Skip to main content
SpringerLink
Log in

Basic Design of MRM Assays for Peptide Quantification

  • Protocol
  • First Online:
LC-MS/MS in Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 658))

Abstract

With the recent availability and accessibility of mass spectrometry for basic and clinical research, the requirement for stable, sensitive, and reproducible assays to specifically detect proteins of interest has increased. Multiple reaction monitoring (MRM) or selective reaction monitoring (SRM) is a highly selective, sensitive, and robust assay to monitor the presence and amount of biomolecules. Until recently, MRM was typically used for the detection of drugs and other biomolecules from body fluids. With increased focus on biomarkers and systems biology approaches, researchers in the proteomics field have taken advantage of this approach. In this chapter, we will introduce the reader to the basic principle of designing and optimizing an MRM workflow. We provide examples of MRM workflows for standard proteomic samples and provide suggestions for the reader who is interested in using MRM for quantification.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
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

Abbreviations

QqQ:

triple quadrupole mass spectrometer

SRM:

selective reaction monitoring

MRM:

multiple reaction monitoring

sMRM:

scheduled MRM

CID:

collision-induced dissociation

Amu:

atomic mass unit

LC:

liquid chromatography

LC-MS/MS:

liquid chromatography mass spectrometry

References

  1. Lange, V., Picotti, P., Domon, B., and Aebersold, R. (2008) Selected reaction monitoring for quantitative proteomics: a tutorial. Mol. Syst. Biol. 4(222), 1–14.

    Google Scholar 

  2. Keshishian, H., Addona, T., Burgess, M., Kuhn, E., and Carr, S. A. (2007) Quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilusion. Mol. Cell Proteomics 6(12), 2212–2229.

    Article  PubMed  CAS  Google Scholar 

  3. Keshishian, H., Addona, T., Burgess, M., Mani, D. R., Shi, X., Kuhn, E., Sabatine, M. S., Gerszten RE., Carr SA. (2009) Quantification of cardiovascular biomarkers in pateint plasma by targeted mass spectrometry and stable isotope dillusion. Mol. Cell Proteomics 8(10), 2339–2349.

    Google Scholar 

  4. Addona, T., Abbatiello, S. E., Schilling, B., Skates, S. J., Mani, D. R., Bunk, D. M., Spiegelman, C. H., Zimmerman, L. J., Ham, A. J., Keshishian, H., Hall, S. C., Allen, S., Blackman, R. K., Borchers, C. H., Buck, C., Cardasis, H. L., Cusack, M. P., Dodder, N. G., Gibson, B. W., Held, J. M., Hiltke, T., Jackson, A., Johansen, E. B., Kisinger, C. R., Li, J., Mesri, M., Neubert, T. A., Niles, R. K., Pulsipher, T. C., Ransohoff, D., Rodriguez, H., Rudnick, P. A., Smith, D., Tabb, D. L., Tegeler, T. J., Variyath, A. M., Vega-Montoto, L. J., Wahlander, A., Waldemarson, S., Wang, M., Whiteaker, J. R., Zhao, L., Anderson, N. L., Fisher, S. J., Liebler, D. C., Paulovich, A. G., Regnier, F. E., Tempst, P., Carr, S. A. (2009) Multi-site asessment of the precision and reproducibility of multiple rection monitoring-based measurements of proteins in plasma. Nat. Biotechnol. 27(7), 633–641.

    Article  PubMed  CAS  Google Scholar 

  5. Wolf-Yadlin, A., Hautaniemi, S., Lauffenburger, D. A., and White, F. M. (2007) Multiple reaction monitoring for robust quantitative proteomic analysis of cellular signaling networks. Proc. Natl. Acad. Sci. USA 104(14), 5860–5865.

    Article  PubMed  CAS  Google Scholar 

  6. Mayya, V., Rezual, K., Wu, L., Fong, M. B., and Han, D. K. (2006) Absolute quantification of multisite phosphorylation by selective reaction monitoring mass spectrometry: determination of inhibitory phosphorylation ststus of cyclin-dependent kinases. Mol. Cell Proteomics 5(6), 1146–1157.

    Article  PubMed  CAS  Google Scholar 

  7. Picotti, P., Bodenmiller, B., Mueller, L. N., Domon, B., and Aebersold, R. (2009) Full dynamic range proteome analysis of S. cerevisiae by targeted proteomics. Cell 138(4), 795–806.

    Article  PubMed  CAS  Google Scholar 

  8. Fusaro, V. A., Mani, D. R., Mesirov, J. P., and Carr, S. A. (2009) Prediction of high-responding peptides for targeted protein assays by mass spectrometry. Nat. Biotechnol. 27(2), 190–198.

    Article  PubMed  CAS  Google Scholar 

  9. Mallick, P., Schirle, M., Chen, S. S., Flory, M. R., Lee, H., Martin, D., Ranish, J., Raught B., Schmitt R., Werner T., Kuster B., Aebersold R. (2007) Computational prediction of proteotypic peptides for quantitative proteomics. Nat. Biotechnol. 25(1), 125–131.

