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Proteomic Strategies to Characterize Signaling Pathways

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Mass Spectrometry Data Analysis in Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1007))

Abstract

Cells respond to external stimuli by transducing signals through a series of intracellular molecules and eliciting an appropriate response. The cascade of events through which the signals are transduced include post-translational modifications such as phosphorylation and ubiquitylation in addition to formation of multi-protein complexes. Improvements in biological mass spectrometry and protein/peptide microarray technology have tremendously improved our ability to probe proteins, protein complexes, and signaling pathways in a high-throughput fashion. Today, a single mass spectrometry-based investigation of a signaling pathway has the potential to uncover the large majority of known signaling intermediates painstakingly characterized over decades in addition to discovering a number of novel ones. Here, we discuss various proteomic strategies to characterize signaling pathways and provide protocols for phosphoproteomic analysis.

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References

  1. Wang JY (1988) Antibodies for phosphotyrosine: analytical and preparative tool for tyrosyl-phosphorylated proteins. Anal Biochem 172:1–7

    Article  PubMed  CAS  Google Scholar 

  2. Frackelton AR Jr, Posner M, Kannan B, Mermelstein F (1991) Generation of monoclonal antibodies against phosphotyrosine and their use for affinity purification of phosphotyrosine-containing proteins. Methods Enzymol 201:79–92

    Article  PubMed  CAS  Google Scholar 

  3. Kamps MP (1991) Generation and use of anti-phosphotyrosine antibodies for immunoblotting. Methods Enzymol 201:101–110

    Article  PubMed  CAS  Google Scholar 

  4. Gronborg M, Kristiansen TZ, Stensballe A, Andersen JS, Ohara O, Mann M, Jensen ON, Pandey A (2002) A mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho-specific antibodies: identification of a novel protein, Frigg, as a protein kinase A substrate. Mol Cell Proteomics 1:517–527

    Article  PubMed  CAS  Google Scholar 

  5. Pandey A, Podtelejnikov AV, Blagoev B, Bustelo XR, Mann M, Lodish HF (2000) Analysis of receptor signaling pathways by mass spectrometry: identification of vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors. Proc Natl Acad Sci U S A 97:179–184

    Article  PubMed  CAS  Google Scholar 

  6. Pandey A, Fernandez MM, Steen H, Blagoev B, Nielsen MM, Roche S, Mann M, Lodish HF (2000) Identification of a novel immunoreceptor tyrosine-based activation motif-containing molecule, STAM2, by mass spectrometry and its involvement in growth factor and cytokine receptor signaling pathways. J Biol Chem 275:38633–38639

    Article  PubMed  CAS  Google Scholar 

  7. Salomon AR, Ficarro SB, Brill LM, Brinker A, Phung QT, Ericson C, Sauer K, Brock A, Horn DM, Schultz PG, Peters EC (2003) Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry. Proc Natl Acad Sci U S A 100:443–448

    Article  PubMed  CAS  Google Scholar 

  8. Kosako H, Nagano K (2011) Quantitative phosphoproteomics strategies for understanding protein kinase-mediated signal transduction pathways. Expert Rev Proteomics 8:81–94

    Article  PubMed  CAS  Google Scholar 

  9. Stasyk T, Huber LA (2012) Mapping in vivo signal transduction defects by phosphoproteomics. Trends Mol Med 18:43–51

    Article  PubMed  CAS  Google Scholar 

  10. Tinti M, Nardozza AP, Ferrari E, Sacco F, Corallino S, Castagnoli L, Cesareni G (2012) The 4G10, pY20 and p-TYR-100 antibody specificity: profiling by peptide microarrays. N Biotechnol 29:571–577

    Article  PubMed  CAS  Google Scholar 

  11. Ficarro SB, McCleland ML, Stukenberg PT, Burke DJ, Ross MM, Shabanowitz J, Hunt DF, White FM (2002) Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat Biotechnol 20:301–305

