title: Targeted analysis of tyrosine phosphorylation by immuno-affinity enrichment of tyrosine phosphorylated peptides prior to mass spectrometric analysis.
abstract: Tyrosine phosphorylation is a key process that regulates seminal biological functions, hence, deregulation of this mechanism is an underlying cause of several diseases including cancer and immunological disorders. Due to its low abundance, tyrosine phosphorylation is typically under-represented in most of the global MS-based phosphoproteomic studies. Here, we describe a selective approach based on immuno-affinity purification using specific antibodies to enrich tyrosine phosphorylated peptides from a complex proteolytic digest. LC-MS/MS analysis is subsequently used for peptide identification allowing the exact localization of the phosphorylated residue within the sequence. Using this approach more than 1000 non-redundant phosphotyrosine peptides can be identified in less than 6h of MS analysis, reflecting the high sensitivity and specificity of the technique. The identified tyrosine phosphorylated peptides can be used to study different biological aspects of tyrosine signaling and disease.
authors.affiliation: Biomolecular Mass and Spectrometry and Proteomics Group, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors.forename: Adja D
authors.initials: AD
authors.lastname: Zoumaro-Djayoon
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
authors.forename: Javier
authors.initials: J
authors.lastname: Muñoz
mesh_terms: D000915:Antibody Affinity; D002853:Chromatography, Liquid; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D046912:Multiprotein Complexes / Q000737:chemistry; D010455:Peptides / Q000032:analysis / Q000737:chemistry*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000737:chemistry; D011505:Protein-Tyrosine Kinases / Q000737:chemistry; D059748:Proteolysis; D012680:Sensitivity and Specificity; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D046912:Multiprotein Complexes; D010455:Peptides; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases