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Quantitative analysis of peptides and proteins in biomedicine by targeted mass spectrometry

Nature Methods volume 10, pages 28–34 (2013)Cite this article

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Abstract

Targeted mass spectrometry (MS) is becoming widely used in academia and in pharmaceutical and biotechnology industries for sensitive and quantitative detection of proteins, peptides and post-translational modifications. Here we describe the increasing importance of targeted MS technologies in clinical proteomics and the potential key roles these techniques will have in bridging biomedical discovery and clinical implementation.

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Figure 1: Comparison of conventional data-dependent analysis to targeted MRM-MS on a triple quadrupole mass spectrometer.
Figure 2: Protein and peptide enrichment strategies to increase sensitivity and specificity of analyte detection in SID-MRM-MS.

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References

  1. Lawson, A.M. The scope of mass spectrometry in clinical chemistry. Clin. Chem. 21, 803–824 (1975).

    CAS  PubMed  Google Scholar 

  2. Zhu, X. & Desiderio, D.M. Peptide quantification by tandem mass spectrometry. Mass Spectrom. Rev. 15, 213–240 (1996).

    CAS  PubMed  Google Scholar 

  3. Grebe, S.K.G. & Singh, R.J.J. LC-MS/MS in the clinical laboratory—where to from here? Clin. Biochem. Rev. 32, 5–31 (2011).

    PubMed  PubMed Central  Google Scholar 

  4. Yost, R.A. & Enke, C.G. Triple quadrupole mass spectrometry for direct mixture analysis and structure elucidation. Anal. Chem. 51, 1251–1264 (1979).

    CAS  PubMed  Google Scholar 

  5. Picotti, P. & Aebersold, R. Selected reaction monitoring-based proteomics: workflows, potential, pitfalls and future directions. Nat. Methods 9, 555–566 (2012).

    CAS  PubMed  Google Scholar 

  6. Barr, J.R. et al. Isotope dilution–mass spectrometric quantification of specific proteins: model application with apolipoprotein A-I. Clin. Chem. 42, 1676–1682 (1996).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  9. Addona, T.A. et al. Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma. Nat. Biotechnol. 27, 633–641 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Brun, V., Masselon, C., Garin, J. & Dupuis, A. Isotope dilution strategies for absolute quantitative proteomics. J. Proteomics 72, 740–749 (2009).

    CAS  PubMed  Google Scholar 

  11. Abbatiello, S.E., Mani, D.R., Keshishian, H. & Carr, S.A. Automated detection of inaccurate and imprecise transitions in quantitative assays of peptides by multiple monitoring mass spectrometry. Clin. Chem. 56, 291–305 (2010).

    CAS  PubMed  Google Scholar 

  12. Reiter, L. et al. mProphet: automated data processing and statistical validation of large-scale SRM experiments. Nat. Methods 8, 430–435 (2011).

    CAS  PubMed  Google Scholar 

  13. Remily-Wood, E.R. et al. A database of reaction monitoring mass spectrometry assays elucidating therapeutic response in cancer. Proteomics Clin. Appl. 5, 383–396 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Hüttenhain, R. et al. Reproducible quantification of cancer-associated proteins in body fluids using targeted proteomics. Sci. Transl. Med. 4, 142ra94 (2012).

    PubMed  PubMed Central  Google Scholar 

  15. Addona, T.A. et al. A pipeline that integrates the discovery and verification of plasma protein biomarkers reveals candidate markers for cardiovascular disease. Nat. Biotechnol. 29, 635–643 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Fortin, T. et al. Clinical quantitation of prostate-specific antigen biomarker in the low nanogram/milliliter range by conventional bore liquid chromatography-tandem mass spectrometry multiple reaction monitoring coupling and correlation with ELISA tests. Mol. Cell. Proteomics 8, 1006–1015 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Shi, T. et al. Antibody-free, targeted mass-spectrometric approach for quantification of proteins at low picogram per milliliter levels in human plasma/serum. Proc. Natl. Acad. Sci. USA 109, 15395–15400 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Berna, M.J. et al. Quantification of NTproBNP in rat serum using immunoprecipitation and LC/MS/MS: a biomarker of drug-induced cardiac hypertrophy. Anal. Chem. 80, 561–566 (2008).

    CAS  PubMed  Google Scholar 

  19. Anderson, N.L. et al. Mass spectrometric quantitation of peptides and proteins using stable isotope standards and capture by anti-peptide antibodies (SISCAPA). J. Proteome Res. 3, 235–244 (2004).

    CAS  PubMed  Google Scholar 

  20. Whiteaker, J.R., et al. Evaluation of large scale quantitative proteomic assay development using peptide affinity-based mass spectrometry. Mol. Cell. Proteomics 10, M110.005645. (2011).

    PubMed  PubMed Central  Google Scholar 

  21. Neubert, H., Gale, J. & Muirhead, D. Online high-flow peptide immunoaffinity enrichment and nanoflow LC-MS/MS: assay development for total salivary pepsin/pepsinogen. Clin. Chem. 56, 1413–1423 (2010).

    CAS  PubMed  Google Scholar 

  22. Whiteaker, J.R. et al. Sequential multiplexed analyte quantification using peptide immunoaffinity enrichment coupled to mass spectrometry. Mol. Cell. Proteomics 11, M111.015347 (2012).

    PubMed  Google Scholar 

  23. Kuhn, E., et al. Inter-laboratory evaluation of automated, multiplexed peptide immunoaffinity enrichment coupled to multiple reaction monitoring mass spectrometry for quantifying proteins in plasma. Mol. Cell. Proteomics 11, M111.013854. (2012).