    Article  PubMed  CAS  Google Scholar 

  12. Muszynska G, Dobrowolska G, Medin A, Ekman P, Porath JO (1992) Model studies on iron(III) ion affinity chromatography. II. Interaction of immobilized iron(III) ions with phosphorylated amino acids, peptides and proteins. J Chromatogr 604:19–28

    Article  PubMed  CAS  Google Scholar 

  13. Posewitz MC, Tempst P (1999) Immobilized gallium(III) affinity chromatography of phosphopeptides. Anal Chem 71:2883–2892

    Article  PubMed  CAS  Google Scholar 

  14. Cao P, Stults JT (1999) Phosphopeptide analysis by on-line immobilized metal-ion affinity chromatography-capillary electrophoresis-electrospray ionization mass spectrometry. J Chromatogr A 853:225–235

    Article  PubMed  CAS  Google Scholar 

  15. Beausoleil SA, Jedrychowski M, Schwartz D, Elias JE, Villen J, Li J, Cohn MA, Cantley LC, Gygi SP (2004) Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc Natl Acad Sci U S A 101:12130–12135

    Article  PubMed  CAS  Google Scholar 

  16. Han G, Ye M, Zhou H, Jiang X, Feng S, Jiang X, Tian R, Wan D, Zou H, Gu J (2008) Large-scale phosphoproteome analysis of human liver tissue by enrichment and fractionation of phosphopeptides with strong anion exchange chromatography. Proteomics 8:1346–1361

    Article  PubMed  CAS  Google Scholar 

  17. McNulty DE, Annan RS (2008) Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection. Mol Cell Proteomics 7:971–980

    Article  PubMed  CAS  Google Scholar 

  18. Alpert AJ (2008) Electrostatic repulsion hydrophilic interaction chromatography for isocratic separation of charged solutes and selective isolation of phosphopeptides. Anal Chem 80:62–76

    Article  PubMed  CAS  Google Scholar 

  19. Andersson L, Porath J (1986) Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography. Anal Biochem 154:250–254

    Article  PubMed  CAS  Google Scholar 

  20. Feng S, Ye M, Zhou H, Jiang X, Jiang X, Zou H, Gong B (2007) Immobilized zirconium ion affinity chromatography for specific enrichment of phosphopeptides in phosphoproteome analysis. Mol Cell Proteomics 6:1656–1665

    Article  PubMed  CAS  Google Scholar 

  21. Gruhler A, Olsen JV, Mohammed S, Mortensen P, Faergeman NJ, Mann M, Jensen ON (2005) Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway. Mol Cell Proteomics 4:310–327

    Article  PubMed  CAS  Google Scholar 

  22. Zhou H, Low TY, Hennrich ML, van der Toorn H, Schwend T, Zou H, Mohammed S, Heck AJ (2011) Enhancing the identification of phosphopeptides from putative basophilic kinase substrates using Ti (IV) based IMAC enrichment. Mol Cell Proteomics 10(M110):006452

    PubMed  Google Scholar 

  23. Villen J, Beausoleil SA, Gerber SA, Gygi SP (2007) Large-scale phosphorylation analysis of mouse liver. Proc Natl Acad Sci U S A 104:1488–1493

    Article  PubMed  CAS  Google Scholar 

  24. Sano A, Nakamura H (2004) Titania as a chemo-affinity support for the column-switching HPLC analysis of phosphopeptides: application to the characterization of phosphorylation sites in proteins by combination with protease digestion and electrospray ionization mass spectrometry. Anal Sci 20:861–864

    Article  PubMed  CAS  Google Scholar 

  25. Larsen MR, Thingholm TE, Jensen ON, Roepstorff P, Jorgensen TJ (2005) Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics 4:873–886

    Article  PubMed  CAS  Google Scholar 

  26. Molina H, Horn DM, Tang N, Mathivanan S, Pandey A (2007) Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry. Proc Natl Acad Sci U S A 104:2199–2204

