitative proteomics. PloS one 2014, 9 (10), e110316.
84. Heroux, M. S.; Chesnik, M. A.; Halligan, B. D.; Al-Gizawiy, M.; Connelly, J. M.; Mueller, W. M.; Rand, S. D.; Cochran, E. J.; LaViolette, P. S.; Malkin, M. G. Comprehensive characterization of glioblastoma tumor tissues for biomarker identification using mass spectrometry-based label-free quantitative proteomics. Physiol. Genomics 2014, 46 (13), 467-481.
67
Appendix A
A) RG2-LKE
B) RG2+LKE
Figure A-1. Comparison of MALDI mass spectra of peptides eluting out at tR of 24.83 min in untreated RG2 cells and LKE treated RG2 cells.
68 A) RG2-LKE
B) RG2+LKE
Figure A-2. Comparison of MALDI mass spectra of peptides eluting out at tR of 73.75 min in untreated RG2 and LKE treated RG2 cells.
69
Figure A-3.Nano-LC-ESI-MS/MS TIC showing separation of peptides obtained by enzymatic digestion of proteins originating from untreated RG2 cells.
Figure A-4. Nano-LC-ESI-MS/MS TIC showing separation of peptides obtained by enzymatic digestion of proteins originating from RG2 cells treated with 10 µM LKE.
70 Appendix B
Table B-1.A list of proteins identified from untreated RG2 cells in two replicatenano-
HPLC MALDI-MS/MSexperiments.*
Protein ID Protein name Mass Unique Cellular peptides location PGK1_RAT Phosphoglycerate kinase 1 44510 2 CYT VIME_RAT Vimentin 53700 4 CYT HS90A_RAT Heat shock protein HSP 90-alpha 84762 6 CYT TBB2A_RAT Tubulin beta-2A chain 49875 4 CYT ALDOA_RAT Fructose-bisphosphate aldolase A 39327 7 CYT ACTA_RAT Actin, aortic smooth muscle 41982 5 CYT DESM_RAT Desmin 53424 2 CYT HSP7C_RAT Heat shock cognate 71 kDa protein 70827 5 CYT PRDX1_RAT Peroxiredoxin-1 22095 3 CYT ACTB_RAT Actin, cytoplasmic 1 41710 3 CYT EF1A1_RAT Elongation factor 1-alpha 1 50082 3 CYT KPYM_RAT Pyruvate kinase PKM 57781 3 CYT H4_RAT Histone H4 11360 3 N, C ENPL_RAT Endoplasmin 92713 4 ER HS90B_RAT Heat shock protein HSP 90-beta 83229 3 CYT GRP78_RAT 78 kDa glucose-regulated protein 72302 3 ER PPIA_RAT Peptidyl-prolyl cis-trans isomerase 17863 2 CYT A G3P_RAT Glyceraldehyde-3-phosphate 35805 2 CYT dehydrogenase
*Abbreviations for cellular locations: Actin cytoskeleton- ACSK, cell membrane- CM, chromosome- C, cytoplasm- CYT, cytoskeleton- CSK, cytosolic small ribosomal unit- CSRU, cytosolic large ribosomal unit- CLRU, endoplasmic reticulum- ER, golgi apparatus- GA, mitochondrion- M, myosin II complex- M II C, nucleus- N, ribosome- R, secreted- S. The locations were determined using UniProt.
71 Table B.2.A list of proteins identified from LKE-treated RG2 cells in two replicatenano- HPLC MALDI-MS/MSexperiments.*
Protein ID Protein name Mass Unique Cellular peptides location H2A1C_RAT Histone H2A type 1-C 14097 3 N,C HS90B_RAT Heat shock protein HSP 90-beta 83229 4 CYT RS3A_RAT 40S ribosomal protein S3a 29926 2 CYT TBA1A_RAT Tubulin alpha-1A chain 50104 2 CYT ACTA_RAT Actin, aortic smooth muscle 41982 2 CYT HSP7C_RAT Heat shock cognate 71 kDa protein 70827 6 CYT ADA_RAT Adenosine deaminase 39874 2 CM EF2_RAT Elongation factor 2 95223 3 CYT ACTB_RAT Actin, cytoplasmic 1 41710 5 CYT HS90A_RAT Heat shock protein HSP 90-alpha 84762 3 CYT KPYM_RAT Pyruvate kinase PKM 57781 3 CYT PRDX1_RAT Peroxiredoxin-1 22095 4 CYT H4_RAT Histone H4 11360 3 N,C NDKA_RAT Nucleoside diphosphate kinase A 17182 2 CYT EF1A1_RAT Elongation factor 1-alpha 1 50082 2 CYT TBB2A_RAT Tubulin beta-2A chain 49875 3 CYT RL13_RAT 60S ribosomal protein L13 24294 2 CLRU PDIA3_RAT Protein disulfide-isomerase A3 56588 3 ER ENPL_RAT Endoplasmin 92713 2 ER GRP78_RAT 78 kDa glucose-regulated protein 72302 2 ER GRP75_RAT Stress-70 protein, mitochondrial 73812 2 M KCRB_RAT Creatine kinase B-type 42698 2 CYT
*Abbreviations for cellular locations: Actin cytoskeleton- ACSK, cell membrane- CM, chromosome- C, cytoplasm- CYT, cytoskeleton- CSK, cytosolic small ribosomal unit- CSRU, cytosolic large ribosomal unit- CLRU, endoplasmic reticulum- ER, golgi apparatus- GA, mitochondrion- M, myosin II complex- M II C, nucleus- N, ribosome- R, secreted- S. The locations were determined using UniProt.
Table B.3.A list of proteins from untreated RG2 cells identified with ≥5 unique peptides in two replicate nano-HPLC-ESI-MS/MS experiments.*
72 Accession Description Mass Unique Cellular Da Peptides location M0R9D5 Uncharacterized protein 571300 56 N F1M779 Clathrin heavy chain 191400 52 CM D4A8D5 Filamin, beta (Predicted) 275100 50 CSK C0JPT7 Filamin alpha 280300 47 CM G3V6P7 Myosin, heavy polypeptide 9, non-muscle 226300 46 CM P12785 Fatty acid synthase 272500 45 C G3V8C3 Vimentin 53700 45 CYT G3V852 Protein Tln1 269500 43 M P05197 Elongation factor 2 95200 37 CYT P21263 Nestin 208700 37 CSK P11980-2 Isoform M2 of Pyruvate kinase PKM 57700 33 CYT G3V7Q7 IQ motif containing GTPase activating protein 1 (Predicted), isoform CRA_b 188700 32 C G3V8L3 Lamin A, isoform CRA_b 74300 32 CYT G3V6S0 Protein Sptbn1 273300 31 N Q9QXQ0 Alpha-actinin-4 104800 30 - F1LRI5 Protein Gcn1l1 292500 30 CYT P63018 Heat shock cognate 71 kDa protein 70800 29 NUC O35821 Myb-binding protein 1A 152200 29 N D4A781 Protein Ipo5 123600 28 CYT P46462 Transitional endoplasmic reticulum ATPase 89300 27 N Q5U300 Ubiquitin-like modifier-activating enzyme 1 117700 27 N P34058 Heat shock protein HSP 90-beta 83200 26 EXT P06761 78 kDa glucose-regulated protein 72300 25 N Q6TXE9 LRRGT00050 166700 25 CYT F1LP60 Moesin (Fragment) 67600 25 CM P06685 Sodium/potassium-transporting ATPase subunit alpha-1 113000 25 CM Q6P502 T-complex protein 1 subunit gamma 60600 25 N P82995 Heat shock protein HSP 90-alpha 84800 24 CYT P16617 Phosphoglycerate kinase 1 44500 24 CYT E9PSZ3 Spectrin alpha chain, non-erythrocytic 1 284500 24 CM P48721 Stress-70 protein, mitochondrial 73800 24 CYT P28480 T-complex protein 1 subunit alpha 60300 24 NUC D4ACB8 Chaperonin subunit 8 (Theta) (Predicted), isoform CRA_a 59600 23 CM F1LRV4 Heat shock 70 kDa protein 4 94000 23 CYT F2Z3Q8 Importin subunit beta-1 97100 23 CYT Q1JU68 Eukaryotic translation initiation factor 3 subunit A 163100 22 CM Q6P3V8 Eukaryotic translation initiation factor 4A1 46100 22 EXT P05065 Fructose-bisphosphate aldolase A 39300 22 -
73 F1M5X1 Protein Rrbp1 156400 22 SC Q3MHS9 Chaperonin containing Tcp1, subunit 6A (Zeta 1) 58000 21 CYT Q68FQ0 T-complex protein 1 subunit epsilon 59500 21 N P63039 60 kDa heat shock protein, mitochondrial] 60900 20 N Q66HD0 Endoplasmin 92700 20 CM F1LRL9 Microtubule-associated protein 1B 269500 20 CYT D4AC23 Protein Cct7 59600 20 CYT D4ACN7 Protein Myof 233200 20 CYT O35814 Stress-induced-phosphoprotein 1 62500 20 - Q5XIM9 T-complex protein 1 subunit beta 57400 20 - Q4FZT9 26S proteasome non-ATPase regulatory subunit 2 100100 19 P P07150 Annexin A1 38800 19 - P62630 Elongation factor 1-alpha 1 50100 19 N P04642 L-lactate dehydrogenase A chain 36400 19 N G3V6T7 Protein disulfide-isomerase A4 72700 19 - P04785 Protein disulfide-isomerase 56900 19 CYT D4AD15 Protein LOC100911431 174900 19 - P50399 Rab GDP dissociation inhibitor beta 50500 19 M P50137 Transketolase 67600 19 CYT M0R5J4 Uncharacterized protein 47000 19 N Q920P6 Adenosine deaminase 39900 18 CM P97536 Cullin-associated NEDD8-dissociated protein 1 136300 18 N F1LM33 Leucine-rich PPR motif-containing protein, mitochondrial 156600 18 M P13383 Nucleolin 77100 18 M Q7TPB1 T-complex protein 1 subunit delta 58100 18 EXT P62909 40S ribosomal protein S3 26700 17 CYT P15999 ATP synthase subunit alpha, mitochondrial 59700 17 CYT P10719 ATP synthase subunit beta, mitochondrial 56300 17 M D4A9D6 DEAH (Asp-Glu-Ala-His) box polypeptide 9 (Predicted) 131600 17 - P47942 Dihydropyrimidinase-related protein 2 62200 17 CYT B2GUZ3 Mthfd1l protein 105800 17 CM Q5U3Z7 Serine hydroxymethyltransferase 55700 17 N G3V8A5 Vacuolar protein sorting-associated protein 35 91700 17 ER Q08163 Adenylyl cyclase-associated protein 1 51600 16 - P00507 Aspartate aminotransferase, mitochondrial 47300 16 C G3V9G4 ATP citrate lyase, isoform CRA_b 119600 16 CM G3V6T1 Coatomer subunit alpha 138300 16 CM P07335 Creatine kinase B-type 42700 16 - Q68FR6 Elongation factor 1-gamma 50000 16 CM P85845 Fascin 54500 16 C
