An Overview of The Phosphoproteomic Workflow

Abstract

Proteomic studies permit the evaluation of pathological or physiological events in more detail through technological developments in the fields of mass spectrometry and chromatography. Protein phosphorylation, an important research area of mass spectrometry-based proteomic studies, plays an active role as a dynamic marker of molecular activity in the pathological alteration of cellular processes. Therefore, phosphoproteomic studies are gaining importance in the elucidation of molecular mechanisms of diseases and the development of effective diagnostic and treatment methods. Identification and quantitative analysis of phosphoproteins requires the use of highly efficient and reproducible methods. The development of existing methods is ongoing in order to maximize the efficiency of phosphoproteomic applications. We hope that this study, which explains the basic methodology of mass spectrometry-based phosphoproteomic research and discusses its practical challenges, will be useful for researchers interested in MS-based phosphoproteomics.

Keywords

• Phosphoproteomics
• sample preparation
• enrichment
• quantitative phosphoproteomics
• mass spectrometry

Fosfoproteomik Uygulama Basamaklarına Genel Bakış

Abstract

Proteomik çalışmalar, kütle spektrometresi ve kromatografi alanındaki teknolojik gelişmeler sayesinde patolojik ya da fizyolojik olayları daha detaylı olarak değerlendirebilmemize imkan sağlamaktadır. Kütle spektrometresi tabanlı proteomik çalışmaların önemli bir araştırma alanı olan protein fosforilasyonları, hücresel işleyişin patolojik değişiminde moleküler aktivitenin dinamik belirteçleri olarak etkin rol oynar. Bu nedenle hastalıkların moleküler mekanizmalarının aydınlatılmasında ve etkin tanı-tedavi yöntemlerinin geliştirilmesinde fosfoproteomik çalışmalar artan öneme sahiptir. Fosfoproteinlerin tanımlanması ve miktar analizlerinin yapılabilmesi oldukça verimli tekrarlanabilir yöntemlerin kullanılmasını gerektirir. Bu amaçla fosfoproteomik uygulamalardaki verimliliğin en üst düzeye çıkarılmasında mevcut yöntemler geliştirilmeye devam etmektedir. Kütle spektrometresi tabanlı fosfoproteomik araştırmaların temel metodolojisinin anlatıldığı ve uygulamalarda karşılaşılan zorlukların tartışıldığı bu çalışmanın, MS tabanlı fosfoproteomik alanına ilgi duyan araştırmacılara yardımcı olacağını umuyoruz.

Keywords

• Fosfoproteomik
• örnek hazırlığı
• zenginleştirme
• niceliksel fosfoproteomik
• kütle spetrometresi

References

1. Abdallah C, Dumas-Gaudot E, Renaut J, Sergeant K. Gel-based and gel-free quantitative proteomics approaches at a glance. Int J Plant Genomics. 2012;2012:494572.
2. Aebersold R, Mann M. Mass spectrometry-based proteomics. Nature. 2003;422(6928):198-207.
3. Aebersold R, Mann M. Mass-spectrometric exploration of proteome structure and func-tion. Nature. 2016 Sep 15;537(7620):347-55.
4. Aryal UK, Ross AR. Enrichment and analysis of phosphopeptides under different expe-rimental conditions using titanium dioxide affinity chromatography and mass spectro-metry. Rapid Commun Mass Spectrom. 2010;24(2):219-31.
5. Bantscheff M, Lemeer S, Savitski MM, Kuster B. Quantitative mass spectrometry in pro-teomics: critical review update from 2007 to the present. Anal Bioanal Chem. 2012;404(4):939-65.
6. Batth TS, Olsen JV. Offline High pH Reversed-Phase Peptide Fractionation for Deep Phosphoproteome Coverage. Methods Mol Biol. 2016;1355:179-92.
7. Beltran L, Cutillas PR. Advances in phosphopeptide enrichment techniques for phosphop-roteomics. Amino Acids. 2012;43(3):1009-24.
8. Blacken GR, Volný M, Diener M, Jackson KE, Ranjitkar P, Maly DJ, Turecek F. Reactive landing of gas-phase ions as a tool for the fabrication of metal oxide surfaces for in situ phosphopeptide enrichment. J Am Soc Mass Spectrom. 2009;20(6):915-26.

