Quantitative Proteomics by Mass Spectrometry

Volume 359 of the series Methods in Molecular Biology pp 71-86

The Absolute Quantification Strategy

Application to Phosphorylation Profiling of Human Separase Serine 1126
  • Scott A. GerberAffiliated withDepartment of Genetics, Norris Cotton Cancer CenterDartmouth Medical School
  • , Arminja N. KettenbachAffiliated withDepartment of Cell Biology, Harvard Medical School
  • , John RushAffiliated withCell Signaling Technology
  • , Steven P. GygiAffiliated withDepartment of Cell Biology, Harvard Medical School

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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