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

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


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • B-Lymphocytes / immunology*
  • Cell Line
  • Chemical Fractionation / methods
  • Dendrimers / chemistry*
  • Humans
  • Immunoglobulin M / immunology
  • Intracellular Signaling Peptides and Proteins / antagonists & inhibitors
  • Intracellular Signaling Peptides and Proteins / chemistry*
  • Intracellular Signaling Peptides and Proteins / immunology*
  • Ions / chemistry
  • Metals / chemistry
  • Phosphopeptides / analysis*
  • Phosphopeptides / isolation & purification
  • Phosphorylation
  • Protein-Tyrosine Kinases / antagonists & inhibitors
  • Protein-Tyrosine Kinases / chemistry*
  • Protein-Tyrosine Kinases / immunology*
  • Proteomics / methods
  • Signal Transduction
  • Syk Kinase
  • Titanium / chemistry
  • Zirconium / chemistry*


  • Dendrimers
  • Immunoglobulin M
  • Intracellular Signaling Peptides and Proteins
  • Ions
  • Metals
  • PAMAM Starburst
  • Phosphopeptides
  • Zirconium
  • Titanium
  • Protein-Tyrosine Kinases
  • SYK protein, human
  • Syk Kinase