9. Boersema PJ, Foong LY, Ding VM, Lemeer S, van Breukelen B, Philp R, Boekhorst J, Snel B, den Hertog J, Choo AB, Heck AJ. In-depth qualitative and quantitative profiling of tyrosine phosphorylation using a combination of phosphopeptide immunoaffinity puri-fication and stable isotope dimethyl labeling. Mol Cell Proteomics. 2010;9(1):84-99.
10. Boersema PJ, Mohammed S, Heck AJ. Hydrophilic interaction liquid chromatography (HILIC) in proteomics. Anal Bioanal Chem. 2008;391(1):151-9.
11. Caprioli RM, Farmer TB, Gile J. Molecular imaging of biological samples: localization of peptides and proteins using MALDI-TOF MS. Anal Chem. 1997;69(23):4751-60.
12. Carruthers NJ, Rosenspire AJ, Caruso JA, Stemmer PM. Low level Hg(2+) exposure mo-dulates the B-cell cytoskeletal phosphoproteome. J Proteomics. 2018;173:107-114.
13. Carson RH, Lewis CR, Erickson MN, Zagieboylo AP, Naylor BC, Li KW, Farnsworth PB, Price JC. Imaging regiospecific lipid turnover in mouse brain with desorption elect-rospray ionization mass spectrometry. J Lipid Res. 2017;58(9):1884-1892.
14. Chen H, Talaty NN, Takáts Z, Cooks RG. Desorption electrospray ionization mass spect-rometry for high-throughput analysis of pharmaceutical samples in the ambient environ-ment. Anal Chem. 2005;77(21):6915-27.
15. Chen SY, Juang YM, Chien MW, Li KI, Yu CS, Lai CC. Magnetic iron oxide nanopartic-le enrichment of phosphopeptides on a radiate microstructure MALDI chip. Analyst. 2011;136(21):4454-9.
16. Chen X, Wei S, Ji Y, Guo X, Yang F. Quantitative proteomics using SILAC: Principles, applications, and developments. Proteomics. 2015;15(18):3175-92.
17. Choudhary C, Mann M. Decoding signalling networks by mass spectrometry-based prote-omics. Nat Rev Mol Cell Biol. 2010;11(6):427-39.
18. Croft D, Mundo AF, Haw R, Milacic M, Weiser J, Wu G, Caudy M, Garapati P, Gillespie M, Kamdar MR, Jassal B, Jupe S, Matthews L, May B, Palatnik S, Rothfels K, Sha-movsky V, Song H, Williams M, Birney E, Hermjakob H, Stein L, D'Eustachio P. The Reactome pathway knowledgebase. Nucleic Acids Res. 2014;42 (Database issue):D472-7.
19. de Hoog CL, Mann M. Proteomics. Annu Rev Genomics Hum Genet. 2004;5:267-93. Drabovich Andrei P., et al. "Proteomic and mass spectrometry technologies for biomarker discovery." Proteomic and metabolomic approaches to biomarker discovery 2013; 17-37.
20. Dunn JD, Reid GE, Bruening ML. Techniques for phosphopeptide enrichment prior to analysis by mass spectrometry. Mass Spectrom Rev. 2010 Jan-Feb;29(1):29-54.
21. Dyballa N, Metzger S. Fast and sensitive coomassie staining in quantitative proteomics. Methods Mol Biol. 2012;893:47-59.
22. Edelmann MJ. Strong cation exchange chromatography in analysis of posttranslational modifications: innovations and perspectives. J Biomed Biotechnol. 2011;2011:936508.
23. Espina V, Edmiston KH, Heiby M, Pierobon M, Sciro M, Merritt B, Banks S, Deng J, VanMeter AJ, Geho DH, Pastore L, Sennesh J, Petricoin EF 3rd, Liotta LA. A portrait of tissue phosphoprotein stability in the clinical tissue procurement process. Mol Cell Proteomics. 2008;7(10):1998-2018.
24. Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM. Electrospray ionization for mass spectrometry of large biomolecules. Science. 1989;246(4926):64-71.
25. Fredens J, Færgeman NJ. Quantitative proteomics by amino acid labeling identifies novel NHR-49 regulated proteins in C. elegans. Worm. 2012;1(1):66-71.
26. Geiger T, Velic A, Macek B, Lundberg E, Kampf C, Nagaraj N, Uhlen M, Cox J, Mann M. Initial quantitative proteomic map of 28 mouse tissues using the SILAC mouse. Mol Cell Proteomics. 2013;12(6):1709-22.