74 Q66X93 Staphylococcal nuclease domain-containing protein 1 101900 16 CM D3ZVQ0 Ubiquitin carboxyl-terminal hydrolase 95700 16 CYT Q6P3V9 60S ribosomal protein L4 47300 15 N O35567 Bifunctional purine biosynthesis protein PURH 64200 15 P18418 Calreticulin 48000 15 EXT D3ZPR0 Chromosome segregation 1-like (S. cerevisiae) (Predicted) 110100 15 N O08651 D-3-phosphoglycerate dehydrogenase] 56500 15 - Q6AYI1 DEAD (Asp-Glu-Ala-Asp) box polypeptide 5 69200 15 CM D4A8A0 DNA fragmentation factor subunit beta 243200 15 V Q80U96 Exportin-1 123000 15 CYT P61980 Heterogeneous nuclear ribonucleoprotein K 50900 15 CYT A7VJC2-4 Isoform B0b of Heterogeneous nuclear ribonucleoproteins A2/B1 33900 15 N Q80Z29 Nicotinamide phosphoribosyltransferase 55400 15 N G3V6H2 Pre-mRNA processing factor 8, isoform CRA_a 273400 15 N Q9JKB7 Guanine deaminase OS=Rattus norvegicus GN=Gda PE=2 SV=1 - [Q9JKB7_RAT] 50900 14 - F1LV13 Heterogeneous nuclear ribonucleoprotein M 73700 14 CYT Q6IMY8 Heterogeneous nuclear ribonucleoprotein U 87700 14 CYT P05708 Hexokinase-1 102300 14 CM Q5M7W5 Microtubule-associated protein 4 110200 14 CYT P25113 Phosphoglycerate mutase 1 28800 14 CM G3V786 Protein Akr1b8 36100 14 N P11598 Protein disulfide-isomerase A3 56600 14 CM Q5XI34 Protein Ppp2r1a 65300 14 N O08629 Transcription intermediary factor 1-beta 88900 14 CYT P68255 14-3-3 protein theta 27800 13 CYT P50475 Alanine--tRNA ligase, cytoplasmic 106700 13 CM Q5U362 Annexin 35900 13 CYT P47860 ATP-dependent 6-phosphofructokinase, platelet type 85700 13 - Q6P7A7 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1] 68400 13 CM P68101 Eukaryotic translation initiation factor 2 subunit 1 36100 13 CM Q6P6V0 Glucose-6-phosphate isomerase 62800 13 - P63245 Guanine nucleotide-binding protein subunit beta-2- like 1 35100 13 N D4AE96 Importin 7 (Predicted), isoform CRA_c 119400 13 ER E9PU28 Inosine-5'-monophosphate dehydrogenase 2 55800 13 CYT F1MA98 Nucleoprotein TPR 267100 13 CYT G3V6Y6 Phosphorylase 96700 13 CM F2Z3R2 Protein Hnrnpl 63900 13 -
75 F1LPV0 Protein Nars 64100 13 CYT G3V7T6 Protein Sf3b1 145700 13 N P48500 Triosephosphate isomerase 26800 13 N Q04462 Valine--tRNA ligase 140300 13 N P62260 14-3-3 protein epsilon 29200 12 CYT F1LMZ8 26S proteasome non-ATPase regulatory subunit 11 47400 12 P P62907 60S ribosomal protein L10a 24800 12 N Q07936 Annexin A2 38700 12 EN P40329 Arginine--tRNA ligase, cytoplasmic 75800 12 CYT Q5U302 Catenin (Cadherin associated protein), alpha 1 100200 12 CYT Q00657 Chondroitin sulfate proteoglycan 4 251800 12 N Q66HL2 Cortactin 56900 12 M B5DFC8 Eukaryotic translation initiation factor 3 subunit C 105400 12 CM M0R961 Far upstream element-binding protein 2 74200 12 CSK P41542 General vesicular transport factor p115 107100 12 - P10860 Glutamate dehydrogenase 1, mitochondrial 61400 12 M Q6URK4- Isoform 2 of Heterogeneous nuclear 2 ribonucleoprotein A3 37100 12 ER Q2PQA9 Kinesin-1 heavy chain 109500 12 - P38656 Lupus La protein homolog 47700 12 N P04636 Malate dehydrogenase, mitochondrial 35700 12 N Q6AYD3 Proliferation-associated 2G4 43600 12 N Q63009 Protein arginine N-methyltransferase 1 40500 12 N F1LN59 Protein Eif4g2 (Fragment) 102000 12 N V9GZ85 Protein LOC100361457 (Fragment) 41600 12 N F1LRJ2 Protein Srrm2 295900 12 CYT E2RUH2 Ribonuclease inhibitor 49900 12 N Q3MIE4 Synaptic vesicle membrane protein VAT-1 homolog 43100 12 N Q64428 Trifunctional enzyme subunit alpha, mitochondrial 82600 12 CSK Q4KM49 Tyrosine--tRNA ligase, cytoplasmic 59100 12 - M3ZCQ2 U5 small nuclear ribonucleoprotein 200 kDa helicase 244400 12 CYT Q9Z2L0 Voltage-dependent anion-selective channel protein 1 30700 12 N P63102 14-3-3 protein zeta/delta 27800 11 N P49242 40S ribosomal protein S3a 29900 11 R P21531 60S ribosomal protein L3 46100 11 N H7C5Y5 60S ribosomal protein L6 33600 11 N Q9ER34 Aconitate hydratase, mitochondrial 85400 11 C P14668 Annexin A5 35700 11 M Q641Y8 ATP-dependent RNA helicase DDX1 82400 11 C P35565 Calnexin 67200 11 M
76 P23514 Coatomer subunit beta 106900 11 C D3ZHP6 Condensin complex subunit 1 155500 11 M G3V9K0 Cysteinyl-tRNA synthetase (Predicted), isoform CRA_b 85500 11 - P81795 Eukaryotic translation initiation factor 2 subunit 3 51000 11 CM Q641X8 Eukaryotic translation initiation factor 3 subunit E 52200 11 N P05369 Farnesyl pyrophosphate synthase 40800 11 CM P41562 Isocitrate dehydrogenase [NADP] cytoplasmic 46700 11 N P56574 Isocitrate dehydrogenase [NADP], mitochondrial 50900 11 CYT Q5PPJ6 Leucyl-tRNA synthetase 134200 11 N Q3KR86 MICOS complex subunit Mic60 (Fragment) 67100 11 CM Q3B8Q1 Nucleolar RNA helicase 2 85900 11 CM Q6DGG0 Peptidyl-prolyl cis-trans isomerase D 40700 11 N P04961 Proliferating cell nuclear antigen 28700 11 N F1M013 Protein LOC100910109 (Fragment) 29800 11 CYT Q9ESN0 Protein Niban 103400 11 CM F1M9V7 Protein Npepps 103300 11 ER F1LZW6 Protein Slc25a13 (Fragment) 74300 11 N Q91Y81 Septin-2 41600 11 C Q6P799 Serine--tRNA ligase, cytoplasmic 58600 11 CYT X1WI37 Uncharacterized protein (Fragment) 29400 11 - P85972 Vinculin 116500 11 N Q63569 26S protease regulatory subunit 6A 49100 10 N G3V8B6 26S proteasome non-ATPase regulatory subunit 1 105600 10 P Q4V7C7 Actin-related protein 3 47300 10 ER P10760 Adenosylhomocysteinase 47500 10 N D4AEP0 Adenylosuccinate synthetase isozyme 2 50100 10 V P15178 Aspartate--tRNA ligase, cytoplasmic 57100 10 CSK P27653 C-1-tetrahydrofolate synthase, cytoplasmic 100900 10 N Q07009 Calpain-2 catalytic subunit 79900 10 CM P25235 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2 69000 10 CM D4A8U7 Dynactin 1, isoform CRA_a 141900 10 N Q4G061 Eukaryotic translation initiation factor 3 subunit B 90900 10 CM Q3T1J1 Eukaryotic translation initiation factor 5A-1 16800 10 C Q5XI32 F-actin-capping protein subunit beta 30600 10 N P27881 Hexokinase-2 102500 10 CYT D4A2D7 Importin 4 (Predicted), isoform CRA_b 118900 10 CYT F1LS86 Isoleucine-tRNA synthetase (Predicted) 144200 10 CM Q66HF9 Leucine-rich repeat flightless-interacting protein 1 80000 10 EX P42123 L-lactate dehydrogenase B chain 36600 10 N Q6AYC4 Macrophage-capping protein 38800 10 CYT
77 P51583 Multifunctional protein ADE2 47100 10 - P13084 Nucleophosmin 32500 10 EXT Q9QVC8 Peptidyl-prolyl cis-trans isomerase FKBP4 51400 10 CYT Q63716 Peroxiredoxin-1 22100 10 - Q6AXS5 Plasminogen activator inhibitor 1 RNA-binding protein 44700 10 - D3ZB30 Polypyrimidine tract binding protein 1, isoform CRA_c 56900 10 N F1M446 Protein AI314180 (Fragment) 203500 10 C D4A133 Protein Atp6v1a 68200 10 CYT F1LM66 Protein Eftud2 109400 10 N M0R3M4 Protein LOC100911178 344200 10 - D3ZFP4 Protein Mcm3 91600 10 N F1LQP9 Protein Tnpo1 (Fragment) 102100 10 CYT G3V699 Protein transport protein Sec31A 135100 10 N P50398 Rab GDP dissociation inhibitor alpha 50500 10 CM O55215 Ribosomal protein S2 27200 10 C P11507 Sarcoplasmic/endoplasmic reticulum calcium ATPase 2 114700 10 C O08623 Sequestosome-1 47700 10 N P10960 Sulfated glycoprotein 1 61100 10 N Q2LAP6 Testin 47600 10 M Q6AYT3 tRNA-splicing ligase RtcB homolog 55200 10 - P81155 Voltage-dependent anion-selective channel protein 2 31700 10 N P62815 V-type proton ATPase subunit B, brain isoform 56500 10 M Q5RKI0 WD repeat-containing protein 1 66100 10 M G3V7L6 26S protease regulatory subunit 7 48600 9 P P62198 26S protease regulatory subunit 8 45600 9 P P62243 40S ribosomal protein S8 24200 9 R P85968 6-phosphogluconate dehydrogenase, decarboxylating 53200 9 N Q5XI22 Acetyl-CoA acetyltransferase, cytosolic 41100 9 CYT D3ZAN3 Alpha glucosidase2 alpha neutral subunit (Predicted) 90500 9 - P30835 ATP-dependent 6-phosphofructokinase, liver type 85300 9 CM D3ZZZ9 Catenin (Cadherin associated protein), delta 1 (Predicted), isoform CRA_a 104000 9 - Q7M0E3 Destrin 18500 9 N P63036 DnaJ homolog subfamily A member 1 44800 9 CYT F1LQI5 E3 ubiquitin-protein ligase UBR4 573400 9 CM Q5U2Q7 Eukaryotic peptide chain release factor subunit 1 49000 9 GA Q6P685 Eukaryotic translation initiation factor 2, subunit 2 (Beta) 38200 9 CM
78 Q5RKI5 Flightless I homolog (Drosophila) 144800 9 N P11762 Galectin-1 14800 9 N P07323 Gamma-enolase 47100 9 CYT P05370 Glucose-6-phosphate 1-dehydrogenase 59300 9 CYT P04906 Glutathione S-transferase P 23400 9 M Q9JIL3 Interleukin enhancer-binding factor 3 95900 9 - G3V7U4 Lamin-B1 66600 9 V P43244 Matrin-3 94400 9 CYT D3ZD89 NMDA receptor-regulated gene 1 (Predicted), isoform CRA_b 100900 9 - Q5FVM4 Non-POU domain-containing octamer-binding protein 54900 9 V O35987 NSFL1 cofactor p47 40700 9 P Q9QZ86 Nucleolar protein 58 60000 9 N D4A7R3 Nucleoporin 205kDa (Predicted) 227100 9 N D3ZEN5 Peroxiredoxin-5, mitochondrial (Fragment) 16900 9 CM E9PU01 Protein Chd4 217500 9 N F1LW91 Protein Numa1 (Fragment) 233300 9 CYT F1MAA5 Protein Rangap1 63100 9 N G3V8T5 Protein Ruvbl2 51100 9 ER F1LS72 Protein Uba2 (Fragment) 63300 9 N D3ZXK9 Purine nucleoside phosphorylase (Fragment) 32100 9 M P09527 Ras-related protein Rab-7a 23500 9 CYT Q5FVC2 Rho guanine nucleotide exchange factor 2 111800 9 N P60123 RuvB-like 1 50200 9 N Q9EQS0 Transaldolase 37400 9 ER Q5XFX0 Transgelin-2 22400 9 N F8WFH8 Tryptophan--tRNA ligase, cytoplasmic 53400 9 M G3V6C4 UDP-glucose 6-dehydrogenase 54900 9 N Q9JLA3 UDP-glucose:glycoprotein glucosyltransferase 1 176300 9 N P10688 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase delta-1 85900 8 CM B0BN93 26S proteasome non-ATPase regulatory subunit 13 42800 8 P P19945 60S acidic ribosomal protein P0 34200 8 R P09895 60S ribosomal protein L5 34400 8 N B0K031 60S ribosomal protein L7 30300 8 R Q5M7U6 Actin-related protein 2 44700 8 N Q5M9G3 Caprin-1 78100 8 M Q9QX80 CArG-binding factor A 30800 8 EN Q6MG61 Chloride intracellular channel protein 1 27000 8 M B1WC02 CTP synthase 66600 8 CYT F1LQT9 Cytosine-specific methyltransferase 182300 8 EXT