    Article  PubMed  CAS  Google Scholar 

  27. Wang Z, Udeshi ND, Slawson C, Compton PD, Sakabe K, Cheung WD, Shabanowitz J, Hunt DF, Hart GW (2010) Extensive crosstalk between O-GlcNAcylation and phosphorylation regulates cytokinesis. Sci Signal 3:ra2

    Article  PubMed  Google Scholar 

  28. Blagoev B, Ong SE, Kratchmarova I, Mann M (2004) Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics. Nat Biotechnol 22:1139–1145

    Article  PubMed  CAS  Google Scholar 

  29. Zhang Y, Wolf-Yadlin A, Ross PL, Pappin DJ, Rush J, Lauffenburger DA, White FM (2005) Time-resolved mass spectrometry of tyrosine phosphorylation sites in the epidermal growth factor receptor signaling network reveals dynamic modules. Mol Cell Proteomics 4:1240–1250

    Article  PubMed  CAS  Google Scholar 

  30. Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P, Mann M (2006) Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127:635–648

    Article  PubMed  CAS  Google Scholar 

  31. Qiao Y, Molina H, Pandey A, Zhang J, Cole PA (2006) Chemical rescue of a mutant enzyme in living cells. Science 311:1293–1297

    Article  PubMed  CAS  Google Scholar 

  32. Luo W, Slebos RJ, Hill S, Li M, Brabek J, Amanchy R, Chaerkady R, Pandey A, Ham AJ, Hanks SK (2008) Global impact of oncogenic Src on a phosphotyrosine proteome. J Proteome Res 7:3447–3460

    Article  PubMed  CAS  Google Scholar 

  33. Amanchy R, Zhong J, Molina H, Chaerkady R, Iwahori A, Kalume DE, Gronborg M, Joore J, Cope L, Pandey A (2008) Identification of c-Src tyrosine kinase substrates using mass spectrometry and peptide microarrays. J Proteome Res 7:3900–3910

    Article  PubMed  CAS  Google Scholar 

  34. Amanchy R, Zhong J, Hong R, Kim JH, Gucek M, Cole RN, Molina H, Pandey A (2009) Identification of c-Src tyrosine kinase substrates in platelet-derived growth factor receptor signaling. Mol Oncol 3:439–450

    Article  PubMed  CAS  Google Scholar 

  35. Martinez-Ferrando I, Chaerkady R, Zhong J, Molina H, Kishore H, Herbst-Robinson K, Dancy BM, Katju V, Bose R, Zhang J, Pandey A, Cole PA (2012) Identification of targets of c-Src tyrosine kinase by chemical complementation and phosphoproteomics. Mol Cell Proteomics 11:355–369

    Article  Google Scholar 

  36. Bose R, Molina H, Patterson AS, Bitok JK, Periaswamy B, Bader JS, Pandey A, Cole PA (2006) Phosphoproteomic analysis of Her2/neu signaling and inhibition. Proc Natl Acad Sci U S A 103:9773–9778

    Article  PubMed  CAS  Google Scholar 

  37. Mukherji M, Brill LM, Ficarro SB, Hampton GM, Schultz PG (2006) A phosphoproteomic analysis of the ErbB2 receptor tyrosine kinase signaling pathways. Biochemistry 45:15529–15540

    Article  PubMed  CAS  Google Scholar 

  38. Kruger M, Kratchmarova I, Blagoev B, Tseng YH, Kahn CR, Mann M (2008) Dissection of the insulin signaling pathway via quantitative phosphoproteomics. Proc Natl Acad Sci U S A 105:2451–2456

    Article  PubMed  Google Scholar 

  39. Zhang G, Spellman DS, Skolnik EY, Neubert TA (2006) Quantitative phosphotyrosine proteomics of EphB2 signaling by stable isotope labeling with amino acids in cell culture (SILAC). J Proteome Res 5:581–588

