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 Cgamma2) 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 Cgamma2 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.