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Quantitation of phosphopeptides using affinity chromatography and stable isotope labeling

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Journal of the American Society for Mass Spectrometry

Abstract

Reversible phosphorylation of proteins represents an important component of cellular signaling pathways. The isolation of phosphoproteins in complex mixtures and the determination of the level of phosphorylation have been and remain a major challenge. It has prompted the development of several strategies, including immobilized metal affinity capture to enrich for phosphorylated peptides. An improved methodology was published (Ficarro, et al., Nature Biotechnology 2002, 20, 301–305) that showed increased selectivity through esterification of amino acid side chain carboxylic groups of enzymatically digested peptides. This method was applied for relative quantitation of phosphopeptides in conjunction with the use of stable isotope labeling. The merits and limits of the approach are discussed and its application to the analysis of the effects of serum starvation on in vitro cultured human lung cells is presented.

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Author notes

  1. Scott D. Patterson

    Present address: Amgen Inc, 91320, Thousand Oaks, CA, USA

Authors and Affiliations

  1. Celera Genomics Group, 5 W. Gude Drive, 20850, Rockville, MD, USA

    Tao He, Kim Alving, Brian Feild, James Norton, Elizabeth G. Joseloff, Scott D. Patterson & Bruno Domon

Authors
  1. Tao He
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  2. Kim Alving
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  3. Brian Feild
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Correspondence to Bruno Domon.

Additional information

Published online January 15, 2004

This article is in honor of Ruedi Aebesold, recipient of the 2002 Biemann Award.

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He, T., Alving, K., Feild, B. et al. Quantitation of phosphopeptides using affinity chromatography and stable isotope labeling. J Am Soc Mass Spectrom 15, 363–373 (2004). https://doi.org/10.1016/j.jasms.2003.11.004

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  • DOI: https://doi.org/10.1016/j.jasms.2003.11.004

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