"Development of Novel LC-MS - based Methods for Comprehensive and Accurate Analysis of Proteins and Protein Phosphorylations"

Ronghu Wu, PhD
Assistant Professor, School of Chemistry & Biochemistry
Georgia Tech

Abstract
Reversible protein phosphorylation has an important role in biological systems and is involved in a variety of cellular functions. In order to comprehensive and accurate analysis of protein phosphorylation, we need not only to identify the modification site and measure the abundance change, but also to differentiate the phosphorylation abundance change from protein expression change and determine the phosphorylation site stoichiometry. I investigated the protein calibration by performing parallel comparisons of protein expression and phosphorylation in yeast (S. cerevisiae). Over 4,000 proteins, which cover almost the whole proteome, were quantified and more than 12,000 unique phosphorylation sites were identified. For the first time, 96% of non-redundant phosphopeptide ratios could be calibrated by protein levels, allowing truly differential phosphorylation to be distinguished from altered protein expression. This revealed a starkly different view, with a quarter of seemingly differential phosphopeptides now attributed to changes in protein expression. As for measuring the absolute stoichiometry of protein phosphorylation on a large scale, it is even more challenging. Recently I developed a MS-based method to measure the absolute stoichiometries of protein phosphorylation on a large scale. This approach requires the measurement of only a single ratio relating phosphatase-treated and mock-treated samples. Using the developed method over 5,000 phosphorylation site stoichiometries were obtained in triplicate analyses from yeast. Some interesting biological properties associated with phosphorylation site stoichiometries were characterized.

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