A sensitive magnetic bead method for the detection and identification of tyrosine phosphorylation in proteins by MALDI-TOF/TOF MS

Mark R Condina, Mark Andrew Guthridge, Shaun R McColl, Peter Hoffmann

Research output: Contribution to journalArticleResearchpeer-review

18 Citations (Scopus)


Phosphorylation 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 ?-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. ? 2009 Wiley-VCH Verlag GmbH Co. KGaA
Original languageEnglish
Pages (from-to)3047 - 3057
Number of pages11
Issue number11
Publication statusPublished - 2009
Externally publishedYes

Cite this