27. Gouw JW, Krijgsveld J, Heck AJ. Quantitative proteomics by metabolic labeling of model organisms. Mol Cell Proteomics. 2010;9(1):11-24.
28. Grandjean M, Sermeus A, Branders S, Defresne F, Dieu M, Dupont P, Raes M, De Ridder M, Feron O. Hypoxia integration in the serological proteome analysis unmasks tumor antigens and fosters the identification of anti-phospho-eEF2 antibodies as potential cancer biomarkers. PLoS One. 2013;8(10):e76508.
29. Han C, Yang P. Two Dimensional Gel Electrophoresis-Based Plant Phosphoproteomics. Methods Mol Biol. 2016;1355:213-23.
30. Han G, Ye M, Zhou H, Jiang X, Feng S, Jiang X, Tian R, Wan D, Zou H, Gu J. Large-scale phosphoproteome analysis of human liver tissue by enrichment and fractionation of phosphopeptides with strong anion exchange chromatography. Proteomics. 2008;8(7):1346-61.
31. Harsha HC, Pandey A. Phosphoproteomics in cancer. Mol Oncol. 2010 Dec;4(6):482-95. Harsha HC, Pinto SM, Pandey A. Proteomic strategies to characterize signaling pathways. Methods Mol Biol. 2013;1007:359-77.
32. Herring LE, Grant KG, Blackburn K, Haugh JM, Goshe MB. Development of a tandem affinity phosphoproteomic method with motif selectivity and its application in analysis of signal transduction networks. J Chromatogr B Analyt Technol Biomed Life Sci. 2015;988:166-74.
33. Ho CS, Lam CW, Chan MH, Cheung RC, Law LK, Lit LC, Ng KF, Suen MW, Tai HL. Electrospray ionisation mass spectrometry: principles and clinical applications. Clin Bioc-hem Rev. 2003;24(1):3-12.
34. Honarvar E, Venter AR. Ammonium Bicarbonate Addition Improves the Detection of Proteins by Desorption Electrospray Ionization Mass Spectrometry. J Am Soc Mass Spectrom. 2017;28(6):1109-1117.
35. Hoos MD, Richardson BM, Foster MW, Everhart A, Thompson JW, Moseley MA, Colton CA. Longitudinal study of differential protein expression in an Alzheimer's Mouse model lacking inducible nitric oxide synthase. J Proteome Res. 2013;12(10):4462-77.
36. Iliuk A, Jayasundera K, Schluttenhofer R, Tao WA. Functionalized soluble nanopolymers for phosphoproteome analysis. Methods Mol Biol. 2011;790:277-85.
37. International Human Genome Sequencing Consortium. Finishing the euchromatic sequence of the human genome. Nature. 2004;431(7011):931-45.
38. Ishihama Y, Oda Y, Tabata T, Sato T, Nagasu T, Rappsilber J, Mann M. Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol Cell Proteomics. 2005;4(9):1265-72.
39. Jersie-Christensen RR, Sultan A, Olsen JV. Simple and Reproducible Sample Preparation for Single-Shot Phosphoproteomics with High Sensitivity. Methods Mol Biol. 2016;1355:251-60.
40. Jiang J, Sun X, Li Y, Deng C, Duan G. Facile synthesis of Fe(3)O(4)@PDA core-shell microspheres functionalized with various metal ions: A systematic comparison of com-monly-used metal ions for IMAC enrichment. Talanta. 2018;178:600-607.
41. Jünger MA, Aebersold R. Mass spectrometry-driven phosphoproteomics: patterning the systems biology mosaic. Wiley Interdiscip Rev Dev Biol. 2014;3(1):83-112.
42. Kanshin E, Giguère S, Jing C, Tyers M, Thibault P. Machine Learning of Global Phosp-hoproteomic Profiles Enables Discrimination of Direct versus Indirect Kinase Substrates. Mol Cell Proteomics. 2017;16(5):786-798.
43. Karas M, Krüger R. Ion formation in MALDI: the cluster ionization mechanism. Chem Rev. 2003;103(2):427-40.
44. Kinoshita-Kikuta E, Kinoshita E, Koike T. Phosphopeptide Detection with Biotin-Labeled Phos-tag. Methods Mol Biol. 2016;1355:17-29.