    Article  PubMed  CAS  Google Scholar 

  40. Zhang G, Fenyo D, Neubert TA (2008) Screening for EphB signaling effectors using SILAC with a linear ion trap-orbitrap mass spectrometer. J Proteome Res 7:4715–4726

    Article  PubMed  CAS  Google Scholar 

  41. Hinsby AM, Olsen JV, Mann M (2004) Tyrosine phosphoproteomics of fibroblast growth factor signaling: a role for insulin receptor substrate-4. J Biol Chem 279:46438–46447

    Article  PubMed  CAS  Google Scholar 

  42. Cunningham DL, Sweet SM, Cooper HJ, Heath JK (2010) Differential phosphoproteomics of fibroblast growth factor signaling: identification of Src family kinase-mediated phosphorylation events. J Proteome Res 9:2317–2328

    Article  PubMed  CAS  Google Scholar 

  43. Ding VM, Boersema PJ, Foong LY, Preisinger C, Koh G, Natarajan S, Lee DY, Boekhorst J, Snel B, Lemeer S, Heck AJ, Choo A (2011) Tyrosine phosphorylation profiling in FGF-2 stimulated human embryonic stem cells. PLoS One 6:e17538

    Article  PubMed  CAS  Google Scholar 

  44. Zoumaro-Djayoon AD, Ding V, Foong LY, Choo A, Heck AJ, Munoz J (2011) Investigating the role of FGF-2 in stem cell maintenance by global phosphoproteomics profiling. Proteomics 11:3962–3971

    Article  PubMed  CAS  Google Scholar 

  45. Osinalde N, Moss H, Arrizabalaga O, Omaetxebarria MJ, Blagoev B, Zubiaga AM, Fullaondo A, Arizmendi JM, Kratchmarova I (2011) Interleukin-2 signaling pathway analysis by quantitative phosphoproteomics. J Proteomics 75:177–191

    Article  PubMed  CAS  Google Scholar 

  46. Ali NA, Molloy MP (2011) Quantitative phosphoproteomics of transforming growth factor-beta signaling in colon cancer cells. Proteomics 11:3390–3401

    Article  PubMed  CAS  Google Scholar 

  47. Zhong J, Kim MS, Chaerkady R, Wu X, Huang TC, Getnet D, Mitchell CJ, Palapetta SM, Sharma J, O’Meally RN, Cole RN, Yoda A, Moritz A, Loriaux MM, Rush J, Weinstock DM, Tyner JW, Pandey A (2012) TSLP Signaling Network Revealed by SILAC-Based Phosphoproteomics. Mol Cell Proteomics 11(M112):017764

    PubMed  Google Scholar 

  48. Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1:376–386

    Article  PubMed  CAS  Google Scholar 

  49. Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, Khainovski N, Pillai S, Dey S, Daniels S, Purkayastha S, Juhasz P, Martin S, Bartlet-Jones M, He F, Jacobson A, Pappin DJ (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 3:1154–1169

    Article  PubMed  CAS  Google Scholar 

  50. Thompson A, Schafer J, Kuhn K, Kienle S, Schwarz J, Schmidt G, Neumann T, Johnstone R, Mohammed AK, Hamon C (2003) Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal Chem 75:1895–1904

    Article  PubMed  CAS  Google Scholar 

  51. Blagoev B, Kratchmarova I, Ong SE, Nielsen M, Foster LJ, Mann M (2003) A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling. Nat Biotechnol 21:315–318

    Article  PubMed  CAS  Google Scholar 

  52. Wu SL, Kim J, Bandle RW, Liotta L, Petricoin E, Karger BL (2006) Dynamic profiling of the post-translational modifications and interaction partners of epidermal growth factor receptor signaling after stimulation by epidermal growth factor using extended range proteomic analysis (ERPA). Mol Cell Proteomics 5:1610–1627

    Article  PubMed  CAS  Google Scholar 

  53. Amanchy R, Periaswamy B, Mathivanan S, Reddy R, Tattikota SG, Pandey A (2007) A curated compendium of phosphorylation motifs. Nat Biotechnol 25:285–286