    PubMed  Google Scholar 

  24. Sparbier, K. et al. Immuno-MALDI-TOF MS: new perspectives for clinical applications of mass spectrometry. Proteomics 9, 1442–1450 (2009).

    CAS  PubMed  Google Scholar 

  25. Rifai, N., Gillette, M.A. & Carr, S.A. Protein biomarker discovery and validation: the long and uncertain path to clinical utility. Nat. Biotechnol. 24, 971–983 (2006).

    CAS  PubMed  Google Scholar 

  26. Anderson, N.L. The clinical plasma proteome: a survey of clinical assays for proteins in plasma and serum. Clin. Chem. 56, 177–185 (2010).

    CAS  PubMed  Google Scholar 

  27. Gonzalez-Angulo, A.M. et al. Functional proteomics can define prognosis and predict pathologic complete response in patients with breast cancer. Clin. Proteomics 8, 11 (2011).

    PubMed  PubMed Central  Google Scholar 

  28. Ng, A.H.C., Uddayasankar, U. & Wheeler, A.R. Immunoassays in microfluidic systems. Anal. Bioanal. Chem. 397, 991–1007 (2010).

    CAS  PubMed  Google Scholar 

  29. Ellington, A.A., Kullo, I.J., Bailey, K.R. & Klee, G.G. Antibody-based protein multiplex platforms: technical and operational challenges. Clin. Chem. 56, 186–193 (2010).

    CAS  PubMed  Google Scholar 

  30. Brody, E.N., Gold, L., Lawn, R.M., Walker, J.J. & Zichi, D. High-content affinity-based proteomics: unlocking protein biomarker discovery. Expert Rev. Mol. Diagn. 10, 1013–1022 (2010).

    CAS  PubMed  Google Scholar 

  31. Thiviyanathan, V. & Gorenstein, D.G. Aptamers and the next generation of diagnostic reagents. Proteomics Clin. Appl. advance online publication 23 October 2012 (doi:10.1002/prca.201200042).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Makawita, S. & Diamandis, E.P. The bottleneck in the cancer biomarker pipeline and protein quantification through mass spectrometry–based approaches: current strategies for candidate verification. Clin. Chem. 56, 212–222 (2010).

    CAS  PubMed  Google Scholar 

  33. Smith, R.D. Mass spectrometry in biomarker applications: from untargeted discovery to targeted verification, and implications for platform convergence and clinical application. Clin. Chem. 58, 528–530 (2012).

    CAS  PubMed  Google Scholar 

  34. Percy, A.J., Chambers, A.G., Yang, J., Domanski, D. & Borchers, C.H. Comparison of standard-flow and nano-flow liquid chromatography systems for MRM-based quantitation of putative plasma biomarker proteins. Anal. Bioanal. Chem. 404, 1089–1101 (2012).

    CAS  PubMed  Google Scholar 

  35. Whiteaker, J.R. et al. A targeted proteomics-based pipeline for verification of biomarkers in plasma. Nat. Biotechnol. 29, 625–634 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Domanski, D. et al. MRM-based multiplexed quantitation of 67 putative cardiovascular disease biomarkers in human plasma. Proteomics 12, 1222–1243 (2012).

    CAS  PubMed  Google Scholar 

  37. Pan, S. et al. Multiplex targeted proteomic assay for biomarker detection in plasma: a pancreatic cancer biomarker case study. J. Proteome Res. 11, 1937–1948 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Wang, Q. et al. Mutant proteins as cancer-specific biomarkers. Proc. Natl. Acad. Sci. USA 108, 2444–2449 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Hoofnagle, A.N., Becker, J.O., Wener, M.H. & Heinecke, J.W. Quantification of thyroglobulin, a low-abundance serum protein, by immunoaffinity peptide enrichment and tandem mass spectrometry. Clin. Chem. 54, 1796–1804 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Jaffe, J.D. et al. Accurate inclusion mass screening: a bridge from unbiased discovery to targeted assay development for biomarker verification. Mol. Cell. Proteomics 7, 1952–1962 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Gillet, L.C. et al. Targeted data extraction of the MS/MS spectra generated by data independent acquisition: a new concept for consistent and accurate proteome analysis. Mol. Cell. Proteomics 11, 1–17 (2012).

    Google Scholar 

  42. Gallien, S. et al. Targeted proteomic quantification on quadrupole Orbitrap mass spectrometer. Mol. Cell. Proteomics advance online publication 7 September 2012 (doi:10.1074/mcp.O112.019802).

    Google Scholar 

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Acknowledgements

We thank L. Gaffney for her help with graphics. This work was supported in part by the Broad Institute of MIT and Harvard, and by grants from the US National Cancer Institute (U24CA160034, part of the Clinical Proteomics Tumor Analysis Consortium initiative, to S.A.C.) and National Heart, Lung, and Blood Institute (HHSN268201000033C and R01HL096738 to S.A.C.). S.A.C. and M.A.G. acknowledge the financial support of the Women's Cancer Research Fund of the Entertainment Industry Foundation and the Komen Foundation for funding portions of this work.

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  1. Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts, USA

    Michael A Gillette & Steven A Carr

  2. Massachusetts General Hospital, Boston, Massachusetts, USA

    Michael A Gillette

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  1. Michael A Gillette
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Correspondence to Steven A Carr.

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Gillette, M., Carr, S. Quantitative analysis of peptides and proteins in biomedicine by targeted mass spectrometry. Nat Methods 10, 28–34 (2013). https://doi.org/10.1038/nmeth.2309

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