45. Kisluk J, Ciborowski M, Niemira M, Kretowski A, Niklinski J. Proteomics biomarkers for non-small cell lung cancer. J Pharm Biomed Anal. 2014;101:40-9.
46. Kuyama H, Sonomura K, Nishimura O. Sensitive detection of phosphopeptides by matrix-assisted laser desorption/ionization mass spectrometry: use of alkylphosphonic acids as matrix additives. Rapid Commun Mass Spectrom. 2008;22(8):1109-16.
47. Larsen MR, Thingholm TE, Jensen ON, Roepstorff P, Jørgensen TJ. Highly selective en-richment of phosphorylated peptides from peptide mixtures using titanium dioxide micro-columns. Mol Cell Proteomics. 2005;4(7):873-86.
48. Leitner A. Enrichment Strategies in Phosphoproteomics. Methods Mol Biol. 2016;1355:105-21. Li L, Wei Y, To C, Zhu CQ, Tong J, Pham NA, Taylor P, Ignatchenko V, Ignatchenko A, Zhang W, Wang D, Yanagawa N, Li M, Pintilie M, Liu G, Muthuswamy L, Shepherd FA, Tsao MS, Kislinger T, Moran MF. Integrated omic analysis of lung cancer reveals meta-bolism proteome signatures with prognostic impact. Nat Commun. 2014;5:5469.
49. Li R, Liao G, Nirujogi RS, Pinto SM, Shaw PG, Huang TC, Wan J, Qian J, Gowda H, Wu X, Lv DW, Zhang K, Manda SS, Pandey A, Hayward SD. Phosphoproteomic Profiling Reveals Epstein-Barr Virus Protein Kinase Integration of DNA Damage Response and Mitotic Signaling. PLoS Pathog. 2015;11(12):e1005346.
50. Macek B, Mann M, Olsen JV. Global and site-specific quantitative phosphoproteomics: principles and applications. Annu Rev Pharmacol Toxicol. 2009;49:199-221.
51. Mann M, Hendrickson RC, Pandey A. Analysis of proteins and proteomes by mass spect-rometry. Annu Rev Biochem. 2001;70:437-73.
52. Martins-de-Souza, Daniel. "Shotgun Proteomics." Methods in Molecular Biology 1156 (2014).May C, Brosseron F, Chartowski P, Meyer HE, Marcus K. Differential proteome analysis using 2D-DIGE. Methods Mol Biol. 2012;893:75-82.
53. May C, Brosseron F, Pfeiffer K, Meyer HE, Marcus K. Proteome analysis with classical 2D-PAGE. Methods Mol Biol. 2012;893:37-46.
54. McLachlin DT, Chait BT. Improved beta-elimination-based affinity purification strategy for enrichment of phosphopeptides. Anal Chem. 2003;75(24):6826-36.
55. Nardiello D, Palermo C, Natale A, Quinto M, Centonze D. Strategies in protein sequen-cing and characterization: multi-enzyme digestion coupled with alternate CID/ETD tan-dem mass spectrometry. Anal Chim Acta. 2015;854:106-17.
56. Negroni L, Claverol S, Rosenbaum J, Chevet E, Bonneu M, Schmitter JM. Comparison of IMAC and MOAC for phosphopeptide enrichment by column chromatography. J Chroma-togr B Analyt Technol Biomed Life Sci. 2012;891-892:109-12.
57. Nolte H, Hölper S, Housley MP, Islam S, Piller T, Konzer A, Stainier DY, Braun T, Krü-ger M. Dynamics of zebrafish fin regeneration using a pulsed SILAC approach. Proteo-mics. 2015;15(4):739-51.
58. Olsen JV, Macek B, Lange O, Makarov A, Horning S, Mann M. Higher-energy C-trap dissociation for peptide modification analysis. Nat Methods. 2007;4(9):709-12.
59. Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics. 2002;1(5):376-86.
60. Ong SE, Mann M. Mass spectrometry-based proteomics turns quantitative. Nat Chem Biol. 2005;1(5):252-62.
61. Pan N, Liu P, Cui W, Tang B, Shi J, Chen H. Highly efficient ionization of phosphopepti-des at low pH by desorption electrospray ionization mass spectrometry. Analyst. 2013;138(5):1321-1324.
62. Piersma SR, Knol JC, de Reus I, Labots M, Sampadi BK, Pham TV, Ishihama Y, Verheul HM, Jimenez CR. Feasibility of label-free phosphoproteomics and application to base-line signaling of colorectal cancer cell lines. J Proteomics.2015;127(Pt B):247-58.
