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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
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
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Bansal, S., and DeStefano, A. (2007) Key elements of bioanalytical method validation for small molecules. AAPS J. 9(1), E109–E114.
Links
AB marketing info: MRMpilot: http://www3.appliedbiosystems.com/cms/groups/psm_marketing/documents/generaldocuments/cms_047953.pdf
Multiquant: http://www3.appliedbiosystems.com/cms/groups/psm_marketing/documents/generaldocuments/cms_047952.pdf
ThermoFisher tool for MRM design: http://www.thermo.com/pinpoint
The GPM: http://www.thegpm.org
The peptide atlas: http://www.peptideatlas.org
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
Editor information
Editors and Affiliations
Rights 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