    Article  PubMed  CAS  Google Scholar 

  10. Walsh, G. M., Lin, S., Evans, D. M., Khosrovi-Eghbal, A., Beavis, R. C., and Kast, J. J. (2009) Implementation of a data repository-driven approach for targeted proteomics experiments by multiple reaction monitoring. Proteomics 72(5), 838–852.

    Article  PubMed  CAS  Google Scholar 

  11. Desiere, F., Deutsch, E. W., Nesvizhskii, A. I., Mallick, P., King, N. L., Eng, J. K., Aderem, A., Boyle R., Brunner, E., Donohoe, S., Fausto, N., Hafen, E., Hood, L., Katze, M. G., Kennedy, K. A., Kregenow, F., Lee, H., Lin, B., Martin, D., Ranish, J. A., Rawlings, D. J., Samelson, L. E., Shiio, Y., Watts, J. D., Wollscheid, B., Wright, M. E., Yan, W., Yang, L., Yi, E. C., Zhang, H., Aebersold, R. (2005) Integration with the human genome of peptide sequences obtained by high-throughput mass spectrometry. Genome Biol. 6(1), R9.

    Article  PubMed  Google Scholar 

  12. Kuster, B., Schirle, M., Mallick, P., and Aebersold, R. (2005) Scoring proteomes with proteotypic peptide probes. Nat. Rev. Mol. Cell Biol. 6(7), 577–583.

    Article  PubMed  CAS  Google Scholar 

  13. Sherwood, C. A., Eastham, A., Lee, L. W., Risler, J., Vitek, O., and Martin, D. B. (2009) Correlation between y-type ions observed in ion trap and triple quadrupole mass spectrometers. J. Proteome Res. 8(9), 4243–4251.

    Article  PubMed  CAS  Google Scholar 

  14. Gerber, S. A., Rush, J., Stemman, O., Kirschner, M. W., and Gygi, S. P. (2003) Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc. Natl. Acad. Sci. USA 100(12), 6940–6945.

    Article  PubMed  CAS  Google Scholar 

  15. Ong, S. E., Blagoev, B., Kratchmarova, I., Kristensen, D. B., Steen, H., Pandey, A., and 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(5), 376–386.

    Article  PubMed  CAS  Google Scholar 

  16. Ross, P. L., Huang, Y. N., Marchese, J. N., 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, D. J. (2004) Multiplexed protein quantification in Saccharomyces cerevisiae using amine-reactive isobaring tagging reagents. Mol. Cell Proteomics 3(12), 1154–1169.

    Article  PubMed  CAS  Google Scholar 

  17. Unwin, R. D., Griffiths, J. R., and Whetton, A. D. (2009) A sensitive mass spectrometric method for hypothesis-driven detection of peptide post-translational modifications: multiple reaction monitoring-initiated detection and sequencing (MIDAS). Nat. Protoc. 4(6), 870–877.

    Article  PubMed  CAS  Google Scholar 

  18. Unwin, R. D., Griffiths, J. R., Leverentz, M. K., Grallert, A., Hagan, I. M., and Whetton, A. D. (2005) Multiple reaction monitoring to identify sites of protein phosphorylation with high sensitivity. Mol. Cell Proteomics 4(8), 1134–1144.

    Article  PubMed  CAS  Google Scholar 

  19. Heftmann, E. (2004) Chromatography: fundamentals and applications of chromatography and related differential migration methods – Part A: Fundamentals and techniques, 6th ed. Elsevier Science, The Netherlands.

    Google Scholar 

  20. Bansal, S., and DeStefano, A. (2007) Key elements of bioanalytical method validation for small molecules. AAPS J. 9(1), E109–E114.

    Article  PubMed  CAS  Google Scholar 

Links

Download references

Acknowledgments

The authors would like to thank Tony Pawson, Brett Larsen, Vivian Nguyen, Ginny Chen, Rune Linding, Steve Tate, Sarah Robinson, and Marilyn Hsiung for helpful discussion, support, and advice. Claus Jorgensen would like to thank the Lundbeck Foundation for generous support.

Author information

Authors and Affiliations

  1. Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada

    Andrew James & Claus Jorgensen

Authors
  1. Andrew James
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Claus Jorgensen
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Bart’s and the London School of Medicine, Institute of Cancer, Queen Mary, Universtiy of London, Charterhouse Square, London, EC1M 6BQ, United Kingdom

    Pedro R. Cutillas

  2. , EGA Institute for Women's Health, University College London, Gower Street, London, WC1E 6BT, United Kingdom

    John F. Timms

Rights and permissions

Reprints and permissions

Copyright information

© 2010 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

James, A., Jorgensen, C. (2010). Basic Design of MRM Assays for Peptide Quantification. In: Cutillas, P., Timms, J. (eds) LC-MS/MS in Proteomics. Methods in Molecular Biology, vol 658. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-780-8_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-780-8_10

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-779-2

  • Online ISBN: 978-1-60761-780-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation