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A sensitive magnetic bead method for the detection and identification of tyrosine phosphorylation in proteins by MALDI-TOF/TOF MS
journal contribution
posted on 2009-06-11, 00:00 authored by Mark R Condina, Mark GuthridgeMark Guthridge, Shaun R McColl, Peter HoffmannPhosphorylation is one of the most important PTMs and is estimated to occur on 30% of the mammalian proteome. Its perturbed regulation has been implicated in many pathologies. The rarity of phosphotyrosine compared with phosphoserine or phosphothreonine is prompting the development of more sensitive approaches because proteomic technologies that are currently used to assess tyrosine phosphorylation in proteins are inadequate, identifying only a fraction of the predicted tyrosine phosphoproteome. Here we describe the development of a reproducible, high‐sensitivity methodology for the detection and mapping of phosphotyrosine residues by MS. The anti‐phosphotyrosine antibody 4G10 was coupled covalently to super para‐magnetic beads or by affinity to super para‐magnetic beads with protein G covalently attached. Using this approach, we successfully enriched phosphotyrosine peptides mixed with non‐phosphorylated peptides at a ratio of up to 1:200, enabling detection at a level representing the highest sensitivity reported for tyrosine phosphorylation. The beads were subsequently used to enrich tyrosine phosphopeptides from a digest of the in vitro‐phosphorylated recombinant β‐intracellular region of the granulocyte‐macrophage colony‐stimulating factor receptor, which was subsequently analysed by MALDI‐TOF/TOF MS. Our results define this methodology as a sensitive approach for tyrosine phosphoproteome analysis.
History
Journal
ProteomicsVolume
9Issue
11Pagination
3047 - 3057Publisher
Wiley-VCH Verlag GmbH & Co. KGaALocation
Weinheim, GermanyPublisher DOI
ISSN
1615-9853eISSN
1615-9861Language
engPublication classification
C1 Refereed article in a scholarly journalCopyright notice
2009, WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimUsage metrics
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Science & TechnologyLife Sciences & BiomedicineBiochemical Research MethodsBiochemistry & Molecular BiologyAntibodyMALDIMSPhosphorylationProteomic methodsMETAL AFFINITY-CHROMATOGRAPHYNONSPECIFIC CYTOTOXIC-CELLSTANDEM MASS-SPECTROMETRYPHOSPHORIC-ACIDHUMAN GENOMEKINASERECEPTORPHOSPHOPEPTIDESENRICHMENTPEPTIDES
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