79 Q6AYH5 Dynactin subunit 2 44100 8 N P13803 Electron transfer flavoprotein subunit alpha, mitochondrial 34900 8 N B5DEN5 Eukaryotic translation elongation factor 1 beta 2 24700 8 N Q68FP1 Gelsolin 86000 8 N Q66H61 Glutaminyl-tRNA synthetase 87600 8 M G3V7G8 Glycine--tRNA ligase 81700 8 ER Q66HA8 Heat shock protein 105 kDa 96400 8 N G3V9R8 Heterogeneous nuclear ribonucleoprotein C (C1/C2) 32800 8 - Q7TP47 Heterogeneous nuclear ribonucleoprotein Q 59700 8 EXT P63159 High mobility group protein B1 24900 8 CM F1LN18 Hypoxia up-regulated protein 1 111200 8 - O35303-5 Isoform 5 of Dynamin-1-like protein 82500 8 - P35213-2 Isoform Short of 14-3-3 protein beta/alpha 27800 8 CM Q62733 Lamina-associated polypeptide 2, isoform beta 50200 8 CYT P15650 Long-chain specific acyl-CoA dehydrogenase, mitochondrial 47800 8 CYT D3ZJH9 Malic enzyme 65300 8 N Q66HF1 NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial 79400 8 N G3V6H9 Nucleosome assembly protein 1-like 1 45300 8 - P10111 Peptidyl-prolyl cis-trans isomerase A 17900 8 CYT P24368 Peptidyl-prolyl cis-trans isomerase B 23800 8 M P38652 Phosphoglucomutase-1 61400 8 CM P54319 Phospholipase A-2-activating protein 87000 8 EXT P62963 Profilin-1 14900 8 - P67779 Prohibitin 29800 8 N Q5XIH7 Prohibitin-2 33300 8 - Q5XIC6 Proteasome (Prosome, macropain) 26S subunit, non-ATPase, 12 52900 8 M Q5U2S7 Proteasome (Prosome, macropain) 26S subunit, non-ATPase, 3 60700 8 CM Q6P9V7 Proteasome (Prosome, macropain) activator subunit 1 28600 8 CM D3ZD73 Protein Ddx6 54200 8 CYT Q63081 Protein disulfide-isomerase A6 48100 8 CM M0R3X6 Protein LOC100912203 30600 8 - F1LPL7 Protein LOC100912427 35700 8 CSK D3Z941 Protein Mars 101500 8 N G3V6W6 Protein Psmc6 45800 8 ER D4A2Z6 Protein Sec63 83500 8 CYT F1LQW3 Protein Sfpq (Fragment) 65700 8 N Q4QQV0 Protein Tubb6 50000 8 N
80 Q5XI73 Rho GDP-dissociation inhibitor 1 23400 8 CYT Q63413 Spliceosome RNA helicase Ddx39b 49000 8 CM F1LQB2 Structural maintenance of chromosomes protein 3 138600 8 - Q5XHY5 Threonine--tRNA ligase, cytoplasmic 80500 8 CM Q5U2N2 Ubiquitin carboxyl-terminal hydrolase 55900 8 CM B2RYG6 Ubiquitin thioesterase OTUB1 31300 8 CSK Q63356 Unconventional myosin-Ie 126700 8 CM P61983 14-3-3 protein gamma 28300 7 CYT O70351 3-hydroxyacyl-CoA dehydrogenase type-2 27200 7 M P62278 40S ribosomal protein S13 17200 7 CYT P62271 40S ribosomal protein S18 O 17700 7 CYT P17074 40S ribosomal protein S19 16100 7 - Q6PDV7 60S ribosomal protein L10 24600 7 N P41123 60S ribosomal protein L13 24300 7 CYT P62718 60S ribosomal protein L18a 20700 7 CM O88656 Actin-related protein 2/3 complex subunit 1B 41000 7 CM Q05962 ADP/ATP translocase 1 33000 7 N P51635 Alcohol dehydrogenase [NADP(+)] 36500 7 N Q9Z1P2 Alpha-actinin-1 102900 7 N Q64057 Alpha-aminoadipic semialdehyde dehydrogenase 58700 7 CYT Q5XI77 Annexin 54100 7 CYT P00787 Cathepsin B 37400 7 N G3V936 Citrate synthase 51800 7 M O35142 Coatomer subunit beta 102500 7 CYT P45592 Cofilin-1 18500 7 - B1WBY1 Cul1 protein 89600 7 N P47875 Cysteine and glycine-rich protein 1 20600 7 CYT Q4QQV4 Dead end homolog 1 (Zebrafish) 57300 7 N D3ZD97 DEAH (Asp-Glu-Ala-His) box polypeptide 15 (Predicted), isoform CRA_b 90900 7 N O35824 DnaJ homolog subfamily A member 2 45700 7 N Q8R3Z7 EH-domain containing 4 61400 7 N Q6AYK8 Eukaryotic translation initiation factor 3 subunit D 63900 7 CSK Q6P9U8 Eukaryotic translation initiation factor 3 subunit H 39900 7 - B0BNA7 Eukaryotic translation initiation factor 3 subunit I 36400 7 EN G3V7G9 Eukaryotic translation initiation factor 3 subunit L 45100 7 M D3ZAZ0 Eukaryotic translation initiation factor 3 subunit M 42500 7 NU F7FEZ6 Heterogeneous nuclear ribonucleoprotein A1 34200 7 CSK Q3KRF2 High density lipoprotein binding protein (Vigilin) 141600 7 CYT D3ZBN0 Histone H1.5 22600 7 N D4A857 Importin 9 (Predicted) 115900 7 EN Q63269 Inositol 1,4,5-trisphosphate receptor type 3 304100 7 C
81 G3V824 Insulin-like growth factor 2 receptor, isoform CRA_b 273400 7 C Q99J82 Integrin-linked protein kinase 51300 7 CYT Q68FR9-2 Isoform 2 of Elongation factor 1-delta 72100 7 EXT P20070-2 Isoform 2 of NADH-cytochrome b5 reductase 3 34800 7 M Q75Q39 Mitochondrial import receptor subunit TOM70 67400 7 CYT G3V9Y1 Myosin, heavy polypeptide 10, non-muscle, isoform CRA_b 228900 7 CM Q66HC5 Nuclear pore complex protein Nup93 93200 7 CYT Q4KLK7 Nucleolar protein 5A 65400 7 ER A0JPJ7 Obg-like ATPase 1] 44500 7 - P52944 PDZ and LIM domain protein 1 35600 7 - G3V741 Phosphate carrier protein, mitochondrial 39500 7 CM P63004 Platelet-activating factor acetylhydrolase IB subunit alpha 46600 7 - Q6AYU5 Poly(RC) binding protein 2 38600 7 N Q9EPH8 Polyadenylate-binding protein 1 70700 7 M O70196 Prolyl endopeptidase 80700 7 SC Q6PCT9 Proteasome (Prosome, macropain) 26S subunit, non-ATPase, 6 45600 7 C P18420 Proteasome subunit alpha type-1 29500 7 CM P60901 Proteasome subunit alpha type-6 27400 7 C D3ZIE9 Protein Aldh18a1 87300 7 CYT D3ZTH8 Protein LOC689899 17700 7 - F1LVV4 Protein Rcc2 (Fragment) 46600 7 N Q63945 Protein SET 33400 7 CYT Q4KLI7 Protein Sf3a3 58800 7 CYT E9PSJ4 Protein Spag9 145900 7 ER F1LM47 Protein Sucla2 50300 7 N I6L9G6 Protein Tardbp 32100 7 MIT G3V7I8 RCG57812, isoform CRA_b 137600 7 N E9PTJ8 Rho guanine nucleotide exchange factor 1 102600 7 N D4A5Q2 Structural maintenance of chromosomes protein 134200 7 N R9PXU4 Thioredoxin reductase 1, cytoplasmic 54400 7 ER Q920J4 Thioredoxin-like protein 1 32200 7 CYT P63029 Translationally-controlled tumor protein 19400 7 N Q64560 Tripeptidyl-peptidase 2 138200 7 CSK Q4KM73 UMP-CMP kinase 22200 7 N O54975 Xaa-Pro aminopeptidase 1 69600 7 ER Q63570 26S protease regulatory subunit 6B 47400 6 P G3V9U2 3-ketoacyl-CoA thiolase, mitochondrial 41800 6 CYT P13471 40S ribosomal protein S14 16200 6 M P62250 40S ribosomal protein S16 16400 6 N
82 B0BN81 40S ribosomal protein S5 22900 6 N P62083 40S ribosomal protein S7 22100 6 CYT P02401 60S acidic ribosomal protein P2 11700 6 N P23358 60S ribosomal protein L12 17800 6 N P61314 60S ribosomal protein L15 24100 6 N P29410 Adenylate kinase 2, mitochondrial 26400 6 N Q3T1L0 Aldehyde dehydrogenase family 16 member A1 85400 6 CYT P07943 Aldose reductase 35800 6 M Q6IRJ7 Annexin 50000 6 N P50430 Arylsulfatase B 58900 6 N P32198 Carnitine O-palmitoyltransferase 1, liver isoform 88100 6 CYT P04762 Catalase 59700 6 N Q66H80 Coatomer subunit delta 57200 6 N Q4AEF8 Coatomer subunit gamma-1 97600 6 N G3V624 Coronin 53100 6 N P39951 Cyclin-dependent kinase 1 34100 6 CM P32551 Cytochrome b-c1 complex subunit 2, mitochondrial 48400 6 CM Q9ESW0 DNA damage-binding protein 1 126800 6 M Q01986 Dual specificity mitogen-activated protein kinase kinase 1 43400 6 CYT F1MAP9 E3 ubiquitin-protein ligase HUWE1 460200 6 - Q68FU3 Electron transfer flavoprotein subunit beta 27700 6 C Q8R4A1 ERO1-like protein alpha 54000 6 C Q5RK09 Eukaryotic translation initiation factor 3 subunit G 35600 6 M Q07205 Eukaryotic translation initiation factor 5 48900 6 EXT P31977 Ezrin 69300 6 N Q3T1K5 F-actin-capping protein subunit alpha-2 32900 6 N P62804 Histone H4 11400 6 N P14408-2 Isoform Cytoplasmic of Fumarate hydratase, mitochondrial 50100 6 - Q99MZ8 LIM and SH3 domain protein 1 30000 6 - Q924S5 Lon protease homolog, mitochondrial OS=Rattus norvegicus GN=Lonp1 PE=2 SV=1 - [LONM_RAT] 105700 6 CYT O88989 Malate dehydrogenase, cytoplasmic 36500 6 N Q66HR2 Microtubule-associated protein RP/EB family member 1 30000 6 - Q6AYN8 Minichromosome maintenance deficient 7 (S. cerevisiae) 81000 6 N G3V8F5 Mitochondrial import receptor subunit TOM40 homolog 37900 6 CYT P63086 Mitogen-activated protein kinase 1 41200 6 - Q64119 Myosin light polypeptide 6 17000 6 CYT
