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


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 


  1. 1.
    de Leenheer, A. P. and Thienpont, L. M. (1992) Applications of isotope-dilution mass-spectrometry in clinical-chemistry, pharmacokinetics, and toxicology. Mass Spectrom. Reviews 11, 249–307.CrossRefGoogle Scholar
  2. 2.
    Aebersold, R. and Mann, M. (2003) Mass spectrometry-based proteomics. Nature 422, 198–207.CrossRefPubMedGoogle Scholar
  3. 3.
    Tao, W. A. and Aebersold, R. (2003) Advances in quantitative proteomics via stable isotope tagging and mass spectrometry. Curr. Opin. Biotechnol. 14, 110–118.CrossRefPubMedGoogle Scholar
  4. 4.
    Sechi, S. and Oda, Y. (2003) Quantitative proteomics using mass spectrometry. Curr. Opin. Chem. Biol. 7, 70–77.CrossRefPubMedGoogle Scholar
  5. 5.
    Gerber, S. A., Rush, J., Stemmann, O., Kirschner, M. W., and Gygi, S. P. (2003) Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc. Natl. Acad. USA 100, 6940–6945.CrossRefGoogle Scholar
  6. 6.
    Stemmann, O., Zou, H., Gerber, S. A., Gygi, S. P., and Kirschner, M. W. (2001) Dual inhibition of sister chromatid separation at metaphase. Cell 107, 715–726.CrossRefPubMedGoogle Scholar
  7. 7.
    Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H., and Aebersold, R. (1999) Quantitative analysis of protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994–999.CrossRefPubMedGoogle Scholar
  8. 8.
    Ong, S. E., Blagoev, B., Kratchmarova, I., et al. (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1, 376–386CrossRefPubMedGoogle Scholar
  9. 9.
    Kirkpatrick, D., Gerber, S. A., and Gygi, S. P. (2005) The absolute quantification strategy: a general procedure for the quantification of proteins and post-translational modifications. Methods 35, 265–273.CrossRefPubMedGoogle Scholar
  10. 10.
    Broenstrup, M. (2005) Absolute quantification strategies in proteomics based on mass spectrometry. Expert Rev. Proteomics 1, 503–512.CrossRefGoogle Scholar
  11. 11.
    Zou, H., McGarry, T. J., Bernal, T., and Kirschner, M. W. (1999) Identification of a vertebrate sister-chromatid separation involved in transformation and tumorigenesis. Science 285, 418–422.CrossRefPubMedGoogle Scholar
  12. 12.
    Waizenegger, I., Gimenez-Abian, J. F., Wernic, D., and Peters, J. M. (2002) Regulation of human separase by securin binding and autocleavage. Cell 12, 1368–1378.Google Scholar
  13. 13.
    Chestukhin, A. and DeCaprio, J. A. (2003) Western blot screening for monoclonal antibodies against human separase. J. Immunol. Methods 274, 105–113.CrossRefPubMedGoogle Scholar
  14. 14.
    Havlis, J. and Shevchenko, A. (2004) Absolute quantification of proteins in solutions and in polyacrylamide gels by mass spectrometry. Anal. Chem. 76, 3029–3036.CrossRefPubMedGoogle Scholar

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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