A Targeted MRM Approach for Tempo-Spatial Proteomics Analyses

  • Annie Moradian
  • Tanya R. Porras-Yakushi
  • Michael J. Sweredoski
  • Sonja Hess
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1394)

Abstract

When deciding to perform a quantitative proteomics analysis, selectivity, sensitivity, and reproducibility are important criteria to consider. The use of multiple reaction monitoring (MRM) has emerged as a powerful proteomics technique in that regard since it avoids many of the problems typically observed in discovery-based analyses. A prerequisite for such a targeted approach is that the protein targets are known, either as a result of previous global proteomics experiments or because a specific hypothesis is to be tested. When guidelines that have been established in the pharmaceutical industry many decades ago are taken into account, setting up an MRM assay is relatively straightforward. Typically, proteotypic peptides with favorable mass spectrometric properties are synthesized with a heavy isotope for each protein that is to be monitored. Retention times and calibration curves are determined using triple-quadrupole mass spectrometers. The use of iRT peptide standards is both recommended and fully integrated into the bioinformatics pipeline. Digested biological samples are mixed with the heavy and iRT standards and quantified. Here we present a generic protocol for the development of an MRM assay.

Key words

MRM Quadrupole mass spectrometry Quantitation 

References

  1. 1.
    Picotti P, Aebersold R (2012) Selected reaction monitoring-based proteomics: workflows, potential, pitfalls and future directions. Nat Methods 9(6):555–566. doi:10.1038/nmeth.2015 CrossRefPubMedGoogle Scholar
  2. 2.
    Carr SA, Abbatiello SE, Ackermann BL et al (2014) Targeted peptide measurements in biology and medicine: best practices for mass spectrometry-based assay development using a fit-for-purpose approach. Mol Cell Proteomics 13(3):907–917. doi:10.1074/mcp.M113.036095 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Huttenhain R, Soste M, Selevsek N et al (2012) Reproducible quantification of cancer-associated proteins in body fluids using targeted proteomics. Sci Transl Med 4(142):142ra194. doi:10.1126/scitranslmed.3003989 CrossRefGoogle Scholar
  4. 4.
    Kuzyk MA, Smith D, Yang J et al (2009) Multiple reaction monitoring-based, multiplexed, absolute quantitation of 45 proteins in human plasma. Mol Cell Proteomics 8(8):1860–1877. doi:10.1074/mcp.M800540-MCP200 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Picotti P, Rinner O, Stallmach R et al (2010) High-throughput generation of selected reaction-monitoring assays for proteins and proteomes. Nat Methods 7(1):43–46. doi:10.1038/nmeth.1408 CrossRefPubMedGoogle Scholar
  6. 6.
    Escher C, Reiter L, MacLean B et al (2012) Using iRT, a normalized retention time for more targeted measurement of peptides. Proteomics 12(8):1111–1121. doi:10.1002/pmic.201100463 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    MacLean B, Tomazela DM, Shulman N et al (2010) Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 26(7):966–968. doi:10.1093/bioinformatics/btq054 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Wisniewski JR, Zougman A, Nagaraj N et al (2009) Universal sample preparation method for proteome analysis. Nat Methods 6(5):359–362. doi:10.1038/nmeth.1322 CrossRefPubMedGoogle Scholar
  9. 9.
    Hess S, Akermann M, Ropte D et al (2001) Rapid and sensitive LC separation of new impurities in trimethoprim. J Pharm Biomed Anal 25(3–4):531–538CrossRefPubMedGoogle Scholar
  10. 10.
    Hess S, Dolker M, Haferburg D et al (2001) Separation, analyses and syntheses of trimethoprim impurities. Pharmazie 56(4):306–310PubMedGoogle Scholar
  11. 11.
    Hess S, Muller CE, Frobenius W et al (2000) 7-deazaadenines bearing polar substituents: structure-activity relationships of new A(1) and A(3) adenosine receptor antagonists. J Med Chem 43(24):4636–4646. doi:10.1021/Jm000967d CrossRefPubMedGoogle Scholar
  12. 12.
    Hess S, Teubert U, Ortwein J et al (2001) Profiling indomethacin impurities using high-performance liquid chromatography and nuclear magnetic resonance. Eur J Pharm Sci 14(4):301–311CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Annie Moradian
    • 1
  • Tanya R. Porras-Yakushi
    • 1
  • Michael J. Sweredoski
    • 1
  • Sonja Hess
    • 1
  1. 1.Proteome Exploration LaboratoryCalifornia Institute of TechnologyPasadenaUSA

Personalised recommendations