83 P13832 Myosin regulatory light chain RLC-A 19900 6 R P00388 NADPH--cytochrome P450 reductase 76900 6 N B4F7E8 Niban-like protein 1 84700 6 CYT F1LS02 Nuclear pore complex protein Nup155 154800 6 - Q5XHZ9 Pachytene checkpoint protein 2 homolog 48400 6 CYT P97852 Peroxisomal multifunctional enzyme type 2 79400 6 - Q9QZA2 Programmed cell death 6-interacting protein 96600 6 N P54001 Prolyl 4-hydroxylase subunit alpha-1 60900 6 N Q63921 Prostaglandin G/H synthase 1 69000 6 N P21670 Proteasome subunit alpha type-4 29500 6 N D3ZWZ6 Protein Igf2bp2 (Fragment) 56.5 6 - D3ZFA8 Protein LOC100362366 15500 6 CYT M0RD20 Protein LOC100911363 25400 6 M D3ZP96 Protein Mcm2 102100 6 - D3ZBL6 Protein Nup160 155400 6 N B1WC34 Protein Prkcsh 59200 6 -- Q6B345 Protein S100-A11 11100 6 CYT Q6P9U0 Protein Serpinb6 43000 6 - B2RYP4 Protein Snx2 58500 6 C D4A4J0 Protein Supt16h 119800 6 M D4AAM0 Protein Tnpo3 104100 6 CYT D4A1Q9 Protein Ttll12 73900 6 - F2Z3T9 Protein U2af2 53500 6 CSK E9PTR4 Protein Ubap2l 116800 6 - Q4QQS7 Protein Umps 52300 6 - F1LY19 Protein Upf1 123900 6 - P49432 Pyruvate dehydrogenase E1 component subunit beta, mitochondrial 39000 6 CYT F1LRB8 S-adenosylmethionine synthase 43600 6 N Q62991 Sec1 family domain-containing protein 1 72200 6 CYT Q4QQT4 Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A beta isoform 66000 6 CM Q5XIG8 Serine-threonine kinase receptor-associated protein 38400 6 N B0BNE5 S-formylglutathione hydrolase 31300 6 N Q925Q9 SH3 domain-containing kinase-binding protein 1 78000 6 C D3ZZ38 Sorting nexin 67800 6 CM Q5U211 Sorting nexin-3 18800 6 CM B0BN85 Suppressor of G2 allele of SKP1 homolog 38100 6 - P11232 Thioredoxin 11700 6 M Q5M7V8 Thyroid hormone receptor-associated protein 3 108200 6 CYT G3V679 Transferrin receptor protein 1 85800 6 M Q60587 Trifunctional enzyme subunit beta, mitochondrial 51400 6 CYT
84 Q6PEC1 Tubulin-specific chaperone A 12700 6 CYT P20417 Tyrosine-protein phosphatase non-receptor type 1 49600 6 - P18645 UDP-glucose 4-epimerase 38200 6 - Q499V1 Uridine phosphorylase 34100 6 N F1LQ81 Vesicle-fusing ATPase (Fragment) 82600 6 CM Q9R1Z0 Voltage-dependent anion-selective channel protein 3 30800 6 N Q5FVI6 V-type proton ATPase subunit C 1 43900 6 N P26772 10 kDa heat shock protein, mitochondrial 10900 5 CYT P68511 14-3-3 protein eta 28200 5 CYT P62282 40S ribosomal protein S11 18400 5 ER P62246 40S ribosomal protein S15a 14800 5 CYT P62755 40S ribosomal protein S6 28700 5 CYT P38983 40S ribosomal protein SA 32800 5 N Q794F9 4F2 cell-surface antigen heavy chain 58000 5 N Q9JLJ3 4-trimethylaminobutyraldehyde dehydrogenase 53600 5 CYYT P62914 60S ribosomal protein L11 20200 5 N Q5RK10 60S ribosomal protein L13a 23400 5 N P84100 60S ribosomal protein L19 23500 5 CYT P18445 60S ribosomal protein L27a 16600 5 CYT D3ZUY8 Adaptor protein complex AP-2, alpha 1 subunit (Predicted) 107600 5 N F1LPQ9 A-kinase anchor protein 2 95900 5 CYT Q6AYS7 Aminoacylase-1A 45800 5 CM O88321 Antisecretory factor 41000 5 N P18484 AP-2 complex subunit alpha-2] 10400 5 CYT B1WC49 Api5 protein OS=Rattus norvegicus GN=Api5 PE=2 SV=1 - [B1WC49_RAT] 56700 5 CM Q5M9F7 ARP10 actin-related protein 10 homolog (S. cerevisiae) 46200 5 - Q9ER24 Ataxin-10 53700 5 N P31399 ATP synthase subunit d, mitochondrial 18800 5 CYT P35435 ATP synthase subunit gamma, mitochondrial 30200 5 CM Q6AY58 B-cell receptor-associated protein 31 27900 5 CYT D3ZL24 Calpastatin 71700 5 CM A0JN30 Canopy 2 homolog (Zebrafish) 20700 5 CYT O70509 CD44 antigen 39700 5 CM Q5RJK5 Chromobox homolog 3 (HP1 gamma homolog, Drosophila- 20800 5 M Q4KM65 Cleavage and polyadenylation specificity factor subunit 5 26200 5 CM D4ABY2 Coatomer subunit gamma-2 80400 5 N P18395 Cold shock domain-containing protein E1 88800 5 N
85 O35796 Complement component 1 Q subcomponent- binding protein, mitochondrial 31000 5 CYT O35828 Coronin-7 100700 5 N Q03114 Cyclin-dependent-like kinase 5 33200 5 EN D4A4T9 Cysteine and histidine-rich domain-containing protein 1 37300 5 CYT D3ZU74 Cytoplasmic dynein 1 intermediate chain 2 68300 5 CYT Q62698 Cytoplasmic dynein 1 light intermediate chain 2 54700 5 EXT P08461 Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial 67100 5 M A9CMB8 DNA replication licensing factor MCM6 92800 5 CYT Q9WUL0 DNA topoisomerase 1 90700 5 C P43138 DNA-(apurinic or apyrimidinic site) lyase 35500 5 M Q641Y0 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit 48900 5 CM Q8VHU4 Elongator complex protein 1 149100 5 N F1M3L7 Epidermal growth factor receptor kinase substrate 8 91900 5 N Q5RKG9 Eukaryotic translation initiation factor 4B 69000 5 EXT B2GUV7 Eukaryotic translation initiation factor 5B 137600 5 CM Q3KRD8 Eukaryotic translation initiation factor 6 26600 5 CM Q5PQK2 Fusion, derived from t(1216) malignant liposarcoma (Human) 52600 5 - G3V8V1 Granulin, isoform CRA_c 65100 5 C Q8K586 GTP-binding nuclear protein Ran, testis-specific isoform 24400 5 N D3ZL86 HEAT repeat containing 1 (Predicted) 241100 5 N D3ZNH4 Histone H2B 15500 5 - P50503 Hsc70-interacting protein 41300 5 N P27605 Hypoxanthine-guanine phosphoribosyltransferase 24500 5 N Q56R18 Importin subunit alpha 57700 5 CM Q7TP98 Interleukin enhancer-binding factor 2 51300 5 - O55012-2 Isoform 2 of Phosphatidylinositol-binding clathrin assembly protein 64600 5 M B3GNI6-3 Isoform 3 of Septin-11 48900 5 N Q63151-2 Isoform Short of Long-chain-fatty-acid--CoA ligase 3 79300 5 EXT Q6MG49 Large proline-rich protein BAG6 119900 5 CYT Q5RJR8 Leucine-rich repeat-containing protein 59 34800 5 ER Q499P2 Leukotriene A(4) hydrolase 69000 5 N B5DEG8 LOC685144 protein 118400 5 CYT Q6QI16 LRRGT00192 33800 5 CYT P14562 Lysosome-associated membrane glycoprotein 1 43900 5 N
86 Q99MI7 NEDD8-activating enzyme E1 catalytic subunit 51700 5 V G3V6P1 Nerve growth factor receptor (TNFR superfamily, member 16) 45400 5 - P04182 Ornithine aminotransferase, mitochondrial 48300 5 - F1SW39 PC4 and SFRS1 interacting protein 1 59600 5 CYT Q63530 Phosphotriesterase-related protein 39100 5 CYT F1LPK7 Plastin 3 (T-isoform), isoform CRA_a 70700 5 N Q9JMJ4 Pre-mRNA-processing factor 19 55200 5 ER Q99ML5 Prenylcysteine oxidase 56300 5 CYT R9PXR7 Prostaglandin E synthase 3 (Fragment) 18600 5 SC G3V8G2 Proteasome (Prosome, macropain) 26S subunit, non-ATPase, 5 (Predicted), isoform CRA_a 55800 5 N Q6IE67 Proteasome subunit alpha type 28300 5 C F1LNN1 Proteasome subunit beta type 23000 5 C F1M8H5 Protein Abcf2 50300 5 C D4ADI5 Protein Arap3 169500 5 CYT F1M949 Protein Ckap5 225300 5 C G3V7M0 Protein Cnot1 266700 5 N D4AD75 Protein Dpy19l1 84300 5 - D3ZJ32 Protein Esyt2 94400 5 - D3ZLC1 Protein Lmnb2 67200 5 N Q99MI5 Protein LOC100912604 34000 5 - F1LT49 Protein Lrrc47 63500 5 - D3ZDK7 Protein Pgp 34600 5 M F1MAH5 Protein Ppp6r3 94200 5 N B0BNK1 Protein Rab5c 23400 5 CM Q5XFW8 Protein SEC13 homolog 35500 5 CYT D3ZQM0 Protein Sf3a1 88500 5 ER M0R402 Protein Tmx3 51700 5 N D3ZRN5 Protein Trove2 60000 5 V D3ZZC1 Protein Txndc5 46300 5 EXT D3ZF39 Protein Uap1 58400 5 N B5DEH4 Protein Uap1l1 56400 5 M Q32PZ3 Protein unc-45 homolog A 103200 5 CM Q6T5E8 RCG55639, isoform CRA_g 59500 5 N D3Z8E0 Ribosomal protein S6 kinase 83700 5 CYT Q4V898 RNA-binding motif protein, X chromosome 42200 5 C P22509 rRNA 2'-O-methyltransferase fibrillarin 34200 5 N P63331 Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform 35600 5 N B2RYI2 Signal recognition particle subunit SRP68 70400 5 M G3V702 Smu-1 suppressor of mec-8 and unc-52 homolog (C. elegans) 57500 5 CM
87 Q920L2 Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial 71600 5 CM Q6AXQ0 SUMO-activating enzyme subunit 1 38500 5 N P24155 Thimet oligopeptidase 78300 5 CYT Q4KLL0 Transcription elongation factor A protein 1 33900 5 N Q5XIP9 Transmembrane protein 43 44700 5 - P09495 Tropomyosin alpha-4 chain 28500 5 - Q00981 Ubiquitin carboxyl-terminal hydrolase isozyme L1 24800 5 CYT F1MAA1 Ubiquitin carboxyl-terminal hydrolase 154600 5 CYT D3ZC84 Ubiquitin carboxyl-terminal hydrolase 289300 5 CM Q9EQX9 Ubiquitin-conjugating enzyme E2 N 17100 5 CM D3ZET9 Uncharacterized protein 125700 5 N F1MAJ2 Uncharacterized protein 13800 5 V
*Abbreviations for cellular locations: Cell membrane- CM, chromosome- C, cytoplasm- CYT, cytoskeleton- CSK, endoplasmic reticulum- ER, endosome- EN, extracellular- EXT, golgi apparatus- GA, mitochondrion- M, nucleus- N, ribosome- R. The locations of some proteins were omitted because they were not provided by Proteome Discoverer.