63. Premsler T, Lewandrowski U, Sickmann A, Zahedi RP. Phosphoproteome analysis of the platelet plasma membrane. Methods Mol Biol. 2011;728:279-90.
64. Qi Y, Volmer DA. Electron-based fragmentation methods in mass spectrometry: An over-view. Mass Spectrom Rev. 2017;36(1):4-15.
65. Ram PT, Mendelsohn J, Mills GB. Bioinformatics and systems biology. Mol Oncol. 2012;6(2):147-54.
66. Ravikumar V, Macek B, Mijakovic I. Resources for Assignment of Phosphorylation Sites on Peptides and Proteins. Methods Mol Biol. 2016;1355:293-306.
67. Rolland D, Basrur V, Conlon K, Wolfe T, Fermin D, Nesvizhskii AI, Lim MS, Elenitoba-Johnson KS. Global phosphoproteomic profiling reveals distinct signatures in B-cell non-Hodgkin lymphomas. Am J Pathol. 2014;184(5):1331-42.
68. Rothenberg DA, Gordon EA, White FM, Lourido S. Identification of Direct Kinase Substrates Using Analogue-Sensitive Alleles. Methods Mol Biol. 2016;1355:71-84.
69. Ruprecht B, Koch H, Medard G, Mundt M, Kuster B, Lemeer S. Comprehensive and rep-roducible phosphopeptide enrichment using iron immobilized metal ion affinity chroma-tography (Fe-IMAC) columns. Mol Cell Proteomics. 2015;14(1):205-15.
70. Rush J, Moritz A, Lee KA, Guo A, Goss VL, Spek EJ, Zhang H, Zha XM, Polakiewicz RD, Comb MJ. Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat Biotechnol. 2005;23(1):94-101.
71. Shenoy A, Geiger T. Super-SILAC: current trends and future perspectives. Expert Rev Proteomics. 2015;12(1):13-9.
72. Shukla HD, Vaitiekunas P, Cotter RJ. Advances in membrane proteomics and cancer biomarker discovery: current status and future perspective. Proteomics. 2012;12(19-20):3085-104.
73. Silberring, J, A. Drabik. Proteomic Profiling and Analytical Chemistry: The Crossroads (2016): 145.Silva AM, Vitorino R, Domingues MR, Spickett CM, Domingues P. Post-translational modifications and mass spectrometry detection. Free Radic Biol Med. 2013;65:925-41.
74. Song L, Wang F, Dong Z, Hua X, Xia Q. Label-free quantitative phosphoproteomic profi-ling of cellular response induced by an insect cytokine paralytic peptide. J Proteomics. 2017;154:49-58. Stensballe A, Jensen ON. Phosphoric acid enhances the performance of Fe(III) affinity chromatography and matrix-assisted laser desorption/ionization tandem mass spectrometry for recovery, detection and sequencing of phosphopeptides. Rapid Commun Mass Spectrom. 2004;18(15):1721-30.
75. Sury MD, Chen JX, Selbach M. In vivo stable isotope labeling by amino acids in Drosop-hila melanogaster. Methods Mol Biol. 2014;1188:85-93.
76. Thaler F, Valsasina B, Baldi R, Xie J, Stewart A, Isacchi A, Kalisz HM, Rusconi L. A new approach to phosphoserine and phosphothreonine analysis in peptides and proteins: chemical modification, enrichment via solid-phase reversible binding, and analysis by mass spectrometry. Anal Bioanal Chem. 2003;376(3):366-73.
77. Thingholm TE, Jensen ON, Larsen MR. Analytical strategies for phosphoproteomics. Pro-teomics. 2009;9(6):1451-68.
78. Thingholm TE, Jensen ON, Robinson PJ, Larsen MR. SIMAC (sequential elution from IMAC), a phosphoproteomics strategy for the rapid separation of monophosphorylated from multiply phosphorylated peptides. Mol Cell Proteomics. 2008;7(4):661-71.
79. Tsai CF, Hsu CC, Hung JN, Wang YT, Choong WK, Zeng MY, Lin PY, Hong RW, Sung TY, Chen YJ. Sequential phosphoproteomic enrichment through complementary metal-directed immobilized metal ion affinity chromatography. Anal Chem. 2014;86(1):685-93.
