Skip to main content
SpringerLink
Log in

Increased Depth and Breadth of Plasma Protein Quantitation via Two-Dimensional Liquid Chromatography/Multiple Reaction Monitoring-Mass Spectrometry with Labeled Peptide Standards

  • Protocol
  • First Online:
Quantitative Proteomics by Mass Spectrometry

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1410))

Abstract

Absolute quantitative strategies are emerging as a powerful and preferable means of deriving concentrations in biological samples for systems biology applications. Method development is driven by the need to establish new—and validate current—protein biomarkers of high-to-low abundance for clinical utility. In this chapter, we describe a methodology involving two-dimensional (2D) reversed-phase liquid chromatography (RPLC), operated under alkaline and acidic pH conditions, combined with multiple reaction monitoring (MRM)-mass spectrometry (MS) (also called selected reaction monitoring (SRM)-MS) and a complex mixture of stable isotope-labeled standard (SIS) peptides, to quantify a broad and diverse panel of 253 proteins in human blood plasma. The quantitation range spans 8 orders of magnitude—from 15 mg/mL (for vitamin D-binding protein) to 450 pg/mL (for protein S100-B)—and includes 31 low-abundance proteins (defined as being <10 ng/mL) of potential disease relevance. The method is designed to assess candidates at the discovery and/or verification phases of the biomarker pipeline and can be adapted to examine smaller or alternate panels of proteins for higher sample throughput. Also detailed here is the application of our recently developed software tool—Qualis-SIS—for protein quantitation (via regression analysis of standard curves) and quality assessment of the resulting data. Overall, this chapter provides the blueprint for the replication of this quantitative proteomic method by proteomic scientists of all skill levels.

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 84.99
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 109.99
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

References

  1. Askenazi M, Li S, Singh S, Marto JA (2010) Pathway Palette: a rich internet application for peptide-, protein- and network-oriented analysis of MS data. Proteomics 10:1880–1885. doi:10.1002/pmic.200900723

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  2. Villanueva J, Carrascal M, Abian J (2014) Isotope dilution mass spectrometry for absolute quantification in proteomics: concepts and strategies. J Proteomics 96:184–199. doi:10.1016/j.jprot.2013.11.004

    Article  CAS  PubMed  Google Scholar 

  3. Gillette MA, Carr SA (2013) Quantitative analysis of peptides and proteins in biomedicine by targeted mass spectrometry. Nat Methods 10:28–34

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Gallien S, Duriez E, Crone C, Kellmann M, Moehring T et al (2012) Targeted proteomic quantification on quadrupole-orbitrap mass spectrometer. Mol Cell Proteomics 11:1709–1723. doi:10.1074/mcp.O112.019802

    Article  PubMed Central  PubMed  Google Scholar 

  5. Gallien S, Bourmaud A, Kim SY, Domon B (2014) Technical considerations for large-scale parallel reaction monitoring analysis. J Proteomics 100:147–159. doi:10.1016/j.jprot.2013.10.029

    Article  CAS  PubMed  Google Scholar 

  6. Peterson AC, Russell JD, Bailey DJ, Westphall MS, Coon JJ (2012) Parallel reaction monitoring for high resolution and high mass accuracy quantitative, targeted proteomics. Mol Cell Proteomics 11:1475–1488. doi:10.1074/mcp.O112.020131

    Article  PubMed Central  PubMed  Google Scholar 

  7. Brun V, Dupuis A, Adrait A, Marcellin M, Thomas D et al (2007) Isotope-labeled protein standards: toward absolute quantitative proteomics. Mol Cell Proteomics 6:2139–2149

    Article  CAS  PubMed  Google Scholar 

  8. Gerber SA, Rush J, Stemman O, Kirschner MW, Gygi SP (2003) Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc Natl Acad Sci U S A 100:6940–6945

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  9. Pratt JM, Simpson DM, Doherty MK, Rivers J, Gaskell SJ et al (2006) Multiplexed absolute quantification for proteomics using concatenated signature peptides encoded by QconCAT genes. Nat Protoc 1:1029–1043

    Article  CAS  PubMed  Google Scholar 

  10. Boja ES, Rodriguez H (2012) Mass spectrometry-based targeted quantitative proteomics: achieving sensitive and reproducible detection of proteins. Proteomics 12:1093–1110. doi:10.1002/pmic.2011003871093