    Article  PubMed  CAS  Google Scholar 

  54. Schwartz D, Chou MF, Church GM (2009) Predicting protein post-translational modifications using meta-analysis of proteome scale data sets. Mol Cell Proteomics 8:365–379

    PubMed  CAS  Google Scholar 

  55. Diella F, Gould CM, Chica C, Via A, Gibson TJ (2008) Phospho.ELM: a database of phosphorylation sites—update 2008. Nucleic Acids Res 36:D240–D244

    Article  PubMed  CAS  Google Scholar 

  56. Hornbeck PV, Kornhauser JM, Tkachev S, Zhang B, Skrzypek E, Murray B, Latham V, Sullivan M (2012) PhosphoSitePlus: a comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse. Nucleic Acids Res 40:D261–D270

    Article  PubMed  CAS  Google Scholar 

  57. Keshava Prasad TS, Goel R, Kandasamy K, Keerthikumar S, Kumar S, Mathivanan S, Telikicherla D, Raju R, Shafreen B, Venugopal A, Balakrishnan L, Marimuthu A, Banerjee S, Somanathan DS, Sebastian A, Rani S, Ray S, Harrys Kishore CJ, Kanth S, Ahmed M, Kashyap MK, Mohmood R, Ramachandra YL, Krishna V, Rahiman BA, Mohan S, Ranganathan P, Ramabadran S, Chaerkady R, Pandey A (2009) Human Protein Reference Database—2009 update. Nucleic Acids Res 37:D767–D772

    Article  PubMed  CAS  Google Scholar 

  58. Obenauer JC, Cantley LC, Yaffe MB (2003) Scansite 2.0: proteome-wide prediction of cell signaling interactions using short sequence motifs. Nucleic Acids Res 31:3635–3641

    Article  PubMed  CAS  Google Scholar 

  59. Miller ML, Jensen LJ, Diella F, Jorgensen C, Tinti M, Li L, Hsiung M, Parker SA, Bordeaux J, Sicheritz-Ponten T, Olhovsky M, Pasculescu A, Alexander J, Knapp S, Blom N, Bork P, Li S, Cesareni G, Pawson T, Turk BE, Yaffe MB, Brunak S, Linding R (2008) Linear motif atlas for phosphorylation-dependent signaling. Sci Signal 1(35):ra2

    Article  PubMed  Google Scholar 

  60. Blom N, Gammeltoft S, Brunak S (1999) Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. J Mol Biol 294:1351–1362

    Article  PubMed  CAS  Google Scholar 

  61. Huang HD, Lee TY, Tzeng SW, Horng JT (2005) KinasePhos: a web tool for identifying protein kinase-specific phosphorylation sites. Nucleic Acids Res 33:W226–W229

    Article  PubMed  CAS  Google Scholar 

  62. Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57–70

    Article  PubMed  CAS  Google Scholar 

  63. Druker BJ, Tamura S, Buchdunger E, Ohno S, Segal GM, Fanning S, Zimmermann J, Lydon NB (1996) Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 2:561–566

    Article  PubMed  CAS  Google Scholar 

  64. Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, Harris PL, Haserlat SM, Supko JG, Haluska FG, Louis DN, Christiani DC, Settleman J, Haber DA (2004) Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 350:2129–2139

    Article  PubMed  CAS  Google Scholar 

  65. Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, Fleming T, Eiermann W, Wolter J, Pegram M, Baselga J, Norton L (2001) Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 344:783–792

    Article  PubMed  CAS  Google Scholar 

  66. Harsha HC, Jimeno A, Molina H, Mihalas AB, Goggins MG, Hruban RH, Schulick RD, Kamath U, Maitra A, Hidalgo M, Pandey A (2008) Activated epidermal growth factor receptor as a novel target in pancreatic cancer therapy. J Proteome Res 7:4651–4658

