Quantitative Proteomics by Mass Spectrometry pp 71-86

Part of the Methods in Molecular Biology book series (MIMB, volume 359)

The Absolute Quantification Strategy

Application to Phosphorylation Profiling of Human Separase Serine 1126
  • Scott A. Gerber
  • Arminja N. Kettenbach
  • John Rush
  • Steven P. Gygi

Abstract

The absolute quantification (AQUA) strategy provides a means to determine the precise protein or modified protein levels directly from cells or tissues. The technique is based on two major principles: stable isotope dilution theory and the use of synthetic peptides containing such stable isotopes to exactly mimic native counterparts after proteolysis. These peptides can be synthesized with modifications such as phosphorylation, methylation, and acetylation to allow for the direct, quantitative analysis of posttranslationally modified proteins. In this chapter, we discuss the development of an AQUA method and demonstrate its usefulness in the measurement of endogenous levels of the human protein separase at a functionally relevant phosphorylation site, serine 1126.

Key Words

Quantitative proteomic Quantitation Proteomics mass spectrometry isotopic labeling absolute quantification phosphoprotein 

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Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  • Scott A. Gerber
    • 1
    • 2
  • Arminja N. Kettenbach
    • 3
  • John Rush
    • 4
  • Steven P. Gygi
    • 3
  1. 1.Department of GeneticsNorris Cotton Cancer CenterHanover
  2. 2.Dartmouth Medical SchoolHanover
  3. 3.Department of Cell BiologyHarvard Medical SchoolBoston
  4. 4.Cell Signaling TechnologyBeverly

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