    Article  CAS  PubMed  Google Scholar 

  11. Picotti P, Aebersold R (2012) Selected reaction monitoring-based proteomics: workflows, potential, pitfalls and future directions. Nat Methods 9:555–566. doi:10.1038/nmeth.2015

    Article  CAS  PubMed  Google Scholar 

  12. Simpson DM, Beynon RJ (2012) QconCATs: design and expression of concatenated protein standards for multiplexed protein quantification. Anal Bioanal Chem 404:977–989. doi:10.1007/s00216-012-6230-1

    Article  CAS  PubMed  Google Scholar 

  13. Percy AJ, Chambers AG, Yang J, Hardie DB, Borchers CH (2014) Advances in multiplexed MRM-based protein biomarker quantitation toward clinical utility. Biochim Biophys Acta 1844:917–926. doi:10.1016/j.bbapap.2013.06.008

    Article  CAS  PubMed  Google Scholar 

  14. Berna M, Ott L, Engle S, Watson D, Solter P et al (2008) Quantification of NTproBNP in rat serum using immunoprecipitation and LC/MS/MS: a biomarker of drug-induced cardiac hypertrophy. Anal Chem 80:561–566. doi:10.1021/ac702311m

    Article  CAS  PubMed  Google Scholar 

  15. Whiteaker JR, Zhao L, Lin C, Yan P, Wang P et al (2012) Sequential multiplexed analyte quantification using peptide immunoaffinity enrichment coupled to mass spectrometry. Mol Cell Proteomics 11:M111.015347. doi:10.1074/mcp.M111

    Article  PubMed Central  PubMed  Google Scholar 

  16. Whiteaker JR, Zhao L, Frisch C, Ylera F, Harth S et al (2014) High-affinity recombinant antibody fragments (Fabs) can be applied in peptide enrichment immuno-MRM assays. J Proteome Res 13:2187–2196. doi:10.1021/pr4009404

    Article  PubMed Central  CAS  PubMed  Google Scholar 

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

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Huttenhain R, Soste M, Selevsek N, Rost H, Sethi A et al (2012) Reproducible quantification of cancer-associated proteins in body fluids using targeted proteomics. Sci Transl Med 4:142ra94. doi:10.1126/scitranslmed.3003989

    Article  PubMed Central  PubMed  Google Scholar 

  19. Liu T, Hossain M, Schepmoes AA, Fillmore TL, Sokoll LJ et al (2012) Analysis of serum total and free PSA using immunoaffinity depletion coupled to SRM: correlation with clinical immunoassay tests. J Proteomics 75:4747–4757. doi:10.1016/j.jprot.2012.01.035

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  20. Rezeli M, Végvári A, Ottervald J, Olsson T, Laurell T et al (2011) MRM assay for quantitation of complement components in human blood plasma—a feasibility study on multiple sclerosis. J Proteomics 75:211–220. doi:10.1016/j.jprot.2011.05.042

    Article  CAS  PubMed  Google Scholar 

  21. Paulovich AG, Whiteaker JR, Hoofnagle AN, Wang P (2008) The interface between biomarker discovery and clinical validation: the tar pit of the protein biomarker pipeline. Proteomics Clin Appl 2:1386–1402

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Percy AJ, Yang J, Chambers AG, Simon R, Hardie DB et al (2014) Multiplexed MRM with internal standards for cerebrospinal fluid candidate protein biomarker quantitation. J Proteome Res 13:3733–3747. doi:10.1021/pr500317d

    Article  CAS  Google Scholar 

  23. Gilar M, Olivova P, Daly AE, Gebler JC (2005) Two-dimensional separation of peptides using RP-RP-HPLC system with different pH in first and second separation dimensions. J Sep Sci 28:1694–1703

    Article  CAS  PubMed  Google Scholar 

  24. Song C, Ye M, Han G, Jiang X, Wang F et al (2010) Reversed-phase-reversed-phase liquid chromatography approach with high orthogonality for multidimensional separation of phosphopeptides. Anal Chem 82:53–56. doi:10.1021/ac9023044

    Article  CAS  PubMed  Google Scholar 

  25. Percy AJ, Simon R, Chambers AG, Borchers CH (2014) Enhanced sensitivity and multiplexing with 2D LC/MRM-MS and labeled standards for deeper and more comprehensive protein quantitation. J Proteomics 106:113–124. doi:10.1016/j.jprot.2014.04.024

