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Molecular & Cellular Proteomics 7:645-660, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Special Issue: 8th International Symposium On Mass Spectrometry In The Life Sciences

Combined Enzymatic and Data Mining Approaches for Comprehensive Phosphoproteome Analyses

Application to Cell Signaling Events of Interferon--Stimulated Macrophages ^*,S

Maria Marcantonio^,, Matthias Trost^,¶, Mathieu Courcelles^,, Michel Desjardins^¶ and Pierre Thibault^,,||,**

From the Institute for Research in Immunology and Cancer and Departments of Biochemistry, ^¶ Pathology and Cellular Biology, and ^|| Chemistry, Université de Montréal, P. O. Box 6128, Station Centre-ville, Montréal H3C 3J7, Canada

Protein phosphorylation is a central cell signaling event that underlies a broad spectrum of key physiological processes. Advances in affinity chromatography and mass spectrometry are now providing the ability to identify and quantitate thousands of phosphorylation sites simultaneously. Comprehensive phosphoproteome analyses present sizable analytical challenges in view of suppression effects of phosphopeptides and the variable quality of MS/MS spectra. This work presents an integrated enzymatic and data mining approach enabling the comprehensive detection of native and putative phosphopeptides following alkaline phosphatase digestion of titanium dioxide (TiO₂)-enriched cell extracts. The correlation of retention times of more than 750 phospho- and dephosphopeptide pairs from J774 macrophage cell extracts indicated that removal of the phosphate groups can impart a gain or a loss in hydrophobicity that is partly explained by the formation of a salt bridge with proximal amino groups. Dephosphorylation also led to an average 2-fold increase in MS sensitivity that facilitated peptide sequencing. More importantly, alkaline phosphatase digestion enhanced the overall population of putative phosphopeptides from TiO₂-enriched cell extracts providing a unique approach to profile multiphosphorylated cognates that would have remained otherwise undetected. The application of this approach is demonstrated for differential phosphoproteome analyses of mouse macrophages exposed to interferon- for 5 min. TiO₂ enrichment enabled the identification of 1143 phosphopeptides from 432 different proteins of which 125 phosphopeptides showed a 2-fold change upon interferon- exposure. The use of alkaline phosphatase nearly doubled the number of putative phosphopeptides assignments leading to the observation of key interferon- signaling events involved in vesicle trafficking, production of reactive oxygen species, and mRNA translation.

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