    Article  PubMed  CAS  Google Scholar 

  67. Walters DK, Goss VL, Stoffregen EP, Gu TL, Lee K, Nardone J, McGreevey L, Heinrich MC, Deininger MW, Polakiewicz R, Druker BJ (2006) Phosphoproteomic analysis of AML cell lines identifies leukemic oncogenes. Leuk Res 30:1097–1104

    Article  PubMed  CAS  Google Scholar 

  68. Bai Y, Li J, Fang B, Edwards A, Zhang G, Bui M, Eschrich S, Altiok S, Koomen J, Haura EB (2012) Phosphoproteomics identifies driver tyrosine kinases in sarcoma cell lines and tumors. Cancer Res 72:2501–2511

    Article  PubMed  CAS  Google Scholar 

  69. Jun HJ, Johnson H, Bronson RT, de Feraudy S, White F, Charest A (2012) The oncogenic lung cancer fusion kinase CD74-ROS activates a novel invasiveness pathway through E-Syt1 phosphorylation. Cancer Res 72(15):3764–3774

    Article  PubMed  CAS  Google Scholar 

  70. Suwaki N, Vanhecke E, Atkins KM, Graf M, Swabey K, Huang P, Schraml P, Moch H, Cassidy AM, Brewer D, Al-Lazikani B, Workman P, De-Bono J, Kaye SB, Larkin J, Gore ME, Sawyers CL, Nelson P, Beer TM, Geng H, Gao L, Qian DZ, Alumkal JJ, Thomas G, Thomas GV (2011) A HIF-regulated VHL-PTP1B-Src signaling axis identifies a therapeutic target in renal cell carcinoma. Sci Transl Med 3(85):ra47

    Article  Google Scholar 

  71. Chumbalkar V, Latha K, Hwang Y, Maywald R, Hawley L, Sawaya R, Diao L, Baggerly K, Cavenee WK, Furnari FB, Bogler O (2011) Analysis of phosphotyrosine signaling in glioblastoma identifies STAT5 as a novel downstream target of DeltaEGFR. J Proteome Res 10:1343–1352

    Article  PubMed  CAS  Google Scholar 

  72. Old WM, Shabb JB, Houel S, Wang H, Couts KL, Yen CY, Litman ES, Croy CH, Meyer-Arendt K, Miranda JG, Brown RA, Witze ES, Schweppe RE, Resing KA, Ahn NG (2009) Functional proteomics identifies targets of phosphorylation by B-Raf signaling in melanoma. Mol Cell 34:115–131

    Article  PubMed  CAS  Google Scholar 

  73. Leroy C, Fialin C, Sirvent A, Simon V, Urbach S, Poncet J, Robert B, Jouin P, Roche S (2009) Quantitative phosphoproteomics reveals a cluster of tyrosine kinases that mediates SRC invasive activity in advanced colon carcinoma cells. Cancer Res 69:2279–2286

    Article  PubMed  CAS  Google Scholar 

  74. Nagano K, Shinkawa T, Mutoh H, Kondoh O, Morimoto S, Inomata N, Ashihara M, Ishii N, Aoki Y, Haramura M (2009) Phosphoproteomic analysis of distinct tumor cell lines in response to nocodazole treatment. Proteomics 9:2861–2874

    Article  PubMed  CAS  Google Scholar 

  75. Huang PH, Mukasa A, Bonavia R, Flynn RA, Brewer ZE, Cavenee WK, Furnari FB, White FM (2007) Quantitative analysis of EGFRvIII cellular signaling networks reveals a combinatorial therapeutic strategy for glioblastoma. Proc Natl Acad Sci U S A 104:12867–12872

    Article  PubMed  CAS  Google Scholar 

  76. Rexer BN, Ham AJ, Rinehart C, Hill S, Granja-Ingram NM, Gonzalez-Angulo AM, Mills GB, Dave B, Chang JC, Liebler DC, Arteaga CL (2011) Phosphoproteomic mass spectrometry profiling links Src family kinases to escape from HER2 tyrosine kinase inhibition. Oncogene 30:4163–4174