Table B.4.A list of proteins from LKE-treated RG2 cells identified with ≥5 unique peptides in two replicate nano-HPLC-ESI-MS/MS experiments.*
88 Accession Description MW # Unique Cellular Da Peptides location C0JPT7 Filamin alpha 280300 65 N M0R9D5 Uncharacterized protein 571300 65 - D4A8D5 Filamin, beta (Predicted) 275100 61 CYT G3V6P7 Myosin, heavy polypeptide 9, non-muscle 226300 58 N G3V852 Protein Tln1 269500 55 CYT F1M779 Clathrin heavy chain 191400 54 M G3V8C3 Vimentin 53700 51 CYT P12785 Fatty acid synthase 272500 48 CYT G3V8F8 Nestin 208800 44 CYT P05197 Elongation factor 2 95200 40 N G3V7Q7 IQ motif containing GTPase activating protein 1 (Predicted), isoform CRA_b 188700 39 N D4ACN7 Protein Myof 233200 36 ME P63018 Heat shock cognate 71 kDa protein 70800 35 N F1LRI5 Protein Gcn1l1 292500 34 - P11980-2 Isoform M2 of Pyruvate kinase PKM 57700 33 - G3V8L3 Lamin A, isoform CRA_b 74300 33 N E9PSZ3 Spectrin alpha chain, non-erythrocytic 1 284500 33 - F1M5X1 Protein Rrbp1 156400 32 - P63039 60 kDa heat shock protein, mitochondrial 60900 29 CYT P06761 78 kDa glucose-regulated protein 72300 29 N P46462 Transitional endoplasmic reticulum ATPase 89300 29 P P34058 Heat shock protein HSP 90-beta 83200 28 CYT D4A781 Protein Ipo5 123600 28 N Q1JU68 Eukaryotic translation initiation factor 3 subunit A 163100 27 N Q6TXE9 LRRGT00050 166700 27 CYT Q5U300 Ubiquitin-like modifier-activating enzyme 1 117700 27 N P13383 Nucleolin 77100 26 N Q9QXQ0 Alpha-actinin-4 104800 25 N F2Z3Q8 Importin subunit beta-1 97100 25 CYT O35821 Myb-binding protein 1A 152200 25 N P16617 Phosphoglycerate kinase 1 44500 25 CYT Q66X93 Staphylococcal nuclease domain-containing protein 1 101900 25 N Q68FQ0 T-complex protein 1 subunit epsilon 59500 25 CYT Q5M7W5 Microtubule-associated protein 4 110200 24 CYT P28480 T-complex protein 1 subunit alpha 60300 24 CYT Q6P502 T-complex protein 1 subunit gamma 60600 24 CYT D4ACB8 Chaperonin subunit 8 (Theta) (Predicted), isoform CRA_a 59600 23 CYT D4AC23 Protein Cct7 59600 23 CYT
89 P11598 Protein disulfide-isomerase A3 56600 23 E P04785 Protein disulfide-isomerase 56900 23 ER G3V6S0 Protein Sptbn1 273300 23 - F1M953 Stress-70 protein, mitochondrial 73700 23 CYT Q5XIM9 T-complex protein 1 subunit beta 57400 23 CYT F1LV13 Heterogeneous nuclear ribonucleoprotein M 73700 22 SC F1LP60 Moesin (Fragment) 67600 22 CYT Q3B8Q1 Nucleolar RNA helicase 2 85900 22 N P50137 Transketolase 67600 22 N M0R5J4 Uncharacterized protein 47000 22 - P97536 Cullin-associated NEDD8-dissociated protein 1 136300 21 N P82995 Heat shock protein HSP 90-alpha 84800 21 N O35814 Stress-induced-phosphoprotein 1 62500 21 N Q4FZT9 26S proteasome non-ATPase regulatory subunit 2 100100 20 P P62630 Elongation factor 1-alpha 1 50100 20 N P50399 Rab GDP dissociation inhibitor beta 50500 20 CYT P06685 Sodium/potassium-transporting ATPase subunit alpha-1 113000 20 EN P50475 Alanine--tRNA ligase, cytoplasmic 106700 19 CYT G3V6D3 ATP synthase subunit beta 56300 19 N P16638 ATP-citrate synthase 120600 19 CYT O35567 Bifunctional purine biosynthesis protein PURH 64200 19 M D4A8A0 DNA fragmentation factor subunit beta 243200 19 N Q66HD0 Endoplasmin 92700 19 N P05065 Fructose-bisphosphate aldolase A 39300 19 N G3V6T7 Protein disulfide-isomerase A4 72700 19 ER D3ZVQ0 Ubiquitin carboxyl-terminal hydrolase 95700 19 - Q6P3V9 60S ribosomal protein L4 47300 18 N P15999 ATP synthase subunit alpha, mitochondrial 59700 18 M Q3MHS9 Chaperonin containing Tcp1, subunit 6A (Zeta 1) 58000 18 CYT Q66HA8 Heat shock protein 105 kDa 96400 18 N P61980 Heterogeneous nuclear ribonucleoprotein K 50900 18 N Q6IMY8 Heterogeneous nuclear ribonucleoprotein U 87700 18 N P04642 L-lactate dehydrogenase A chain 36400 18 N P38656 Lupus La protein homolog 47700 18 N G3V6H2 Pre-mRNA processing factor 8, isoform CRA_a 273400 18 N Q5U3Z7 Serine hydroxymethyltransferase 55700 18 N Q7TPB1 T-complex protein 1 subunit delta 58100 18 CYT Q5RKI0 WD repeat-containing protein 1 66100 18 CYT G3V6T1 Coatomer subunit alpha 138300 17 CM D4A8U7 Dynactin 1, isoform CRA_a 141900 17 C F1LNF1 Heterogeneous nuclear ribonucleoproteins A2/B1 37300 17 N
90 (Fragment) Q2PQA9 Kinesin-1 heavy chain 109500 17 CYT D4AD15 Protein LOC100911431 174900 17 CYT F1LPV0 Protein Nars 64100 17 CYT F1LW91 Protein Numa1 (Fragment) 233300 17 G3V8A5 Vacuolar protein sorting-associated protein 35 91700 17 CYT G3V7L6 26S protease regulatory subunit 7 48600 16 P P62909 40S ribosomal protein S3 26700 16 N P07150 Annexin A1 38800 16 CSK P00507 Aspartate aminotransferase, mitochondrial 47300 16 M D4A9D6 DEAH (Asp-Glu-Ala-His) box polypeptide 9 (Predicted) 131600 16 N P47942 Dihydropyrimidinase-related protein 2 62200 16 CYT P85845 Fascin 54500 16 CYT Q66HF9 Leucine-rich repeat flightless-interacting protein 1 80000 16 N B2GUZ3 Mthfd1l protein 105800 16 M D4A133 Protein Atp6v1a 68200 16 M F1LM66 Protein Eftud2 109400 16 N Q5XHY5 Threonine--tRNA ligase, cytoplasmic] 80500 16 CYT Q63569 26S protease regulatory subunit 6A 49100 15 P F1LQS3 60S ribosomal protein L6 33500 15 R P15178 Aspartate--tRNA ligase, cytoplasmic 57100 15 CYT P35565 Calnexin 67200 15 CYT D3ZPR0 Chromosome segregation 1-like (S. cerevisiae) (Predicted) 110100 15 CYT Q6AYI1 DEAD (Asp-Glu-Ala-Asp) box polypeptide 5 69200 15 N Q6P7A7 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1 68400 15 ER Q4G061 Eukaryotic translation initiation factor 3 subunit B 90900 15 CYT M0R961 Far upstream element-binding protein 2 74200 15 N Q6URK4 Heterogeneous nuclear ribonucleoprotein A3 39600 15 N F1LSL3 Inositol 1,4,5-trisphosphate receptor type 3 303700 15 CM G3V7U4 Lamin-B1 66600 15 N F1LM33 Leucine-rich PPR motif-containing protein, mitochondrial 156600 15 C Q5PPJ6 Leucyl-tRNA synthetase 134200 15 N G3V9Y1 Myosin, heavy polypeptide 10, non-muscle, isoform CRA_b 228900 15 N Q6AYD3 Proliferation-associated 2G4 43600 15 N F1LQW3 Protein Sfpq (Fragment) 65700 15 - P48500 Triosephosphate isomerase 26800 15 N Q4KM49 Tyrosine--tRNA ligase, cytoplasmic 59100 15 N F1LNJ2 U5 small nuclear ribonucleoprotein 200 kDa 244700 15 N
91 helicase P62260 14-3-3 protein epsilon 29200 14 CYT Q920P6 Adenosine deaminase 39900 14 CYT P40329 Arginine--tRNA ligase, cytoplasmic 75800 14 CYT Q641Y8 ATP-dependent RNA helicase DDX1 82400 14 N P18418 Calreticulin 48000 14 N P23514 Coatomer subunit beta 106900 14 CM Q66HL2 Cortactin 56900 14 CYT O08651 D-3-phosphoglycerate dehydrogenase 56500 14 - Q68FR6 Elongation factor 1-gamma 50000 14 N B5DFC8 Eukaryotic translation initiation factor 3 subunit C 105400 14 CYT Q80U96 Exportin-1 123000 14 C Q68FP1 Gelsolin 86000 14 EXT Q9JKB7 Guanine deaminase 50900 14 - Q6AXS5-2 Isoform 2 of Plasminogen activator inhibitor 1 RNA-binding protein 43000 14 N P43244 Matrin-3 94400 14 N Q5FVM4 Non-POU domain-containing octamer-binding protein 54900 14 N F1MA98 Nucleoprotein TPR 267100 14 C Q499Q4 Phosphoglucomutase 1 61400 14 CSK P25113 Phosphoglycerate mutase 1 28800 14 N F2Z3R2 Protein Hnrnpl 63900 14 N M0R3M4 Protein LOC100911178 344200 14 M E2RUH2 Ribonuclease inhibitor 49900 14 CYT P62198 26S protease regulatory subunit 8 45600 13 P P21531 60S ribosomal protein L3 46100 13 N P10760 Adenosylhomocysteinase 47500 13 N Q9Z1P2 Alpha-actinin-1 102900 13 N P14668 Annexin A5 35700 13 N Q4QQV4 Dead end homolog 1 (Zebrafish) 57300 13 CYT F1LQI5 E3 ubiquitin-protein ligase UBR4 573400 13 CYT P68101 Eukaryotic translation initiation factor 2 subunit 1 36100 13 N P81795 Eukaryotic translation initiation factor 2 subunit 3 51000 13 N Q6P3V8 Eukaryotic translation initiation factor 4A1 46100 13 N P63245 Guanine nucleotide-binding protein subunit beta-2- like 1 35100 13 N P27881 Hexokinase-2 102500 13 M D4AE96 Importin 7 (Predicted), isoform CRA_c 119400 13 CYT E9PU28 Inosine-5'-monophosphate dehydrogenase 2 55800 13 N P11507-2 Isoform 2 of Sarcoplasmic/endoplasmic reticulum calcium ATPase 2 109600 13 ER O35303-5 Isoform 5 of Dynamin-1-like protein 82500 13 CYT
92 G3V786 Protein Akr1b8 36100 13 CSK Q63009 Protein arginine N-methyltransferase 1 40500 13 N Q9ESN0 Protein Niban 103400 13 CYT Q5XI34 Protein Ppp2r1a 65300 13 CSK G3V7T6 Protein Sf3b1 145700 13 C Q2LAP6 Testin 47600 13 N P62815 V-type proton ATPase subunit B, brain isoform 56500 13 CYT P63102 14-3-3 protein zeta/delta 27800 12 N G3V8B6 26S proteasome non-ATPase regulatory subunit 1 105600 12 P P19945 60S acidic ribosomal protein P0 34200 12 N P62907 60S ribosomal protein L10a 24800 12 N Q08163 Adenylyl cyclase-associated protein 1 OS=Rattus norvegicus GN=Cap1 PE=1 SV=3 - [CAP1_RAT] 51600 12 CYT Q6IRJ7 Annexin 50000 12 N Q07009 Calpain-2 catalytic subunit 79900 12 C Q5U302 Catenin (Cadherin associated protein), alpha 1 100200 12 CYT P07323 Gamma-enolase 47100 12 CYT P04797 Glyceraldehyde-3-phosphate dehydrogenase 35800 12 N G3V7G8 Glycine--tRNA ligase 81700 12 CYT P05708 Hexokinase-1 102300 12 M F1LS86 Isoleucine-tRNA synthetase (Predicted) 144200 12 CYT P04636 Malate dehydrogenase, mitochondrial 35700 12 N A0JPJ7 Obg-like ATPase 44500 12 N G3V6Y6 Phosphorylase 96700 12 CYT F1M949 Protein Ckap5 225300 12 CM F1LN59 Protein Eif4g2 (Fragment) 102000 12 G3V699 Protein transport protein Sec31A 135100 12 CYT Q91Y81 Septin-2 41600 12 N Q5U2N2 Ubiquitin carboxyl-terminal hydrolase 55900 12 CYT G3V6C4 UDP-glucose 6-dehydrogenase 54900 12 N P85972 Vinculin 116500 12 CYT Q9Z2L0 Voltage-dependent anion-selective channel protein 1 30700 12 N F1LMZ8 26S proteasome non-ATPase regulatory subunit 11 47400 11 P Q9ER34 Aconitate hydratase, mitochondrial 85400 11 N Q07936 Annexin A2 38700 11 E Q5U362 Annexin OS=Rattus norvegicus GN=Anxa4 PE=2 SV=1 - [Q5U362_RAT] 35900 11 N P07335 Creatine kinase B-type 42700 11 CYT G3V8T4 DNA damage-binding protein 1 126900 11 N
