title: Global phosphoproteomics of activated B cells using complementary metal ion functionalized soluble nanopolymers.
abstract: 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.
authors.affiliation: 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.
authors.forename: Keerthi B
authors.initials: KB
authors.lastname: Jayasundera
authors.forename: Anton B
authors.initials: AB
authors.lastname: Iliuk
authors.forename: Andrew
authors.initials: A
authors.lastname: Nguyen
authors.forename: Renee
authors.initials: R
authors.lastname: Higgins
authors.forename: Robert L
authors.initials: RL
authors.lastname: Geahlen
authors.forename: W Andy
authors.initials: WA
authors.lastname: Tao
mesh_terms: D001402:B-Lymphocytes / Q000276:immunology*; D002460:Cell Line; D005591:Chemical Fractionation / Q000379:methods; D050091:Dendrimers / Q000737:chemistry*; D006801:Humans; D007075:Immunoglobulin M / Q000276:immunology; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000276:immunology*; D007477:Ions / Q000737:chemistry; D008670:Metals / Q000737:chemistry; D010748:Phosphopeptides / Q000032:analysis* / Q000302:isolation & purification; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000276:immunology*; D040901:Proteomics / Q000379:methods; D015398:Signal Transduction; D000072377:Syk Kinase; D014025:Titanium / Q000737:chemistry; D015040:Zirconium / Q000737:chemistry*
chemical_list: D050091:Dendrimers; D007075:Immunoglobulin M; D047908:Intracellular Signaling Peptides and Proteins; D007477:Ions; D008670:Metals; C104700:PAMAM Starburst; D010748:Phosphopeptides; D015040:Zirconium; D014025:Titanium; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase