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Antibody-Based Proteomic Analysis of Apoptosis Signaling

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Apoptosis Methods in Toxicology

Part of the book series: Methods in Pharmacology and Toxicology ((MIPT))

Abstract

Reagents that assess activation of apoptosis and associated signaling pathways are critical for greater understanding of the molecular basis of programmed cell death. The advent of proteomic technologies to probe these events allows monitoring of hundreds to thousands of proteins, as well as sites of posttranslational modification involved in apoptosis at one time. This view of apoptosis at a network level is a powerful tool in studying known apoptotic pathways, as well as elucidating novel signaling events that affect or are affected by apoptotic signaling. The following is a detailed method for successful proteomic profiling of apoptosis using antibody-based enrichment methods along with a liquid chromatography–tandem mass spectrometry analytical platform.

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Abbreviations

IAP:

Immunoaffinity purification

LC:

Liquid chromatography

MS/MS:

Tandem mass spectrometry

MeCN:

Acetonitrile

MS:

Mass spectrometry

PTM:

Posttranslational modification

TFA:

Trifluoroacetic acid

References

  1. Favaloro B, Allocati N, Graziano V, Di Ilio C, De Laurenzi V (2012) Role of apoptosis in disease. Aging 4:330–349

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Kaufmann T, Strasser A, Jost PJ (2012) Fas death receptor signalling: roles of Bid and XIAP. Cell Death Differ 19:42–50

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Fuchs Y, Steller H (2011) Programmed cell death in animal development and disease. Cell 147:742–758

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Degterev A, Yuan J (2008) Expansion and evolution of cell death programmes. Nat Rev Mol Cell Biol 9:378–390

    Article  CAS  PubMed  Google Scholar 

  5. Jacobson MD, Evan GI (1994) Apoptosis. Breaking the ICE. Curr Biol 4:337–340

    Article  CAS  PubMed  Google Scholar 

  6. Cohen GM (1997) Caspases: the executioners of apoptosis. Biochem J 326(Pt 1):1–16

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Fischer U, Janicke RU, Schulze-Osthoff K (2003) Many cuts to ruin: a comprehensive update of caspase substrates. Cell Death Differ 10:76–100

    Article  CAS  PubMed  Google Scholar 

  8. Alenzi FQ, Lotfy M, Wyse R (2010) Swords of cell death: caspase activation and regulation. Asian Pac J Cancer Prev 11:271–280

    PubMed  Google Scholar 

  9. Guo A, Gu H, Zhou J, Mulhern D, Wang Y, Lee KA, Yang V, Aguiar M, Kornhauser J, Jia X, Ren J, Beausoleil SA, Silva JC, Vemulapalli V, Bedford MT, Comb MJ (2014) Immunoaffinity enrichment and mass spectrometry analysis of protein methylation. Mol Cell Proteomics 13:372–387

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Lee KA, Hammerle LP, Andrews PS, Stokes MP, Mustelin T, Silva JC, Black RA, Doedens JR (2011) Ubiquitin ligase substrate identification through quantitative proteomics at both the protein and peptide levels. J Biol Chem 286(48):41530–41538

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Moritz A, Li Y, Guo A, Villen J, Wang Y, MacNeill J, Kornhauser J, Sprott K, Zhou J, Possemato A, Ren JM, Hornbeck P, Cantley LC, Gygi SP, Rush J, Comb MJ (2010) Akt-RSK-S6 kinase signaling networks activated by oncogenic receptor tyrosine kinases. Sci Signal 3:ra64

    Article  PubMed  PubMed Central  Google Scholar 

  12. Rikova K, Guo A, Zeng Q, Possemato A, Yu J, Haack H, Nardone J, Lee K, Reeves C, Li Y, Hu Y, Tan Z, Stokes M, Sullivan L, Mitchell J, Wetzel R, Macneill J, Ren JM, Yuan J, Bakalarski CE, Villen J, Kornhauser JM, Smith B, Li D, Zhou X, Gygi SP, Gu TL, Polakiewicz RD, Rush J, Comb MJ (2007) Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell 131:1190–1203

    Article  CAS  PubMed  Google Scholar 

  13. Rush J, Moritz A, Lee KA, Guo A, Goss VL, Spek EJ, Zhang H, Zha XM, Polakiewicz RD, Comb MJ (2005) Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat Biotechnol 23:94–101

    Article  CAS  PubMed  Google Scholar 

  14. Stokes MP, Comb MJ (2008) A wide-ranging cellular response to UV damage of DNA. Cell Cycle 7:2097–2099

    Article  CAS  PubMed  Google Scholar 

  15. Stokes MP, Farnsworth CL, Moritz A, Silva JC, Jia X, Lee KA, Guo A, Polakiewicz RD, Comb MJ (2012) PTMScan direct: identification and quantification of peptides from critical signaling proteins by immunoaffinity enrichment coupled with LC-MS/MS. Mol Cell Proteomics 11:187–201