    Article  CAS  PubMed  Google Scholar 

  26. Shi T, Sun X, Gao Y, Fillmore TL, Schepmoes AA et al (2013) Targeted quantification of low ng/mL level proteins in human serum without immunoaffinity depletion. J Proteome Res 12:3353–3361. doi:10.1021/pr400178v

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  27. Percy AJ, Chambers AG, Yang J, Domanski D, Borchers CH (2012) Comparison of standard- and nano-flow liquid chromatography platforms for MRM-based quantitation of putative plasma biomarker proteins. Anal Bioanal Chem 404:1089–1101. doi:10.1007/s00216-012-6010-y

    Article  CAS  PubMed  Google Scholar 

  28. Mohammed Y, Percy AJ, Chambers AG, Borchers CH (2015) Qualis-SIS: automated standard curve generation and quality assessment for multiplexed targeted quantitative proteomic experiments with labeled standards. J Proteome Res 14:1137–1146. doi:10.1021/pr5010955

    Article  CAS  PubMed  Google Scholar 

  29. Domanski D, Percy AJ, Yang J, Chambers AG, Hill JS et al (2012) MRM-based multiplexed quantitation of 67 putative cardiovascular disease biomarkers in human plasma. Proteomics 12:1222–1243. doi:10.1002/pmic.201100568

    Article  CAS  PubMed  Google Scholar 

  30. Proc JL, Kuzyk MA, Hardie DB, Yang J, Smith DS et al (2010) A quantitative study of the effects of chaotropic agents, surfactants, and solvents on the digestion efficiency of human plasma proteins by trypsin. J Proteome Res 9:5422–5437

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  31. Kuzyk MA, Parker CE, Domanski D, Borchers CH (2013) Development of MRM-based assays for the absolute quantitation of plasma proteins. Methods Mol Biol 1023:53–82. doi:10.1007/978-1-4614-7209-4_4

    Article  CAS  PubMed  Google Scholar 

  32. Percy AJ, Chambers AG, Yang J, Borchers CH (2013) Multiplexed MRM-based quantitation of candidate cancer biomarker proteins in undepleted and non-enriched human plasma. Proteomics 13:2202–2215. doi:10.1002/pmic.201200316

    Article  CAS  PubMed  Google Scholar 

  33. Percy AJ, Chambers AG, Smith DS, Borchers CH (2013) Standardized protocols for quality control of MRM-based plasma proteomic workflow. J Proteome Res 12:222–233. doi:10.1021/pr300893w

    Article  CAS  PubMed  Google Scholar 

  34. MacLean B, Tomazela DM, Shulman N, Chambers M, Finney GL et al (2010) Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 26:966–968. doi:10.1093/bioinformatics/btq054

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We wish to thank Genome Canada and Genome BC for STIC (Science and Technology Innovation Centre) funding and support. Carol Parker (UVic-Genome BC Proteomics Centre) is acknowledged for assisting in the manuscript editing process.

Competing Interests: Christoph Borchers is the director of the Centre and the Chief Scientific Officer of MRM Proteomics, which has commercialized the performance kits noted above for system/platform assessment.

Author information

Authors and Affiliations

  1. University of Victoria—Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101-4464 Markham St., Victoria, BC, V8Z 7X8, Canada

    Andrew J. Percy Ph.D., Juncong Yang, Andrew G. Chambers & Christoph H. Borchers Ph.D.

  2. Department of Biochemistry and Microbiology, University of Victoria, Petch Building Room 207, 3800 Finnerty Rd., Victoria, BC, V8P 5C2, Canada

    Christoph H. Borchers Ph.D.

Authors
  1. Andrew J. Percy Ph.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Juncong Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Andrew G. Chambers
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Christoph H. Borchers Ph.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Andrew J. Percy Ph.D. or Christoph H. Borchers Ph.D. .

Editor information

Editors and Affiliations

  1. NIDDK, National Institutes of Health, BETHESDA, Maryland, USA

    Salvatore Sechi

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Percy, A.J., Yang, J., Chambers, A.G., Borchers, C.H. (2016). Increased Depth and Breadth of Plasma Protein Quantitation via Two-Dimensional Liquid Chromatography/Multiple Reaction Monitoring-Mass Spectrometry with Labeled Peptide Standards. In: Sechi, S. (eds) Quantitative Proteomics by Mass Spectrometry. Methods in Molecular Biology, vol 1410. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3524-6_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3524-6_1

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3522-2

  • Online ISBN: 978-1-4939-3524-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation