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Int J Mass Spectrom. 2015 Feb 1;377:744-753.

In-Depth Analyses of B Cell Signaling Through Tandem Mass Spectrometry of Phosphopeptides Enriched by PolyMAC.

Author information

1
Department of Biochemistry, Purdue University, West Lafayette, IN 47906 ; Tymora Analytical Operations, LLC. 1281 Win Hentschel Blvd., West Lafayette, IN 47906.
2
Department of Chemistry, Purdue University, West Lafayette, IN 47907.
3
Department of Medicinal Chemistry & Molecular Pharmacology, Purdue University, West Lafayette, IN 47907.
4
Department of Biochemistry, Purdue University, West Lafayette, IN 47906.
5
Department of Biochemistry, Purdue University, West Lafayette, IN 47906 ; Tymora Analytical Operations, LLC. 1281 Win Hentschel Blvd., West Lafayette, IN 47906 ; Department of Chemistry, Purdue University, West Lafayette, IN 47907 ; Department of Medicinal Chemistry & Molecular Pharmacology, Purdue University, West Lafayette, IN 47907.

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.

KEYWORDS:

Proteomics; Tandem mass spectrometry; kinase; protein phosphorylation

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