title: 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.
authors.affiliation: Department of Biochemistry, Purdue University, West Lafayette, IN 47906 ; Tymora Analytical Operations, LLC. 1281 Win Hentschel Blvd., West Lafayette, IN 47906.
authors.forename: Anton
authors.initials: A
authors.lastname: Iliuk
authors.affiliation: Department of Chemistry, Purdue University, West Lafayette, IN 47907.
authors.forename: Keerthi
authors.initials: K
authors.lastname: Jayasundera
authors.affiliation: Department of Medicinal Chemistry & Molecular Pharmacology, Purdue University, West Lafayette, IN 47907.
authors.forename: Wen-Horng
authors.initials: WH
authors.lastname: Wang
authors.affiliation: Department of Biochemistry, Purdue University, West Lafayette, IN 47906.
authors.forename: Rachel
authors.initials: R
authors.lastname: Schluttenhofer
authors.affiliation: Department of Medicinal Chemistry & Molecular Pharmacology, Purdue University, West Lafayette, IN 47907.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Geahlen
authors.affiliation: 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.
authors.forename: W Andy
authors.initials: WA
authors.lastname: Tao
keywords: Proteomics; Tandem mass spectrometry; kinase; protein phosphorylation