Skip to main content
SpringerLink
Log in

SRM-MS for Posttranslational Modification Analysis

  • Chapter
  • First Online:
Selected Reaction Monitoring Mass Spectrometry (SRM-MS) in Proteomics

Abstract

Posttranslational modifications (PTMs) have an important role in the regulation of protein function, localization, or interaction with other molecules, and are emerging as important biomarkers for certain disease states. Selected reaction monitoring mass spectrometry (SRM-MS) has become a powerful technique for the quantification and validation of PTMs in complex biological samples where signature peptide must include the amino acid residues of PTM(s). Several strategies have been utilized for efficient measurement of PTMs, which include reduction of sample complexity by the purification of organelles/protein complexes or depletion of high abundance proteins, enrichment of modified proteins/peptides with PTM-based affinity chromatography or immunoprecipitation, multidimension chromatography, or the usage of multiple proteases. Phosphorylation, ubiquitination, glycosylation, acetylation, and several other PTMs in protein molecules can be detected and quantified using SRM-MS.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Change history

  • 16 January 2021

    The section heading 5.4.5 was inadvertently published incorrectly in Chapter 5. The same was corrected throughout the book to read as “Automated SRM-MS Data Analysis Workflow

References

  • Balasubramaniam D, Eissler CL, Stauffacher CV, Hall MC. Use of selected reaction monitoring data for label-free quantification of protein modification stoichiometry. Proteomics. 2010;10:4301–5.

    CAS  PubMed  Google Scholar 

  • Bazoti FN, Tsarbopoulos A. Post-translationally modified proteins: Glycosylation and disulfide bond formation. In: Chen G (ed.), Characterization of protein therapeutics using mass spectrometry, @Springer Science+Business Media New York 2013. https://doi.org/10.1007/978-1-4419-7862-2_4.

  • Choudhary C, Kumar C, Gnad F, Nielsen ML, Rehman M, Walther TC, Olsen JV, Mann M. Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science. 2009;325:834–40.

    CAS  PubMed  Google Scholar 

  • Ciccimaro E, Hevko J, Blair IA. Analysis of phosphorylation sites on focal adhesion kinase using nanospray liquid chromatography/multiple reaction monitoring mass spectrometry. Rapid Commun Mass Spectrom. 2006;20:3681–92.

    CAS  PubMed  Google Scholar 

  • Cohen LA. and Guan JL. Mechanism of focal adhesion kinase regulation. Curr. Cancer Drug Targets. 2005;5:629–43.

    Google Scholar 

  • Cox DM, Zhong F, Du M, Duchoslav E, Sakuma T, McDermott JC. Multipe reaction monitoring as a method for identifying protein posttranslational modifications. J Biomol Tech. 2005;16:83–90.

    PubMed  PubMed Central  Google Scholar 

  • Cummings RD, Pierce JM. The challenges and promise of glycomics. Chem Biol. 2014;21:1–15.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Dephoure N, Gould KL, Gygi SP, Kellogg DR. Mapping and analysis of phosphorylation sites: a quick guide for cell biologists. Mol Biol Cell. 2013;24:535–42.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Domanski D, Murphy LC, Borchers CH. Assay development for the determination of phosphorylation stoichiometry using multiple reaction monitoring methods with and without phosphatase treatment: application to breast cancer signaling pathways. Anal Chem. 2010;82:5610–20.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Eissler CL, Bremmer SC, Martinez JS, Parker LL, Charbonneau H, Hall MC. A general strategy for studying multisite protein phosphorylation using label-free selected reaction monitoring mass spectrometry. Anal Biochem. 2011;418:267–75.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Garcia BA, Shabanowitz J, Hunt D. Analysis of protein phosphorylation by mass spectrometry. Methods. 2005;35:256–64.

    CAS  PubMed  Google Scholar 

  • Goldman R, Sanda M. Targeted methods for quantitative analysis of protein glycosylation. Proteomics Clin Appl. 2015;9:17–32.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Griffiths JR, Unwin RD, Evans CA, Leech SH, et al. The application of a hypothesis-driven strategy to the sensitive detection and location of acetylated lysine residues. Am Soc Mass Spectrom. 2007;18:1423–8.

    CAS  Google Scholar 

  • Hong Q, Lebrilla CB, Miyamoto S, Ruhaak LR. Absolute quantification of immunoglobulin G and its glycoforms using multiple reaction monitoring. Anal Chem. 2013;85:8585–93.

