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Molecular & Cellular Proteomics 4:1038-1051, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Quantification of Gel-separated Proteins and Their Phosphorylation Sites by LC-MS Using Unlabeled Internal Standards

Analysis of Phosphoprotein Dynamics in a B Cell Lymphoma Cell Line^*,S

Pedro R. Cutillas^,, Barbara Geering^,¶, Mike D. Waterfield^|| and Bart Vanhaesebroeck^,¶

From the Cell Signalling Group, Ludwig Institute for Cancer Research, 91 Riding House Street, London, W1W 7BS, ^|| Proteomics Unit, Ludwig Institute for Cancer Research, Cruciform Building, London, W1C 6BT, and the ^¶ Department of Biochemistry and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, United Kingdom

Protein phosphorylation plays a critical role in normal cellular function and is often subverted in disease. Although major advances have recently been made in identification and quantitation of protein phosphorylation sites by MS, current methodological limitations still preclude routine, easily usable, and comprehensive quantitative analysis of protein phosphorylation. Here we report a simple LC-MS method to quantify gel-separated proteins and their sites of phosphorylation; in this approach, integrated chromatographic peak areas of peptide analytes from proteins under study are normalized to those of a non-isotopically labeled internal standard protein spiked into the excised gel samples just prior to in-gel digestion. The internal standard intensities correct for differences in enzymatic activities and sample losses that may occur during the processes of in-gel digestion and peptide extraction from the gel pieces. We used this method of peak area measurement with an internal standard to investigate the effects of pervanadate on protein phosphorylation in the WEHI-231 B cell lymphoma cell line and to assess the role of phosphoinositide 3-kinase (PI3K) in these phosphorylation events. Phosphoproteins, isolated from total cell lysates using IMAC or by immunoprecipitation using Tyr(P) antibodies, were analyzed using this method, leading to identification of >400 proteins, several of which were found at higher levels in phosphoprotein fractions after pervanadate treatment. Pretreatment of cells with the PI3K inhibitor wortmannin reduced the phosphorylation level of certain proteins (e.g. STAT1 and phospholipase C2) while increasing the phosphorylation of several others. Peak area measurement with an internal standard was also used to follow the dynamics of PI3K-dependent and -independent changes in the post-translational modification of both known and novel phospholipase C2 phosphorylation sites. Our results illustrate the capacity of this conceptually simple LC-MS method for quantification of gel-separated proteins and their phosphorylation sites and for quantitative profiling of biological systems.

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