93 P25235 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2 69000 11 - Q6AYH5 Dynactin subunit 2 44100 11 C Q5U2Q7 Eukaryotic peptide chain release factor subunit 1 49000 11 N Q6AYK8 Eukaryotic translation initiation factor 3 subunit D 63900 11 CYT Q3T1J1 Eukaryotic translation initiation factor 5A-1 168000 11 N Q3KRF2 High density lipoprotein binding protein (Vigilin) 141600 11 N Q68FR9-2 Isoform 2 of Elongation factor 1-delta 72100 11 Q3KR86 MICOS complex subunit Mic60 (Fragment) 67100 11 M Q80Z29 Nicotinamide phosphoribosyltransferase 55400 11 EXT Q66HC5 Nuclear pore complex protein Nup93 93200 11 N Q4KLK7 Nucleolar protein 5A 65400 11 - P13084 Nucleophosmin 32500 11 NC D3ZB30 Polypyrimidine tract binding protein 1, isoform CRA_c 56900 11 NUC Q9JMJ4 Pre-mRNA-processing factor 19 55200 11 N P67779 Prohibitin 29800 11 N O70196 Prolyl endopeptidase 80700 11 NUC Q5U2S7 Proteasome (Prosome, macropain) 26S subunit, non-ATPase, 3 60700 11 PRO E9PU01 Protein Chd4 217500 11 - Q63081 Protein disulfide-isomerase A6 48100 11 ER V9GZ85 Protein LOC100361457 (Fragment) 41600 11 - D3ZFP4 Protein Mcm3 91600 11 N F1M9V7 Protein Npepps 103300 11 CYT F1LZW6 Protein Slc25a13 (Fragment) 74300 11 - F1LRJ2 Protein Srrm2] 295900 11 SC F1LS72 Protein Uba2 (Fragment) 63300 11 - P24155 Thimet oligopeptidase 78300 11 CYT O08629 Transcription intermediary factor 1-beta 88900 11 C Q04462 Valine--tRNA ligase 140300 11 M P61983 14-3-3 protein gamma 28300 10 CYT P68255 14-3-3 protein theta 27800 10 CYT F1LPH1 Calpastatin 77200 10 N P04762 Catalase 59700 10 M O35142 Coatomer subunit beta' 102500 10 CM Q4AEF8 Coatomer subunit gamma-1 97600 10 CM G3V624 Coronin 53100 10 CSK P39951 Cyclin-dependent kinase 1 34100 10 N D3ZU74 Cytoplasmic dynein 1 intermediate chain 2 68300 10 - D3ZD97 DEAH (Asp-Glu-Ala-His) box polypeptide 15 (Predicted), isoform CRA_b 90900 10 N Q68FU3 Electron transfer flavoprotein subunit beta 27700 10 M
94 B5DEN5 Eukaryotic translation elongation factor 1 beta 2 24700 10 N G3V7G9 Eukaryotic translation initiation factor 3 subunit L 45100 10 CYT F1LND7 Farnesyl pyrophosphate synthase 40800 10 - P05370 Glucose-6-phosphate 1-dehydrogenase 59300 10 N Q6P6V0 Glucose-6-phosphate isomerase 62800 10 EXT P04906 Glutathione S-transferase P 23400 10 N G3V9R8 Heterogeneous nuclear ribonucleoprotein C (C1/C2) 32800 10 N Q499P2 Leukotriene A(4) hydrolase 69000 10 N Q66HF1 NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial 79400 10 M F1LS02 Nuclear pore complex protein Nup155 154800 10 - P52944 PDZ and LIM domain protein 1 35600 10 CYT Q6DGG0 Peptidyl-prolyl cis-trans isomerase D 40700 10 N Q63716 Peroxiredoxin-1 22100 10 N P54319 Phospholipase A-2-activating protein 87000 10 - D3ZYS7 Protein G3bp1 51800 10 CYT D3ZWZ6 Protein Igf2bp2 (Fragment) 56500 10 - M0R9U5 Protein LOC100911422 (Fragment) 37800 10 - D4AB17 Protein LOC100912917 137000 10 - D3Z941 Protein Mars 101500 10 CYT B1WC34 Protein Prkcsh 59200 10 ER G3V6W6 Protein Psmc6 45800 10 P P10960 Sulfated glycoprotein 1 61100 10 EXT Q6AXQ0 SUMO-activating enzyme subunit 1 38500 10 N Q9EQS0 Transaldolase 37400 10 N Q64560 Tripeptidyl-peptidase 2 138200 10 N Q9JLA3 UDP-glucose:glycoprotein glucosyltransferase 1 1763000 10 ER P68511 14-3-3 protein eta 28200 9 CYT P10688 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase delta-1 85900 9 N G3V9U2 3-ketoacyl-CoA thiolase, mitochondrial 41800 9 - P49242 40S ribosomal protein S3a 29900 9 N P29314 40S ribosomal protein S9 22600 9 N D4AEP0 Adenylosuccinate synthetase isozyme 2 50100 9 CYT P51635 Alcohol dehydrogenase [NADP(+)] 36500 9 CSK G3V6S5 C-1-tetrahydrofolate synthase, cytoplasmic 100900 9 CYT Q5M9G3 Caprin-1 78100 9 CYT P45592 Cofilin-1 18500 9 N B1WC02 CTP synthase 66600 9 CM D4A4T9 Cysteine and histidine-rich domain-containing protein 1 37300 9 - Q7M0E3 Destrin 18500 9 CYT
95 Q9WUL0 DNA topoisomerase 1 90700 9 C P63036 DnaJ homolog subfamily A member 1 44800 9 N P13803 Electron transfer flavoprotein subunit alpha, mitochondrial 34900 9 M P31977 Ezrin 69300 9 CYT Q5RKI5 Flightless I homolog (Drosophila) 144800 9 CYT P11762 Galectin-1 14800 9 EXT P41542 General vesicular transport factor p115 107100 9 CM P10860 Glutamate dehydrogenase 1, mitochondrial 61400 9 CYT F7FEZ6 Heterogeneous nuclear ribonucleoprotein A1 34200 9 - Q794E4 Heterogeneous nuclear ribonucleoprotein F 45700 9 N P17425 Hydroxymethylglutaryl-CoA synthase, cytoplasmic 57400 9 N Q63617 Hypoxia up-regulated protein 1 111200 9 ER Q6P6T9 Importin subunit alpha 57800 9 N D3ZD89 NMDA receptor-regulated gene 1 (Predicted), isoform CRA_b 100900 9 N Q6AYU5 Poly(RC) binding protein 2 38600 9 N Q5XIH7 Prohibitin-2 33300 9 N Q5XIC6 Proteasome (Prosome, macropain) 26S subunit, non-ATPase, 12 52900 9 P D4A9T3 Protein Hmg1l1 24700 9 N D4ADW6 Protein Ktn1 145600 9 ER D3ZM33 Protein LOC100362298 (Fragment) 18000 9 R F1M013 Protein LOC100910109 (Fragment) 29800 9 - F1LPL7 Protein LOC100912427 35700 9 - D3ZP96 Protein Mcm2 102100 9 C D3ZMS1 Protein Sf3b2 98100 9 N E9PT66 Protein Sf3b3 135500 9 N E9PSJ4 Protein Spag9 145900 9 CYT D4A4J0 Protein Supt16h 119800 9 N I6L9G6 Protein Tardbp 32100 9 N Q4QQV0 Protein Tubb6 50000 9 N F1LY19 Protein Upf1 123900 9 C P50398 Rab GDP dissociation inhibitor alpha 50500 9 CYT P09527 Ras-related protein Rab-7a 23500 9 CM Q6P799 Serine--tRNA ligase, cytoplasmic 58600 9 CYT Q3MIE4 Synaptic vesicle membrane protein VAT-1 homolog 43100 9 CYT Q64428 Trifunctional enzyme subunit alpha, mitochondrial 82600 9 M D3ZFD0 Uncharacterized protein 232500 9 CYT E9PT76 Uncharacterized protein 28700 9 R P81155 Voltage-dependent anion-selective channel protein 2 31700 9 N
96 O54975 Xaa-Pro aminopeptidase 1 69600 9 CYT P35213 14-3-3 protein beta/alpha 28000 8 N P13233 2',3'-cyclic-nucleotide 3'-phosphodiesterase 47200 8 N Q63570 26S protease regulatory subunit 6B 47400 8 P Q5XI78 2-oxoglutarate dehydrogenase, mitochondrial 116200 8 M P38983 40S ribosomal protein SA 32800 8 N P61314 60S ribosomal protein L15 24100 8 N P09895 60S ribosomal protein L5 34400 8 N B0K031 60S ribosomal protein L7 30300 8 N Q5XIE0 Acidic leucine-rich nuclear phosphoprotein 32 family member E 29400 8 N D3ZAN3 Alpha glucosidase 2 alpha neutral subunit (Predicted) 90500 8 CM Q6MG08 ATP-binding cassette sub-family F member 1 95200 8 N Q9QX80 CArG-binding factor A 30800 8 N D3ZZZ9 Catenin (Cadherin associated protein), delta 1 (Predicted), isoform CRA_a OS=Rattus norvegicus GN=Ctnnd1 PE=1 SV=1 - [D3ZZZ9_RAT] 104000 8 N G3V936 Citrate synthase 51800 8 N O35828 Coronin-7 100700 8 CM Q62698 Cytoplasmic dynein 1 light intermediate chain 2 54700 8 CYT F1LQT9 Cytosine-specific methyltransferase 182300 8 N Q6P6R2 Dihydrolipoyl dehydrogenase, mitochondrial 54000 8 M A9CMB8 DNA replication licensing factor MCM6 92800 8 N Q8R4A1 ERO1-like protein alpha 54000 8 ER Q6P685 Eukaryotic translation initiation factor 2, subunit 2 (Beta) 38200 8 N Q641X8 Eukaryotic translation initiation factor 3 subunit E 52200 8 N Q07205 Eukaryotic translation initiation factor 5 48900 8 N Q5XI32 F-actin-capping protein subunit beta 30600 8 CYT P14408 Fumarate hydratase, mitochondrial 54400 8 CYT Q4V7C6 GMP synthase [glutamine-hydrolyzing] 76700 8 CYT G3V6A4 Heterogeneous nuclear ribonucleoprotein D, isoform CRA_b 32700 8 N R9PXZ2 Heterogeneous nuclear ribonucleoprotein Q (Fragment) 57400 8 - D4A2D7 Importin 4 (Predicted), isoform CRA_b 118900 8 CYT G3V824 Insulin-like growth factor 2 receptor, isoform CRA_b 273400 8 N Q9JHL4-2 Isoform 2 of Drebrin-like protein 48300 8 CM Q9WV25-2 Isoform 2 of Poly(U)-binding-splicing factor PUF60 58500 8 N Q925Q9-2 Isoform 2 of SH3 domain-containing kinase- binding protein 1 73200 8 CSK
