SpringerProtocols: Abstract: The Absolute Quantification Strategy: Application to Phosphorylation Profiling of Human Separase Serine 1126

The Absolute Quantification Strategy: Application to Phosphorylation Profiling of Human Separase Serine 1126

By: Scott A. Gerber^{2 3}, Arminja N. Kettenbach⁴, John Rush⁵, Steven P. Gygi⁴

Abstract

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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.

Affiliation(s): (2) Department of Genetics, Norris Cotton Cancer Center, Hanover, NH
(3) Dartmouth Medical School, Hanover, NH
(4) Department of Cell Biology, Harvard Medical School, Boston, MA
(5) Cell Signaling Technology, Beverly, MA

Book Title: Quantitative Proteomics by Mass Spectrometry

Series: Methods in Molecular Biology | Volume: 359 | Pub. Date: Feb-05-2007 | Page Range: 71-86 | DOI: 10.1007/978-1-59745-255-7_5

Subject: Biochemistry

Key Words: Quantitative proteomic - Quantitation - Proteomics - mass spectrometry - isotopic labeling - absolute quantification - phosphoprotein

References

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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.

2.	Aebersold, R. and Mann, M. (2003) Mass spectrometry-based proteomics. Nature 422, 198–207.

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. <Occurrence Type="DOI"><Handle>10.1016/S0958-1669(02)00018-6</Handle></Occurrence>

4.	Sechi, S. and Oda, Y. (2003) Quantitative proteomics using mass spectrometry. Curr. Opin. Chem. Biol. 7, 70–77. <Occurrence Type="DOI"><Handle>10.1016/S1367-5931(02)00010-8</Handle></Occurrence>

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.

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. <Occurrence Type="DOI"><Handle>10.1016/S0092-8674(01)00603-1</Handle></Occurrence>

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.

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, 376–386

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.

10.	Broenstrup, M. (2005) Absolute quantification strategies in proteomics based on mass spectrometry. Expert Rev. Proteomics 1, 503–512.

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.

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.

13.	Chestukhin, A. and DeCaprio, J. A. (2003) Western blot screening for monoclonal antibodies against human separase. J. Immunol. Methods 274, 105–113. <Occurrence Type="DOI"><Handle>10.1016/S0022-1759(02)00508-2</Handle></Occurrence>

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.

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