    Article  PubMed  CAS  Google Scholar 

  77. Andersen JN, Sathyanarayanan S, Di Bacco A, Chi A, Zhang T, Chen AH, Dolinski B, Kraus M, Roberts B, Arthur W, Klinghoffer RA, Gargano D, Li L, Feldman I, Lynch B, Rush J, Hendrickson RC, Blume-Jensen P, Paweletz CP (2010) Pathway-based identification of biomarkers for targeted therapeutics: personalized oncology with PI3K pathway inhibitors. Sci Transl Med 2(43):ra55

    Article  Google Scholar 

  78. De la Mota-Peynado A, Chernoff J, Beeser A (2011) Identification of the atypical MAPK Erk3 as a novel substrate for p21-activated kinase (Pak) activity. J Biol Chem 286:13603–13611

    Article  PubMed  Google Scholar 

  79. Zach S, Felk S, Gillardon F (2010) Signal transduction protein array analysis links LRRK2 to Ste20 kinases and PKC zeta that modulate neuronal plasticity. PLoS One 5:e13191

    Article  PubMed  CAS  Google Scholar 

  80. Kaushansky A, Gordus A, Chang B, Rush J, MacBeath G (2008) A quantitative study of the recruitment potential of all intracellular tyrosine residues on EGFR, FGFR1 and IGF1R. Mol Biosyst 4:643–653

    Article  PubMed  CAS  Google Scholar 

  81. Wilson B, Liotta LA, Petricoin E 3rd (2010) Monitoring proteins and protein networks using reverse phase protein arrays. Dis Markers 28:225–232

    Article  PubMed  CAS  Google Scholar 

  82. Chan SM, Ermann J, Su L, Fathman CG, Utz PJ (2004) Protein microarrays for multiplex analysis of signal transduction pathways. Nat Med 10:1390–1396

    Article  PubMed  CAS  Google Scholar 

  83. Paweletz CP, Charboneau L, Bichsel VE, Simone NL, Chen T, Gillespie JW, Emmert-Buck MR, Roth MJ, Petricoin IE, Liotta LA (2001) Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front. Oncogene 20:1981–1989

    Article  PubMed  CAS  Google Scholar 

  84. Wulfkuhle JD, Aquino JA, Calvert VS, Fishman DA, Coukos G, Liotta LA, Petricoin EF 3rd (2003) Signal pathway profiling of ovarian cancer from human tissue specimens using reverse-phase protein microarrays. Proteomics 3:2085–2090

    Article  PubMed  CAS  Google Scholar 

  85. Chan SM, Weng AP, Tibshirani R, Aster JC, Utz PJ (2007) Notch signals positively regulate activity of the mTOR pathway in T-cell acute lymphoblastic leukemia. Blood 110:278–286

    Article  PubMed  CAS  Google Scholar 

  86. van Agthoven T, Godinho MF, Wulfkuhle JD, Petricoin EF 3rd, Dorssers LC (2012) Protein pathway activation mapping reveals molecular networks associated with antiestrogen resistance in breast cancer cell lines. Int J Cancer 131(9):1998–2007

    Article  PubMed  Google Scholar 

  87. Lavezzari G, Lackner MR (2012) Monitoring phosphoproteomic response to targeted kinase inhibitors using reverse-phase protein microarrays. Methods Mol Biol 795:203–215

    Article  PubMed  CAS  Google Scholar 

  88. Frederick MJ, VanMeter AJ, Gadhikar MA, Henderson YC, Yao H, Pickering CC, Williams MD, El-Naggar AK, Sandulache V, Tarco E, Myers JN, Clayman GL, Liotta LA, Petricoin EF 3rd, Calvert VS, Fodale V, Wang J, Weber RS (2011) Phosphoproteomic analysis of signaling pathways in head and neck squamous cell carcinoma patient samples. Am J Pathol 178:548–571

