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Anal Chem. 2014 Jul 1;86(13):6363-71. doi: 10.1021/ac500599r. Epub 2014 Jun 20.

Global phosphoproteomics of activated B cells using complementary metal ion functionalized soluble nanopolymers.

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Department of Chemistry, ‡Department of Biochemistry, §School of Chemical Engineering, ∥Department of Medicinal Chemistry and Molecular Pharmacology, and ⊥the Purdue Center for Cancer Research, Purdue University , West Lafayette, Indiana 47907, United States.


Engagement of the B cell receptor for antigen (BCR) leads to immune responses through a cascade of intracellular signaling events. Most studies to date have focused on the BCR and protein tyrosine phosphorylation. Because spleen tyrosine kinase, Syk, is an upstream kinase in multiple BCR-regulated signaling pathways, it also affects many downstream events that are modulated through the phosphorylation of proteins on serine and threonine residues. Here, we report a novel phosphopeptide enrichment strategy and its application to a comprehensive quantitative phosphoproteomics analysis of Syk-dependent downstream signaling events in B cells, focusing on serine and threonine phosphorylation. Using a combination of the Syk inhibitor piceatannol, SILAC quantification, peptide fractionation, and complementary PolyMAC-Ti and PolyMAC-Zr enrichment techniques, we analyzed changes in BCR-stimulated protein phosphorylation that were dependent on the activity of Syk. We identified and quantified over 13,000 unique phosphopeptides, with a large percentage dependent on Syk activity in BCR-stimulated B cells. Our results not only confirmed many known functions of Syk, but more importantly, suggested many novel roles, including in the ubiquitin proteasome pathway, that warrant further exploration.

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