In-depth analyses of B cell signaling through tandem mass spectrometry of phosphopeptides enriched by PolyMAC

Abstract

Tandem mass spectrometry (MS/MS) has enabled researchers to analyze complex biological samples since the original concept inception. It facilitates the identification and quantification of modifications within tens of thousands of proteins in a single large-scale proteomic experiment. Phosphorylation analysis, as one of the most common and important post-translational modifications, has particularly benefited from such progress in the field. Here we showcase the technique through in-depth analyses of B cell signaling based on quantitative phosphoproteomics. As a complement to the previously described PolyMAC-Ti (polymer-based metal ion affinity capture using titanium) reagent, we introduce here PolyMAC-Fe, which utilizes a different metal ion, Fe(III). An extensive comparison using the different available MS/MS fragmentations techniques was made between PolyMAC-Fe, PolyMAC-Ti and IMAC (immobilized metal ion affinity chromatography) reagents in terms of specificity, reproducibility and type of phosphopeptides being enriched. PolyMAC-Fe based chelation demonstrated good selectivity and unique specificity toward phosphopeptides, making it useful in specialized applications. We have combined PolyMAC-Ti and PolyMAC-Fe, along with SILAC-based quantitation and large-scale fractionation, for quantitative B cell phosphoproteomic analyses. The complementary approach allowed us to identify a larger percentage of multiply phosphorylated peptides than with PolyMAC-Ti alone. Overall, out of 13,794 unique phosphorylation sites identified, close to 20% were dependent on BCR signaling. These sites were further mapped to a variety of major signaling networks, offering more detailed information about the biochemistry of B cell receptor engagement.

New Opportunities and Challenges of Smart Polymers in Post‐Translational Modification Proteomics

“…a–c) Polymer‐modified IMAC and MOAC phosphopeptide (PP) enrichment materials: Ti 4+ ‐immobilized polymer bead (a) and polydopamine coated graphene (b), polymer‐grafted capillary (c) . d,e) Schematic representation of enrichment strategies for polymer‐supported metal ion affinity capture‐Fe 3+ (d) and polymer brush–oxotitanium hybrid material (e) . In these examples, because of the sufficient binding sites provided by the functional polymers, the grafting densities of metal ions or TiO 2 on these polymeric matrices are substantially improved, resulting in more favorable PP enrichment.…”

Recent advances in phosphoproteomics and application to neurological diseases

“…To address the drawbacks of solid-phase phosphopeptide extraction, our group developed a PolyMAC strategy by immobilizing the metal ions on the surface of the water-soluble dendrimer to capture phosphopeptides in the homogenous aqueous environment. 59, 60 This strategy has demonstrated outstanding selectivity, sensitivity, and recovery when compared to Fe-IMAC and TiO 2 for complex samples, and this strategy has been applied to study the phosphoproteome of animals and plants. 61–64 …”

The current state of the art of quantitative phosphoproteomics and its applications to diabetes research

“…One main limitation of MOAC is related to its specificity due to the potential binding to acidic peptides. Larsen et al [73] in 2005 described an approach to improve the selectivity towards phosphopeptides by loading the sample in 2,5-dihydroxybenzoic acid (DHB) solution containing acetonitrile and TFA so that DHB competes with the binding of acidic peptides on TiO 2 beads, thus achieving improved overall specificity for enriching phosphopeptides. Phosphopeptides were eluted using an alkaline solution at pH 10.5 using ammonium hydroxide.…”

“…In addition, polymer-based metal-ion affinity capture (PolyMAC) has emerged to as a novel chemical strategy based on water soluble, globular dentrimers multifunctionalized with metal ions (e.g., Ti 4+ , Fe 3+ ) for capturing phosphate groups [72,73]. Comparing to TiO 2 , PolyMAC displayed excellent reproducibility, selectivity, and high recovery of phosphopeptides from complex mixtures.…”