97 Q9Z1W6-4 Isoform 4 of Protein LYRIC 57900 8 - Q924S5 Lon protease homolog, mitochondrial 105700 8 N Q5XIM7 Lysine--tRNA ligase 71600 8 CYT Q6AYC4 Macrophage-capping protein 38800 8 EXT D3ZJH9 Malic enzyme 65300 8 M Q66HR2 Microtubule-associated protein RP/EB family member 1 30000 8 CYT Q6AYN8 Minichromosome maintenance deficient 7 (S. cerevisiae) OS=Rattus norvegicus GN=Mcm7 PE=2 SV=1 - [Q6AYN8_RAT] 81000 8 N R9PXR4 Mitochondrial import receptor subunit TOM70 61900 8 M P51583 Multifunctional protein ADE2 47100 8 CSK O35987 NSFL1 cofactor p47 40700 8 N D4A7R3 Nucleoporin 205kDa (Predicted) 227100 8 CYT G3V6H9 Nucleosome assembly protein 1-like 1 45300 8 N F1SW39 PC4 and SFRS1 interacting protein 1 59600 8 N P10111 Peptidyl-prolyl cis-trans isomerase A 17900 8 EXT P24368 Peptidyl-prolyl cis-trans isomerase B 23800 8 N Q9R063 Peroxiredoxin-5, mitochondrial 22200 8 N G3V918 Phosphoribosylglycinamide formyltransferase, isoform CRA_a 107500 8 CYT P63004 Platelet-activating factor acetylhydrolase IB subunit alpha 46600 8 C Q9EPH8 Polyadenylate-binding protein 1 70700 8 N P04961 Proliferating cell nuclear antigen 28700 8 N Q66HK3 Prostaglandin G/H synthase 1] 69000 8 N Q6P9V7 Proteasome (Prosome, macropain) activator subunit 1 28600 8 P G3V829 Protein Fubp3 61400 8 N G3V681 Protein Mcm4 96500 8 CM D3ZBL6 Protein Nup160 155400 8 - Q5HZV9 Protein phosphatase 1 regulatory subunit 7 41300 8 N F1MAA5 Protein Rangap1 63100 8 C G3V8T5 Protein Ruvbl2 51100 8 N F1LNL2 Protein Smarca5 116600 8 C F1LM47 Protein Sucla2 50300 8 M D4A8H3 Protein Uba6 117900 8 CYT E9PTR4 Protein Ubap2l 116800 8 - Q4QQS7 Protein Umps 52300 8 N D3ZXK9 Purine nucleoside phosphorylase (Fragment) 32100 8 - F1LQN3 Reticulon 126300 8 ER P22509 rRNA 2'-O-methyltransferase fibrillarin 34200 8 N P60123 RuvB-like 1 50200 8 N
98 D4A0F5 Septin-7 50600 8 - O08623 Sequestosome-1 47700 8 N Q6AYB5 Signal recognition particle 54 kDa protein 55700 8 N P13668 Stathmin 17300 8 CYT P07632 Superoxide dismutase [Cu-Zn] 15900 8 EXT Q920J4 Thioredoxin-like protein 1 32200 8 P Q5M7V8 Thyroid hormone receptor-associated protein 3 108200 8 N Q5XFX0 Transgelin-2 22400 8 - P09495 Tropomyosin alpha-4 chain 28500 8 CYT F8WFH8 Tryptophan--tRNA ligase, cytoplasmic 53400 8 CYT Q5RJR2 Twinfilin-1 40100 8 CYT Q00981 Ubiquitin carboxyl-terminal hydrolase isozyme L1 24800 8 CYT P63326 40S ribosomal protein S10 18900 7 NUC P17074 40S ribosomal protein S19 16100 7 CYT M0RD75 40S ribosomal protein S6 (Fragment) 28400 7 R P62083 40S ribosomal protein S7 22100 7 N P62243 40S ribosomal protein S8 OS=Rattus norvegicus GN=Rps8 PE=1 SV=2 - [RS8_RAT] 24200 7 N Q6PDV7 60S ribosomal protein L10 24600 7 CYT P23358 60S ribosomal protein L12 17800 7 N P17077 60S ribosomal protein L9 21900 7 N P85968 6-phosphogluconate dehydrogenase, decarboxylating 53200 7 N P85971 6-phosphogluconolactonase 27200 7 CYT Q5XI22 Acetyl-CoA acetyltransferase, cytosolic 41100 7 N Q5XI77 Annexin 54100 7 CYT Q6QI09 ATP synthase subunit gamma, mitochondrial 67700 7 - P30835 ATP-dependent 6-phosphofructokinase, liver type 85300 7 CYT Q9Z1Y3 Cadherin-2 99600 7 CYT P47727 Carbonyl reductase [NADPH] 1 30600 7 N O70509 CD44 antigen 39700 7 N Q6MG61 Chloride intracellular channel protein 1 27000 7 N Q5RJK5 Chromobox homolog 3 (HP1 gamma homolog, Drosophila) 20800 7 C Q66H80 Coatomer subunit delta 57200 7 CM Q02874 Core histone macro-H2A.1 39500 7 C B1WBY1 Cul1 protein 89600 7 - Q6Q0N1 Cytosolic non-specific dipeptidase 52700 7 C P43138 DNA-(apurinic or apyrimidinic site) lyase 35500 7 N Q6TUG0 DnaJ homolog subfamily B member 11 40500 7 N Q01986 Dual specificity mitogen-activated protein kinase kinase 1 43400 7 N F1MAP9 E3 ubiquitin-protein ligase HUWE1 460200 7 -
99 Q8R3Z7 EH-domain containing 4 61400 7 ER B5DF91 ELAV (Embryonic lethal, abnormal vision, Drosophila)-like 1 (Hu antigen R) 36100 7 N D3ZJR1 Epidermal growth factor receptor pathway substrate 15-like 1 96400 7 CYT D3ZUV3 Eukaryotic translation initiation factor 2 subunit 1 60200 7 CYT Q6P9U8 Eukaryotic translation initiation factor 3 subunit H 39900 7 CYT D3ZAZ0 Eukaryotic translation initiation factor 3 subunit M 42500 7 CYT Q5RKG9 Eukaryotic translation initiation factor 4B 69000 7 - Q32PX7 Far upstream element-binding protein 1 67200 7 N Q5XI19 Fermitin family homolog 2 (Drosophila) 77800 7 N Q6AYD5 G1 to S phase transition 1 68700 7 CSK Q9Z339 Glutathione S-transferase omega-1 27700 7 CYT Q6AYB4 Heat shock 70 kDa protein 14 54400 7 CYT D3ZBN0 Histone H1.5 22600 7 C P62804 Histone H4 11400 7 C Q63692 Hsp90 co-chaperone Cdc37 44500 7 CYT D4A857 Importin 9 (Predicted) 115900 7 CYT G3V667 Integrin, alpha 6, isoform CRA_a 119400 7 CM Q7TP98 Interleukin enhancer-binding factor 2 51300 7 N Q9JIL3 Interleukin enhancer-binding factor 3 95900 7 N P41562 Isocitrate dehydrogenase [NADP] cytoplasmic 46700 7 CYT P56574 Isocitrate dehydrogenase [NADP], mitochondrial 50900 7 M Q5U2Y1-2 Isoform 2 of General transcription factor II-I 103000 7 N B3GNI6-3 Isoform 3 of Septin-11 48900 7 CYT Q6MG49 Large proline-rich protein BAG6 119900 7 N Q99MZ8 LIM and SH3 domain protein 1 30000 7 CYT Q64119 Myosin light polypeptide 6 17000 7 - P52590 Nuclear pore complex protein Nup107 107100 7 C Q9QZ86 Nucleolar protein 58 60000 7 N Q9QVC8 Peptidyl-prolyl cis-trans isomerase FKBP4 51400 7 N F1LPK7 Plastin 3 (T-isoform), isoform CRA_a 70700 7 CYT P62963 Profilin-1 14900 7 N R9PXR7 Prostaglandin E synthase 3 (Fragment) 18600 7 - E9PST5 Protein Acin1 150900 7 N D3ZD73 Protein Ddx6 54200 7 CYT D4ABT8 Protein Hnrnpul2 84800 7 N Q5GFD9 Protein IMPACT 36000 7 CYT D3ZFA8 Protein LOC100362366 155000 7 R M0RD20 Protein LOC100911363 254000 7 - Q99MI5 Protein LOC100912604 34000 7 - D4A2G9 Protein Ranbp1 23600 7 N
100 F1LVV4 Protein Rcc2 (Fragment) 46600 7 N F1LM55 Protein RGD1309922 102700 7 N D3ZQM0 Protein Sf3a1 88500 7 SC B0BMT9 Protein Sqrdl 50200 7 M D4A9L2 Protein Srsf1 27700 7 N F1LQP9 Protein Tnpo1 (Fragment) 102100 7 - D4AAM0 Protein Tnpo3 104100 7 CYT D3ZRN5 Protein Trove2 60000 7 NUC F2Z3T9 Protein U2af2 53500 7 SC O35509 Ras-related protein Rab-11B 24500 7 CYT Q5XI73 Rho GDP-dissociation inhibitor 1 23400 7 N O88453 Scaffold attachment factor B1 104500 7 N Q5XIG8 Serine-threonine kinase receptor-associated protein 38400 7 N Q920L2 Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial 71600 7 M P63029 Translationally-controlled tumor protein 19400 7 N Q6AYT3 tRNA-splicing ligase RtcB homolog 55200 7 N X1WI37 Uncharacterized protein (Fragment) 29400 7 R M0R7Z0 Uncharacterized protein 137500 7 - F1LS04 Unconventional myosin-Ie (Fragment) 114100 7 - Q6AY86 Vacuolar protein sorting-associated protein 26A 38100 7 CYT P26772 10 kDa heat shock protein, mitochondrial 10900 6 CYT P62193 26S protease regulatory subunit 4 49200 6 P Q62785 28 kDa heat- and acid-stable phosphoprotein 20600 6 - O70351 3-hydroxyacyl-CoA dehydrogenase type-2 27200 6 M P62250 40S ribosomal protein S16 16400 6 R P62268 40S ribosomal protein S23 15800 6 CYT B0BN81 40S ribosomal protein S5 22900 6 R P62914 60S ribosomal protein L11 20200 6 N D3ZRM9 60S ribosomal protein L13 24200 6 R P62718 60S ribosomal protein L18a 20700 6 R P84100 60S ribosomal protein L19 23500 6 CYT Q5M7U6 Actin-related protein 2 44700 6 CYT O88656 Actin-related protein 2/3 complex subunit 1B 41000 6 CYT P85970 Actin-related protein 2/3 complex subunit 2 34400 6 CYT Q05962 ADP/ATP translocase 1 33000 6 N Q64057 Alpha-aminoadipic semialdehyde dehydrogenase 58700 6 N G3V9N8 AP-1 complex subunit beta-1 103800 6 GA Q9ER24 Ataxin-10 53700 6 CYT B3DMA1 Atxn2l protein 112800 6 CYT Q6AY58 B-cell receptor-associated protein 31 27900 6 CM Q9WU82 Catenin beta-1 85455 6 N
101 P00787 Cathepsin B 37400 6 EXT O08837 Cell division cycle 5-like protein 92200 6 N G3V8C4 Chloride intracellular channel 4, isoform CRA_b 28700 6 N P32551 Cytochrome b-c1 complex subunit 2, mitochondrial 48400 6 M G3V6S2 Cytoplasmic aconitate hydratase 98100 6 CYT Q5XHY0 DEAD (Asp-Glu-Ala-Asp) box polypeptide 18 75500 6 CM G3V6P2 Dihydrolipoamide S-succinyltransferase (E2 component of 2-oxo-glutarate complex), isoform CRA_a 48900 6 N O88791 Dual specificity mitogen-activated protein kinase kinase 1 11700 6 C Q8VHU4 Elongator complex protein 1 149100 6 N Q3B8Q2 Eukaryotic initiation factor 4A-III 46800 6 N P55053 Fatty acid-binding protein, epidermal 15000 6 CYT Q5XI81 Fragile X mental retardation syndrome-related protein 1 63900 6 N Q66H61 Glutaminyl-tRNA synthetase 87600 6 CYT Q4QQW4 Histone deacetylase 1 55100 6 C M0R4L7 Histone H2B 13900 6 C F1LNF7 Isocitrate dehydrogenase [NAD] subunit alpha, mitochondrial 39600 6 - Q6TRW4-3 Isoform 3 of Sister chromatid cohesion protein PDS5 homolog B 164100 6 - Q62733 Lamina-associated polypeptide 2, isoform beta 50200 6 C Q64654 Lanosterol 14-alpha demethylase 56700 6 ER Q5XIN6 LETM1 and EF-hand domain-containing protein 1, mitochondrial 83000 6 MM Q5RJR8 Leucine-rich repeat-containing protein 59 34800 6 N P42123 L-lactate dehydrogenase B chain 36600 6 CYT B5DEG8 LOC685144 protein 118400 6 - O88989 Malate dehydrogenase, cytoplasmic 36500 6 CYT Q63560 Microtubule-associated protein 6 100400 6 CYT P62076 Mitochondrial import inner membrane translocase subunit Tim13 10500 6 M F1LMW7 Myristoylated alanine-rich C-kinase substrate 29900 6 - P20070 NADH-cytochrome b5 reductase 3 34200 6 CYT Q5I0M2 Nicotinate-nucleotide pyrophosphorylase [carboxylating] 31300 6 CYT D4A3S8 NOL1/NOP2/Sun domain family, member 2 (Predicted) 88000 6 N P49793 Nuclear pore complex protein Nup98-Nup96 197200 6 C F1LPS3 Nucleolar and coiled-body phosphoprotein 1 73500 6 - P35704 Peroxiredoxin-2 21800 6 CYT P97852 Peroxisomal multifunctional enzyme type 2 79400 6 M