    CAS  PubMed  Google Scholar 

  • Jin LL, Tong J, Prakash A, Peterman SM, St-Germain JR, Taylor P, Trudel S, Moran MF. Measurement of protein phosphorylation stoichiometry by selected reaction monitoring mass spectrometry. J Proteome Res. 2010;9:2752–61.

    CAS  PubMed  Google Scholar 

  • Jin LL, Sydorskyy Y, Tong J, Taylor P, Moran MF, St-Germain JR. Measurement of protein phosphorylation stoichiometry by SRM-MS. Curr Protoc Chem Biol. 2012;4:65–81.

    Google Scholar 

  • Kerscher O, Felberbaum R, Hochstrasser M. Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu Rev Cell Dev Biol. 2006;22:159–80.

    CAS  PubMed  Google Scholar 

  • Kim SC, Sprung R, Chen Y, Xu Y, et al. Substrate and functional diversity of lysine acetylation revealed by a proteomics survey. Mol Cell. 2006;23:607–18.

    CAS  PubMed  Google Scholar 

  • Komander D. The emerging complexity of protein ubiquitination. Biochem Soc Trans. 2009;37:937–53.

    CAS  PubMed  Google Scholar 

  • Kurogochi M, Matsushista T, Amano M, Furukawa J, Shinohara Y, Aoshima M, Nishimura SI. Sialic acid-focused quantitative mouse serum glycoproteomics by multiple reaction monitoring assay. Mol Cell Proteomics. 2010;9:2354–68.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Larsen MR, Trelle MB, Thingholm TE, Jensen OL. Analysis of posttranslational modifications of proteins by tandem mass spectrometry. BioTechniques. 2006;40(6):790–7.

    CAS  PubMed  Google Scholar 

  • Liu X, Jin Z, O’brian R, Bathon J, Dietz HC, Grote E, Van Eyk JE. Constrained selected reaction monitoring: Quantification of selected post-translational modifications and protein isoforms. Methods. 2013;61:304–12.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Mann M, Ong SE, Gronborg H, Steen H, Jensen ON, Pandey A. Analysis of protein phosphorylation using mass spectrometry:deciphering the phosphoproteome. Trends Biotechnol. 2002;20:261–8.

    CAS  PubMed  Google Scholar 

  • Mayya V, Rezual K, Wu L, Fong MB, et al. Absolute quantification of multisite phosphorylation by selective reaction monitoring mass spectrometry. Mol Cell Proteomics. 2006;5:1146–57.

    CAS  PubMed  Google Scholar 

  • McNulty DE, Annan RS. Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection. Mol Cell Proteomics. 2008;7:971–80.

    CAS  PubMed  Google Scholar 

  • Mollah S, Wertz IE, Phung Q, Arnott D, Dixit VM, Lill JR. Targeted mass spectrometric strategy for global mapping of ubiquitination on proteins. Rapid Commun Mass Spectrom. 2007;21:3357–64.

    CAS  PubMed  Google Scholar 

  • Morelle W, Michalski JC. Analysis of protein glycosylation by mass spectrometry. Nat Protoc. 2007;2:1585–602.

    CAS  PubMed  Google Scholar 

  • Narumi R, Murakami T, Kuga T, Adachi J, Shiromizu T, Muraoka S, Kume H, Kodera Y, Matsumoto M, Nakayama K, Miyamoto Y, Ishitobi M, Inaji H, Kato K, Tomonaga T. A strategy for large-scale phosphoproteomics and SRM-based validation of human breast cancer tissue samples. J Proteome. 2012;11:5311–22.

    CAS  Google Scholar 

  • Peng J, Schwartz D, Elias JE, Thoreen CC, et al. A proteomics approach to understanding protein ubiquitination. Nat Biotechnol. 2003;21:921–6.

    CAS  PubMed  Google Scholar 

  • Pinkse MW. uitto, PM; Hillhorst, MJ; Ooms, B; and Heck, AJ. Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns. Anal Chem. 2004;76:3935–43.

    CAS  PubMed  Google Scholar 

  • Roth Z, Yehezkel G, Khalaila I, et al. Int J Carb Chem. 2012, Article ID 640923:10 pages.

    Google Scholar 

  • Roux RD, Griffiths JR, Leverentz MK, Grallert A, Hagan IM, Whetton AD. The coming of age of phosphoproteomics – from large data sets to inference of protein functions. Mol Cell Proteomics. 2013:3453–64.