    Article  PubMed  CAS  Google Scholar 

  89. Amanchy R, Kandasamy K, Mathivanan S, Periaswamy B, Reddy R, Yoon WH, Joore J, Beer MA, Cope L, Pandey A (2011) Identification of novel phosphorylation motifs through an integrative computational and experimental analysis of the human phosphoproteome. J Proteomics Bioinform 4:22–35

    Article  PubMed  CAS  Google Scholar 

  90. Harsha HC, Molina H, Pandey A (2008) Quantitative proteomics using stable isotope labeling with amino acids in cell culture. Nat Protoc 3:505–516

    Article  PubMed  CAS  Google Scholar 

  91. Bian Y, Ye M, Song C, Cheng K, Wang C, Wei X, Zhu J, Chen R, Wang F, Zou H (2012) Improve the coverage for the analysis of phosphoproteome of HeLa cells by a tandem digestion approach. J Proteome Res 11:2828–2837

    Article  PubMed  CAS  Google Scholar 

  92. Beausoleil SA, Villen J, Gerber SA, Rush J, Gygi SP (2006) A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat Biotechnol 24:1285–1292

    Article  PubMed  CAS  Google Scholar 

  93. Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26:1367–1372

    Article  PubMed  CAS  Google Scholar 

  94. MacLean D, Burrell MA, Studholme DJ, Jones AM (2008) PhosCalc: a tool for evaluating the sites of peptide phosphorylation from mass spectrometer data. BMC Res Notes 1:30

    Article  PubMed  Google Scholar 

  95. Taus T, Kocher T, Pichler P, Paschke C, Schmidt A, Henrich C, Mechtler K (2011) Universal and confident phosphorylation site localization using phosphoRS. J Proteome Res 10:5354–5362

    Article  PubMed  CAS  Google Scholar 

  96. Ogata H, Goto S, Sato K, Fujibuchi W, Bono H, Kanehisa M (1999) KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res 27:29–34

    Article  PubMed  CAS  Google Scholar 

  97. Kandasamy K, Mohan SS, Raju R, Keerthikumar S, Kumar GS, Venugopal AK, Telikicherla D, Navarro JD, Mathivanan S, Pecquet C, Gollapudi SK, Tattikota SG, Mohan S, Padhukasahasram H, Subbannayya Y, Goel R, Jacob HK, Zhong J, Sekhar R, Nanjappa V, Balakrishnan L, Subbaiah R, Ramachandra YL, Rahiman BA, Prasad TS, Lin JX, Houtman JC, Desiderio S, Renauld JC, Constantinescu SN, Ohara O, Hirano T, Kubo M, Singh S, Khatri P, Draghici S, Bader GD, Sander C, Leonard WJ, Pandey A (2010) NetPath: a public resource of curated signal transduction pathways. Genome Biol 11:R3

    Article  PubMed  Google Scholar 

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Authors and Affiliations

  1. Institute of Bioinformatics, International Technology Park, Bangalore, India

    H. C. Harsha & Sneha M. Pinto

  2. Departments of Biological Chemistry, Pathology, and Oncology, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, Baltimore, MD, USA

    Akhilesh Pandey

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  1. H. C. Harsha
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  2. Sneha M. Pinto
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  3. Akhilesh Pandey
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Editors and Affiliations

  1. Inst. Molecular Pathology & Immunology, Universidade do Porto Inst. Molecular Pathology & Immunology, Porto, Portugal

    Rune Matthiesen

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Harsha, H.C., Pinto, S.M., Pandey, A. (2013). Proteomic Strategies to Characterize Signaling Pathways. In: Matthiesen, R. (eds) Mass Spectrometry Data Analysis in Proteomics. Methods in Molecular Biology, vol 1007. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-392-3_16

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  • DOI: https://doi.org/10.1007/978-1-62703-392-3_16

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-391-6

  • Online ISBN: 978-1-62703-392-3

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