Targeted analysis of tyrosine phosphorylation by immuno-affinity enrichment of tyrosine phosphorylated peptides prior to mass spectrometric analysis

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 6 h 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.