Measurement of Phosphorylated Peptides with Absolute Quantification

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


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 


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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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