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Measurement of Phosphorylated Peptides with Absolute Quantification

  • Raven J. Reddy
  • Timothy G. Curran
  • Yi Zhang
  • Forest M. WhiteEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1410)

Abstract

Mass spectrometry, when coupled to on-line separation such as liquid chromatography or capillary electrophoresis, enables the identification and quantification of protein expression and post-translational modification changes under diverse conditions. To date most of the methods for mass spectrometry-based quantification have either provided relative quantification information (e.g., comparison to a selected condition) or utilized one-point calibration curves, or calibration curves in a different biological matrix. Although these quantitative methods have been used to generate insight into the differences between biological samples, additional biological insight could be gained by accurately measuring the absolute quantity of selected proteins and protein modifications. To address this challenge, we have developed the MARQUIS (Multiplex Absolute Regressed Quantification with Internal Standards) method, designed to provide absolute quantification for potentially hundreds of peptides across multiple samples in a single analysis, using a multi-point internal calibration curve derived from synthetic, isotopically distinct standard peptides.

Key words

Absolute quantification Phosphorylation EGFR IMAC LC-MS/MS 

References

  1. 1.
    Morris M, Chi A, Melas I et al (2014) Phosphoproteomics in drug discovery. Drug Discov Today 19:425–432CrossRefPubMedGoogle Scholar
  2. 2.
    Jones R, Gordus A, Krall J et al (2005) A quantitative protein interaction network for the ErbB receptor using protein microarrays. Nature 439:168–174CrossRefPubMedGoogle Scholar
  3. 3.
    Liang S, Xu Z, Xu X et al (2012) Quantitative proteomics for cancer biomarker discovery. Comb Chem High Throughput Screen 15:221–231CrossRefPubMedGoogle Scholar
  4. 4.
    Wasinger V, Zeng M, Yau Y (2013) Current status and advances in quantitative proteomic mass spectrometry. Int J Proteomics 2013:1–12CrossRefGoogle Scholar
  5. 5.
    Wolf-Yadlin A, Kumar N, Zhang Y et al (2006) Effects of HER2 overexpression on cell signaling networks governing proliferation and migration. Mol Syst Biol 2:1–15CrossRefGoogle Scholar
  6. 6.
    Gerber S, Rush J, Stemman O et al (2003) Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc Natl Acad Sci U S A 100:6940–6945PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Curran T, Zhang Y, Ma D et al (2015) MARQUIS: a multiplex method for absolute quantification of peptides and posttranslational modifications. Nat Commun 6:1–11CrossRefGoogle Scholar
  8. 8.
    Yarden Y (2001) The EGFR family and its ligands in human cancer: signalling mechanisms and therapeutic opportunities. Eur J Cancer 37:S3–S8CrossRefPubMedGoogle Scholar
  9. 9.
    Gan H, Cvrljevic A, Johns T (2013) The epidermal growth factor receptor variant III (EGFRvIII): where wild things are altered. FEBS J 280:5350–5370CrossRefPubMedGoogle Scholar
  10. 10.
    Schulze W, Deng L, Mann M (2005) Phosphotyrosine interactome of the ErbB-receptor kinase family. Mol Syst Biol 1:42–54CrossRefGoogle Scholar
  11. 11.
    Moser K, White F (2006) Phosphoproteomic analysis of rat liver by high capacity IMAC and LC-MS/MS. J Proteome Res 5:98–104CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Raven J. Reddy
    • 1
    • 2
  • Timothy G. Curran
    • 1
    • 2
  • Yi Zhang
    • 3
  • Forest M. White
    • 1
    • 2
    Email author
  1. 1.Department of Biological EngineeringMassachusetts Institute of TechnologyCambridgeUSA
  2. 2.Koch Institute for Integrative Cancer ResearchMassachusetts Institute of TechnologyCambridgeUSA
  3. 3.Thermo-Fisher ScientificSan JoseUSA

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