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.

Comparing Multistep Immobilized Metal Affinity Chromatography and Multistep TiO2 Methods for Phosphopeptide Enrichment

“…There are several approaches for enriching phosphopeptides from the peptide mixtures, including immobilized metal affinity chromatography (IMAC) with Fe 3+ or Ti 4+ 7,8,9 , metal oxide affinity chromatography (MOAC) with TiO 2 10,11 , and immuno-affinity based methods 12 . Other methods such as polymer-based metal ion affinity capture (PolyMAC) 13,14 and polymer-oxotitanium-modified gold wafer (Au-P-oxoTi) 15 result in successful phosphopeptide enrichment.…”

Multiplexed Imaging of Protein Phosphorylation on Membranes Based on TiIV Functionalized Nanopolymers

“…[6] The water-soluble nanopolymer is functionalized with titanium ions, thereby enabling strong and specific binding to phosphoproteins, and with “handle groups” for enrichment before MS analyses [6b, 7] or for detection. [6a, c, d] Multiple titanium ions and handle groups on each nanopolymer improve binding efficacy and enhance the signal, thus allowing efficient isolation or detection of phosphoproteins in low abundance.…”

Understanding Epstein-Barr Virus Life Cycle with Proteomics: A Temporal Analysis of Ubiquitination During Virus Reactivation

“…Previously, proteomic studies were performed to examine the ubiquitination events shortly after IgG cross-linking of mouse or human B cell lines ( Jayasundera et al, 2014;Satpathy et al, 2015). To obtain a global view of the long-term effects on protein ubiquitination, we utilized SILAC-based proteomics to survey the dynamic changes in ubiquitination in EBV + B cells upon IgG cross-linking (Harsha et al, 2008;Ong et al, 2002).…”

“…The cellular response to BCR activation involves the assembly of BCR signaling proteins that regulate a cascade of downstream signaling events, including protein phosphorylation and ubiquitination ( Jayasundera et al, 2014;Matsumoto et al, 2009;Satpathy et al, 2015). System-wide identification of downstream signaling events has revealed important roles for phosphorylation and ubiquitination within minutes of the BCR activation process.…”