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A systematic approach to the analysis of protein phosphorylation

Nature Biotechnology volume 19, pages 375–378 (2001)Cite this article

Abstract

Reversible protein phosphorylation has been known for some time to control a wide range of biological functions and activities1,2,3. Thus determination of the site(s) of protein phosphorylation has been an essential step in the analysis of the control of many biological systems. However, direct determination of individual phosphorylation sites occurring on phosphoproteins in vivo has been difficult to date, typically requiring the purification to homogeneity of the phosphoprotein of interest before analysis4,5,6. Thus, there has been a substantial need for a more rapid and general method for the analysis of protein phosphorylation in complex protein mixtures. Here we describe such an approach to protein phosphorylation analysis. It consists of three steps: (1) selective phosphopeptide isolation from a peptide mixture via a sequence of chemical reactions, (2) phosphopeptide analysis by automated liquid chromatography–tandem mass spectrometry (LC-MS/MS), and (3) identification of the phosphoprotein and the phosphorylated residue(s) by correlation of tandem mass spectrometric data with sequence databases. By utilizing various phosphoprotein standards and a whole yeast cell lysate, we demonstrate that the method is equally applicable to serine-, threonine- and tyrosine-phosphorylated proteins, and is capable of selectively isolating and identifying phosphopeptides present in a highly complex peptide mixture.

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Figure 1: Phosphopeptide isolation strategy and validation with phosphoprotein β-casein.
Figure 2: Phosphopeptide isolation from LCK–MBP kinase reaction mixture.
Figure 3: Phosphopeptide isolation from yeast cell lysate.

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Acknowledgements

The National Science Foundation Science and Technology Center for Molecular Biotechnology, the National Institutes of Health (RO1 A141109 and 1R33 CA84698), the NIH Research Resource Center (RR11823), and the Merck Genome Research Institute provided support for this work. We thank Drs. Beate Rist, Steven P. Gygi, and David R. Goodlett for helpful discussions.

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  1. Huilin Zhou, Julian D. Watts & Ruedi Aebersold

    Present address: Institute for Systems Biology, 4225 Roosevelt Way NE, Suite 200, Seattle, WA, 98105

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  1. Department of Molecular Biotechnology, University of Washington, Seattle, 98195, WA

    Huilin Zhou, Julian D. Watts & Ruedi Aebersold

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Correspondence to Ruedi Aebersold.

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Zhou, H., Watts, J. & Aebersold, R. A systematic approach to the analysis of protein phosphorylation. Nat Biotechnol 19, 375–378 (2001). https://doi.org/10.1038/86777

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