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Stokes MP, Silva JC, Jia X, Lee KA, Polakiewicz RD, Comb MJ (2012) Quantitative profiling of DNA damage and apoptotic pathways in UV damaged cells using PTMScan direct. Int J Mol Sci 14:286–307

    Article  PubMed  PubMed Central  Google Scholar 

  17. Dayon L, Hainard A, Licker V, Turck N, Kuhn K, Hochstrasser DF, Burkhard PR, Sanchez JC (2008) Relative quantification of proteins in human cerebrospinal fluids by MS/MS using 6-plex isobaric tags. Anal Chem 80:2921–2931

    Article  CAS  PubMed  Google Scholar 

  18. Hsu JL, Huang SY, Chow NH, Chen SH (2003) Stable-isotope dimethyl labeling for quantitative proteomics. Anal Chem 75:6843–6852

    Article  CAS  PubMed  Google Scholar 

  19. Ibarrola N, Kalume DE, Gronborg M, Iwahori A, Pandey A (2003) A proteomic approach for quantitation of phosphorylation using stable isotope labeling in cell culture. Anal Chem 75:6043–6049

    Article  CAS  PubMed  Google Scholar 

  20. 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  CAS  PubMed  Google Scholar 

  21. Paardekooper Overman J, Yi JS, Bonetti M, Soulsby M, Preisinger C, Stokes MP, Hui L, Silva JC, Overvoorde J, Giansanti P, Heck AJ, Kontaridis MI, den Hertog J, Bennett AM (2014) PZR coordinates Shp2 Noonan and LEOPARD syndrome signaling in zebrafish and mice. Mol Cell Biol 34:2874–2889

    Article  PubMed  PubMed Central  Google Scholar 

  22. Unwin RD, Pierce A, Watson RB, Sternberg DW, Whetton AD (2005) Quantitative proteomic analysis using isobaric protein tags enables rapid comparison of changes in transcript and protein levels in transformed cells. Mol Cell Proteomics 4:924–935

    Article  CAS  PubMed  Google Scholar 

  23. Viner RI, Zhang T, Second T, Zabrouskov V (2009) Quantification of post-translationally modified peptides of bovine alpha-crystallin using tandem mass tags and electron transfer dissociation. J Proteomics 72:874–885

    Article  CAS  PubMed  Google Scholar 

  24. Wiese S, Reidegeld KA, Meyer HE, Warscheid B (2007) Protein labeling by iTRAQ: a new tool for quantitative mass spectrometry in proteome research. Proteomics 7:340–350

    Article  CAS  PubMed  Google Scholar 

  25. Zhang H, Zha X, Tan Y, Hornbeck PV, Mastrangelo AJ, Alessi DR, Polakiewicz RD, Comb MJ (2002) Phosphoprotein analysis using antibodies broadly reactive against phosphorylated motifs. J Biol Chem 277:39379–39387

    Article  CAS  PubMed  Google Scholar 

  26. Pham VC, Pitti R, Anania VG, Bakalarski CE, Bustos D, Jhunjhunwala S, Phung QT, Yu K, Forrest WF, Kirkpatrick DS, Ashkenazi A, Lill JR (2012) Complementary proteomic tools for the dissection of apoptotic proteolysis events. J Proteome Res 11:2947–2954

    Article  CAS  PubMed  Google Scholar 

  27. Rappsilber J, Ishihama Y, Mann M (2003) Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal Chem 75:663–670

    Article  CAS  PubMed  Google Scholar 

  28. Lundgren DH, Martinez H, Wright ME, Han DK (2009) Protein identification using Sorcerer 2 and SEQUEST. Curr Protoc Bioinformatics Chapter 13, Unit 13 13

    Google Scholar 

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Author information

Authors and Affiliations

  1. Cell Signaling Technology, Inc., 3 Trask Lane, Danvers, MA, 01923, USA

    Matthew P. Stokes, Hongbo Gu & Jeffrey C. Silva

Authors
  1. Matthew P. Stokes
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  2. Hongbo Gu
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  3. Jeffrey C. Silva
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Corresponding author

Correspondence to Matthew P. Stokes .

Editor information

Editors and Affiliations

  1. Department of Biology, North Carolina A&T State University, Greensboro, North Carolina, USA

    Perpetua M. Muganda

1 Electronic Supplementary Material

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Supplemental Table S1

(XLSX 609 kb)

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Stokes, M.P., Gu, H., Silva, J.C. (2016). Antibody-Based Proteomic Analysis of Apoptosis Signaling. In: Muganda, P. (eds) Apoptosis Methods in Toxicology. Methods in Pharmacology and Toxicology. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3588-8_9

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  • DOI: https://doi.org/10.1007/978-1-4939-3588-8_9

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3586-4

  • Online ISBN: 978-1-4939-3588-8

  • eBook Packages: Springer Protocols

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