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

Abstract

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 nonisotopically 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 C␥2) 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 phospho- Reversible covalent modification of proteins is a key molecular mechanism by which membrane receptor activation is converted into intracellular signaling cascades that mediate physiological change. Engagement of ligands with their receptors results in rapid and transient phosphorylation of downstream protein targets, which as a consequence modify their catalytic properties, cellular localization, and/or binding partners. As a consequence, intracellular signaling pathways can control critical aspects of metabolism, cell cycle progression, migration, differentiation, and protein synthesis. Many diseases are a direct consequence of deregulation in these processes (1, 2); these changes may be detectable by quantitative phosphoprotein analysis.The assessment of the phosphorylation status of specific proteins is commonly performed by immunochemical methods using antisera raised against a peptide containing the site of phosphorylation of the target protein. This approach is restricted to the study of known phosphorylation sites and cannot be used to monitor and discover novel phosphorylated proteins and their sites of phosphorylation. To overcome this proble...

Quantitative Profile of Five Murine Core Proteomes Using Label-free Functional Proteomics

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Ultrasensitive and absolute quantification of the phosphoinositide 3-kinase/Akt signal transduction pathway by mass spectrometry

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“…This fact means that even the most sensitive mass spectrometers struggle to detect sites of modification on regulatory proteins (quantification being even more challenging). Thus, MS-based proteomic studies aimed at investigating the mechanisms of signal transduction have used large numbers of cells (typically Ϸ10 8 ) per data point (16,17,27), overexpressed the protein under study (18), or performed analyses on recombinant proteins (31). Clearly, these strategies are not applicable to the analysis of all biological specimens, and it is extremely challenging for current mass spectrometric methods to detect regulatory proteins and their sites of modification in clinically relevant tissues such as biopsies.…”

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