102 G3V741 Phosphate carrier protein, mitochondrial 39500 6 N P31044 Phosphatidylethanolamine-binding protein 1 20800 6 N E9PSV5 Phosphoserine aminotransferase 40500 6 F1MAL6 Plectin (Fragment) 518000 6 CSK G3V8G2 Proteasome (Prosome, macropain) 26S subunit, non-ATPase, 5 (Predicted), isoform CRA_a 55800 6 P Q6PCT9 Proteasome (Prosome, macropain) 26S subunit, non-ATPase, 6 45600 6 P Q4G079 Protein Aimp1 34600 6 CYT D4A450 Protein Bclaf1 184500 6 D3ZDC1 Protein Drg2 40700 6 CYT F1M3H8 Protein Hnrnpa0 30300 6 - M0R9D0 Protein LOC100909983 28300 6 - Q5BJP4 Protein LOC100910882 58600 6 N B0BNK1 Protein Rab5c 23400 6 CM Q6B345 Protein S100-A11 11100 6 N D3ZJU5 Protein Smarcc1 120400 6 C Q3ZB99 Protein Tjp2 131300 6 N Q5XI21 Protein Tom1 54100 6 CYT D3ZAI0 Protein U2surp 118200 6 N B5DEH4 Protein Uap1l1 56400 6 CSK D3ZF89 Protein Ubap2 117500 6 - B2RZA9 Protein Ube2l3 17900 6 N G3V9H0 RAS p21 protein activator 1, isoform CRA_c 115400 6 CSK Q6T5E8 RCG55639, isoform CRA_g 59500 6 CM F1LRB8 S-adenosylmethionine synthase 43600 6 CSK Q09167 Serine/arginine-rich splicing factor 5 30900 6 N B2RYI2 Signal recognition particle subunit SRP68 70400 6 CYT O70593 Small glutamine-rich tetratricopeptide repeat- containing protein alpha 34100 6 CYT Q07647 Solute carrier family 2, facilitated glucose transporter member 3 53500 6 CM D3ZZ38 Sorting nexin 67800 6 EN Q63413 Spliceosome RNA helicase Ddx39b 49000 6 N F1LQB2 Structural maintenance of chromosomes protein 3 138600 6 N D4A5Q2 Structural maintenance of chromosomes protein 134200 6 C B0BN85 Suppressor of G2 allele of SKP1 homolog 38100 6 N R9PXU4 Thioredoxin reductase 1, cytoplasmic 54400 6 N G3V679 Transferrin receptor protein 1 85800 6 EXT F1M951 Tyrosine-protein phosphatase non-receptor type 23 182100 6 N Q9EQX9 Ubiquitin-conjugating enzyme E2 N 17100 6 N Q4KM73 UMP-CMP kinase 22200 6 N E9PT22 Uncharacterized protein 13900 6 -
103 Q9Z270 Vesicle-associated membrane protein-associated protein A 27800 6 CM Q9R1Z0 Voltage-dependent anion-selective channel protein 3 30800 6 N P62282 40S ribosomal protein S11 18400 5 CYT Q6PDW1 40S ribosomal protein S12 14500 5 R P62246 40S ribosomal protein S15a 14800 5 CYT P62853 40S ribosomal protein S25 13700 5 N Q794F9 4F2 cell-surface antigen heavy chain 58000 5 N D4A914 5'-3' exoribonuclease 2 (Predicted), isoform CRA_c 63800 5 N P83732 60S ribosomal protein L24 17800 5 CYT P12749 60S ribosomal protein L26 17300 5 R P62919 60S ribosomal protein L8 28000 5 CYT B2RZ72 Actin-related protein 2/3 complex, subunit 4 (Predicted), isoform CRA_a 19700 5 CYT Q5M9H2 Acyl-Coenzyme A dehydrogenase, very long chain 70800 5 N Q9QYL8 Acyl-protein thioesterase 2 24800 5 CYT P36972 Adenine phosphoribosyltransferase 19500 5 CYT F1LPQ9 A-kinase anchor protein 2 95900 5 - P12711 Alcohol dehydrogenase class-3 39600 5 N P07943 Aldose reductase 35800 5 CYT Q6AYS7 Aminoacylase-1A 45800 5 CYT O88321 Antisecretory factor 41000 5 P P18484 AP-2 complex subunit alpha-2 104000 5 GA P62944 AP-2 complex subunit beta 104500 5 CM P84092 AP-2 complex subunit mu 49600 5 M B1WC49 Api5 protein 56700 5 N P20673 Argininosuccinate lyase 51500 5 N P49088 Asparagine synthetase [glutamine-hydrolyzing] 64200 5 CSK F1LR13 Atlastin-3 60600 5 - Q06647 ATP synthase subunit O, mitochondrial 23400 5 N D3ZD23 ATP-binding cassette, sub-family E (OABP), member 1 67300 5 CYT P47860 ATP-dependent 6-phosphofructokinase, platelet type 85700 5 N Q9WTT7 Basic leucine zipper and W2 domain-containing protein 2 48000 5 CM Q5U2U8 Bcl2-associated athanogene 3 61500 5 CYT P70645 Bleomycin hydrolase 52300 5 N Q05175 Brain acid soluble protein 1 21800 5 N P62161 Calmodulin 16800 5 N P62634 Cellular nucleic acid-binding protein 19400 5 N D3ZHP6 Condensin complex subunit 1 155500 5 C
104 Q4KM69 COP9 (Constitutive photomorphogenic) homolog, subunit 5 (Arabidopsis thaliana) 37600 5 N Q68FW9 COP9 signalosome complex subunit 3 47800 5 N P47875 Cysteine and glycine-rich protein 1 20600 5 N G3V9K0 Cysteinyl-tRNA synthetase (Predicted), isoform CRA_b] 85500 5 CYT G3V7G0 Cytoplasmic dynein 1 light intermediate chain 1 56600 5 C F1M624 DNA ligase (Fragment) 101700 5 - Q5M9H7 DnaJ (Hsp40) homolog, subfamily A, member 2 45700 5 N Q641Y0 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit 48900 5 ER G3V7V4 Ectonucleotide pyrophosphatase/phosphodiesterase family member 1 102700 5 ER P85834 Elongation factor Tu, mitochondrial 49500 5 M G3V982 Engulfment and cell motility 2, ced-12 homolog (C. elegans), isoform CRA_b 83700 5 CYT Q5RK09 Eukaryotic translation initiation factor 3 subunit G 35600 5 N B0BNA7 Eukaryotic translation initiation factor 3 subunit I 36400 5 CYT Q3KRD8 Eukaryotic translation initiation factor 6 26600 5 N Q9Z1X1 Extended synaptotagmin-1 121100 5 ER Q04931 FACT complex subunit SSRP1 80900 5 N Q5RKH2 Galactokinase 1 42300 5 CYT Q5FVM2 Glucose-6-phosphatase 29500 5 P P82808 Glutamine--fructose-6-phosphate aminotransferase [isomerizing] 1 76800 5 - P40615 H/ACA ribonucleoprotein complex subunit 4 56600 5 N Q5XHZ0 Heat shock protein 75 kDa, mitochondrial 80400 5 M P62749 Hippocalcin-like protein 1 22300 5 CM P50503 Hsc70-interacting protein 41300 5 CYT D3ZXS8 Huntingtin interacting protein 2 (Predicted), isoform CRA_a 22400 5 N P27605 Hypoxanthine-guanine phosphoribosyltransferase 24500 5 CYT D4ACG2 IlvB (Bacterial acetolactate synthase)-like (Predicted), isoform CRA_c 72200 5 CM P29410-2 Isoform 2 of Adenylate kinase 2, mitochondrial 25500 5 - Q9JJ50-2 Isoform 2 of Hepatocyte growth factor-regulated tyrosine kinase substrate 85700 5 CYT Q9QY17-4 Isoform 4 of Protein kinase C and casein kinase substrate in neurons 2 protein 51300 5 CYT Q63768-2 Isoform Crk-I of Adapter molecule crk 22800 5 - Q07266-2 Isoform E1 of Drebrin 72600 5 CYT Q91V33 KH domain-containing, RNA-binding, signal transduction-associated protein 1 48300 5 N D3ZHG2 Kinesin light chain 1 61600 5 - F1M8L1 Kinesin-like protein KIF2A 79600 5 N
105 B0BN63 LOC681996 protein 38100 5 CYT P15650 Long-chain specific acyl-CoA dehydrogenase, mitochondrial 47800 5 M P18163 Long-chain-fatty-acid--CoA ligase 1 78100 5 M P0C5W1 Microtubule-associated protein 1S 102700 5 N D4A4P1 Myc box-dependent-interacting protein 1 61100 5 CYT D3ZQ59 Nardilysin 140600 5 M Q99MI7 NEDD8-activating enzyme E1 catalytic subunit 51700 5 N F1M7W7 NEDD8-activating enzyme E1 regulatory subunit 60600 5 - G3V6P1 Nerve growth factor receptor (TNFR superfamily, member 16) 45400 5 N B4F7E8 Niban-like protein 1 84700 5 N Q9EPJ0 Nuclear ubiquitous casein and cyclin-dependent kinase substrate 1 27100 5 N G3V6F4 O-linked N-acetylglucosamine (GlcNAc) transferase (UDP-N- acetylglucosamine:polypeptide-N- acetylglucosaminyl transferase), isoform CRA_b 115700 5 N Q5XHZ9 Pachytene checkpoint protein 2 homolog 48400 5 N O35244 Peroxiredoxin-6 24800 5 CYT E9PTD2 Phosphatidylinositol-binding clathrin assembly protein 64600 5 - D3ZUF9 Pitrilysin metallepetidase 1 (Predicted) 108500 5 M Q99ML5 Prenylcysteine oxidase 56300 5 V Q9QZA2 Programmed cell death 6-interacting protein 96600 5 EXT P54001 Prolyl 4-hydroxylase subunit alpha-1 60900 5 M Q63798 Proteasome activator complex subunit 2 26800 5 P F1M446 Protein AI314180 (Fragment) 203500 5 - D3ZIE9 Protein Aldh18a1 87300 5 CYT D3ZC56 Protein Dst (Fragment) 827800 5 CSK D4A3E1 Protein Hnrnpll 64300 5 N D3ZZK1 Protein LOC100359563 13300 5 R D3ZPN7 Protein LOC100360604 18700 5 R D4ADF5 Protein LOC100912106 14200 5 N D3ZFY8 Protein LOC100912618 16300 5 N M0RCY2 Protein LOC683961 17800 5 R G3V928 Protein Lrp1 504600 5 CYT F1LT49 Protein Lrrc47 63500 5 - F1LX70 Protein Papss1 67900 5 - F7EPH4 Protein Ppa1 32800 5 CYT F1MAH5 Protein Ppp6r3 94200 5 - Q6MG48 Protein PRRC2A 228900 5 N E9PTI6 Protein Raly 30000 5 -
106 D3ZJD3 Protein RGD1565183 15600 5 R Q6P9U0 Protein Serpinb6 43000 5 N F1LM37 Protein Sf1 59700 5 SC D4AE49 Protein Skiv2l2 (Fragment) 103300 5 - E9PU13 Protein Snx4 51900 5 CYT Q5BJN3 Protein Tial1 43400 5 N D3ZF26 Protein Tnks1bp1 179900 5 M P61621 Protein transport protein Sec61 subunit alpha isoform 1 52200 5 ER F1M7B8 Protein Ube3a 99700 5 N D3ZQE8 Protein Xpo5 136800 5 N P06302 Prothymosin alpha 12400 5 N B0BMW0 RAB14, member RAS oncogene family 23900 5 M Q5FVC2 Rho guanine nucleotide exchange factor 2 111800 5 CYT Q62991 Sec1 family domain-containing protein 1 72200 5 CYT P63331 Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform 35600 5 N B0BNE5 S-formylglutathione hydrolase 31300 5 CYT Q5PQX1 Torsin-1A-interacting protein 1 65600 5 N Q4KLL0 Transcription elongation factor A protein 1 33900 5 N Q66HG5 Transmembrane 9 superfamily member 2 75500 5 EN Q63584 Transmembrane emp24 domain-containing protein 10 24800 5 CM Q60587 Trifunctional enzyme subunit beta, mitochondrial 51400 5 M Q6PEC1 Tubulin-specific chaperone A 12700 5 C B2RYG6 Ubiquitin thioesterase OTUB1 31300 5 N M0R735 Uncharacterized protein (Fragment) 17000 5 - E9PT29 Uncharacterized protein 47400 5 - D3ZET9 Uncharacterized protein 125700 5 - Q499V1 Uridine phosphorylase 34100 5 CYT
*Abbreviations for cellular locations: Cell membrane- CM, chromosome- C, cytoplasm- CYT, cytoskeleton- CSK, endoplasmic reticulum- ER, endosome- EN, extracellular- EXT, golgi apparatus- GA, mitochondrion- M, nucleus- N, ribosome- R. The locations of some proteins were ommiteed because they were not provided by Proteome Discoverer.
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