    Google Scholar 

  • Sandra M, Pompach P, Brnakova Z, Wu J. Quantitative liquid chromatography-mass spectrometry-multiple reaction monitoring (LC-MS-MRM) analysis of site-specific glycoforms in liver disease. Mol Cell Proteomics. 2013a;12:1294–305.

    Google Scholar 

  • Sandra M, Pompach P, Benicky J, Goldman R. LC-MS3 quantification of O-glycopeptides in human serum. Electrophoresis. 2013b;34:2342–9.

    Google Scholar 

  • Song E, Pyreddy S, Mechref Y. Quantification of glycopeptides my multiple reaction monitoring liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom. 2012;26:1941–54.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Steen H, Jebanathirajah JA, Springer M, Kirschner MW. Stable isotope-free relative and absolute quantification of protein phosphorylation stoichiometry by MS. PNAS. 2005;102:3948–53.

    CAS  PubMed  Google Scholar 

  • Steen H, Jebanathirajah JA, Rush J, Morrice N, Kirschner MW. Phosphorylation analysis by mass spectrometry – Myths, facts, and the consequences for quantitative and quantitative measurements. Mol Cell Proteomics. 2006;5:172–81.

    CAS  PubMed  Google Scholar 

  • Strieter ER, Korasick DA. Unraveling the complexity of ubiquitin signaling. ACS Chem Biol. 2012;7:52–63.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Thingholm TE, Jensen ON. Enrichment and characterization of phosphopeptides by immobilized metal affinity chromatography (IMAC) and mass spectrometry. Methods Mol Biol. 2009;527:47–56.

    CAS  PubMed  Google Scholar 

  • Toyama A, Nakagawa H, Matsuda K, Sato TA, Nakamura Y, Ueda K. Quantitative structural characterization of local N-glycan microheterogeneity in therapeutic antibodies by energy-resolved oxonium ion monitoring. Anal Chem. 2012;84:9655–62.

    CAS  PubMed  Google Scholar 

  • Unwin RD, Griffiths JR, Leverentz MK, Grallert A, Hagan IM, Whetton AD. Multiple reaction monitoring to identify sites of protein phosphorylation with high sensitivity. Mol Cell Proteomics. 2005;4:1134–44.

    CAS  PubMed  Google Scholar 

  • Vardin E, Ott M. 50 years of protein acetylation: from gene regulation to epigenetics, metabolism and beyond. Nat Rev Mol Cell Biol. 2015;16:258–64.

    Google Scholar 

  • Varki A. Biological roles of oligosaccharides: all of the theories are correct. Glycobiology. 1993;2:97–130.

    Google Scholar 

  • Verdin E, Melanic O. 50 years of protein acetylation: from gene regulation to epigenetics, metabolism and beyond. Nat Rev Mol Cell Biol. 2015;16:258–64.

    CAS  PubMed  Google Scholar 

  • Villen J, Gygi SP. The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry. Nat Protoc. 2008;3:1630–8.

    PubMed  PubMed Central  Google Scholar 

  • Walsh CT, Garneau-Tsodikova S, Gatto JG Jr. Protein posttranslational modifications: The chemistry of protein diversifications. Angew Chem Int Ed. 2005;44:7342–737.

    CAS  Google Scholar 

  • Witze ES, Old WM, Resing KA, Ahn NG. Mapping protein post-translational modifications with mass spectrometry. Nat Methods. 2007;4:798–806.

    CAS  PubMed  Google Scholar 

  • Wolf-Yadlin A, Hautaniem S, Lauffenburger DA, White FM. Multiple reaction monitoring for robust quantitative proteomic analysis of cellular signaling networks. PNAS. 2007;104:5860–5.

    CAS  PubMed  Google Scholar 

  • Zhao Y, Jia W, Wang J, Ying W. Fragmentation and site-specificc quantification of core fucosylated glycoprotein by multiple reaction monitoring mass spectrometry. Anal Chem. 2011;83:8802–9.

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Sanofi Genzyme, Framingham, MA, USA

    Mahmud Hossain

Authors
  1. Mahmud Hossain
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Nature Switzerland AG

About this chapter

Check for updates. Verify currency and authenticity via CrossMark

Cite this chapter

Hossain, M. (2020). SRM-MS for Posttranslational Modification Analysis. In: Selected Reaction Monitoring Mass Spectrometry (SRM-MS) in Proteomics. Springer, Cham. https://doi.org/10.1007/978-3-030-53433-2_8

Download citation

Publish with us

Policies and ethics

Search

Navigation