Gene Regulatory Networks (GRN) play a vital role in nearly every process of life (e.g., metabolism, cell cycle, signal transduction, etc.). Consequently, understanding GRN structure and dynamics has the potential to: (i) shed light on human disease processes, (ii) identify therapeutic drug-targets in pathogens, and (iii) direct improvement of microbes used in the biotechnology industry. Because nearly every GRN contains at least one protein kinase, it is of critical importance to understand the phosphorylation state of a cell - i.e., which proteins are, or are not, phosphorylated and the location of these phosphorylations on the target protein. Our aim is to develop a robust tool to quantify these phosphorylation changes in a cell, on a global and comprehensive level. We demonstrate a novel and cost-effective approach to quantify the phosphorylation state of a cell using Deuterium isobaric Amine Reactive Tag � DiART. DiART is chemically similar to commercially available mass-tags (e.g.,  iTRAQ, TMT), but shows significantly better analytical sensitivity and accuracy, and costs approximately 75% less. The combination of better performance and dramatically lower cost, enables proteomic workflows which have been, up to now, economically prohibitive. For example, using DiART-tagged mixtures of two synthetic, phosphorylated peptides, and their non-phosphorylated counterparts, we demonstrate the compatibility of DiART with TiO2-affinity purification of phosphorylated peptides. Comparison of theoretical vs experimental reporter ion ratios reveals accurate quantification of phosphorylated peptides over a dynamic range of more than 15 fold. Using DiART labelling and TiO2 enrichment (DiART-TiO2) with large quantities (8mg) of protein from model fungus Aspergillus nidulans cell lysate, we quantified 744 unique phosphopeptides. Overlap of TiO2-enriched phosphopeptide median and theoretical values demonstrates our ability to accurately quantify phosphoproteins in a complex mixture. We will discuss these data, and our efforts to use DiART to elucidate an important gene regulatory network in A. nidulans.

New tool for studying gene regulatory networks: DiART - a novel, cost-effective approach to quantify phosphorylation state of a cell

247th National Meeting (2014)

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