80. Tsumoto H, Ra M, Samejima K, Taguchi R, Kohda K. Chemical derivatization of peptides containing phosphorylated serine/threonine for efficient ionization and quantification in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom. 2008;22(7):965-72.
81. van der Mijn JC, Labots M, Piersma SR, Pham TV, Knol JC, Broxterman HJ, Verheul HM, Jiménez CR. Evaluation of different phospho-tyrosine antibodies for label-free phosphoproteomics. J Proteomics. 2015;127(Pt B):259-63.
82. van der Wal Lennart, and Jeroen AA Demmers. "Quantitative Mass Spectrometry-based Proteomics." Recent Advances in Proteomics Research. InTech, 2015.
83. Wan H, Yan J, Yu L, Zhang X, Xue X, Li X, Liang X. Zirconia layer coated mesoporous silica microspheres used for highly specific phosphopeptide enrichment. Talanta. 2010;82(5):1701-7.
84. Wang G, Wu WW, Zeng W, Chou CL, Shen RF. Label-free protein quantification using LC-coupled ion trap or FT mass spectrometry: Reproducibility, linearity, and application with complex proteomes. J Proteome Res. 2006;5(5):1214-23
85. Wang J, Gao L, Lee YM, Kalesh KA, Ong YS, Lim J, Jee JE, Sun H, Lee SS, Hua ZC, Lin Q. Target identification of natural and traditional medicines with quantitative chemi-cal proteomics approaches. Pharmacol Ther. 2016;162:10-22.
86. Wiśniewski JR. Filter-Aided Sample Preparation: The Versatile and Efficient Method for Proteomic Analysis. Methods Enzymol. 2017;585:15-27.
87. Yu F, F. Qiu, and J. Meza. "Desıgn And Statistical Analysis Of Mass-Spectrometry-Based Quantitative Proteomics Data." Proteomic Profiling and Analytical Chemistry: The Cross-roads (2016): 211.
88. Zahari MS, Wu X, Pinto SM, Nirujogi RS, Kim MS, Fetics B, Philip M, Barnes SR, Godfrey B, Gabrielson E, Nevo E, Pandey A. Phosphoproteomic profiling of tumor tissues identifies HSP27 Ser82 phosphorylation as a robust marker of early ischemia. Sci Rep. 2015;5:13660.
89. Zarei M, Sprenger A, Gretzmeier C, Dengjel J. Rapid combinatorial ERLIC-SCX solid-phase extraction for in-depth phosphoproteome analysis. J Proteome Res. 2013;12(12):5989-95.
90. Zarei M, Sprenger A, Metzger F, Gretzmeier C, Dengjel J. Comparison of ERLIC-TiO2, HILIC-TiO2, and SCX-TiO2 for global phosphoproteomics approaches. J Proteome Res. 2011;10(8):3474-83.
91. Zarei M, Sprenger A, Rackiewicz M, Dengjel J. Fast and easy phosphopeptide fractiona-tion by combinatorial ERLIC-SCX solid-phase extraction for in-depth phosphoproteome analysis. Nat Protoc. 2016;11(1):37-45.
92. Zawadzka AM, Schilling B, Cusack MP, Sahu AK, Drake P, Fisher SJ, Benz CC, Gibson BW. Phosphoprotein secretome of tumor cells as a source of candidates for breast cancer biomarkers in plasma. Mol Cell Proteomics. 2014;13(4):1034-49.
93. Zhang H, Xu Y, Filipovic A, Lit LC, Koo CY, Stebbing J, Giamas G. SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1. Br J Cancer. 2013;109(10):2675-84.
94. Zhang Y, Zhang Y, Yu Y. Global Phosphoproteomic Analysis of insulin/Akt/mTORC1/S6K Signaling in Rat Hepatocytes. J Proteome Res. 2017 4;16(8):2825-2835.Zhang Z, Wu S, Stenoien DL, Paša-Tolić L. High-throughput proteomics. Annu Rev Anal Chem (Palo Alto Calif). 2014;7:427-54.
95. Zhao Z, Wu F, Ding S, Sun L, Liu Z, Ding K, Lu J. Label-free quantitative proteomic analysis reveals potential biomarkers and pathways in renal cell carcinoma. Tumour Biol. 2015;36(2):939-51.
96. Zubarev RA. Electron-capture dissociation tandem mass spectrometry. Curr Opin Bio-technol. 2004;15(1):12-6.