Specific visualization and identification of phosphoproteome in gels

The applicability of gel-based proteomic strategies in phosphoproteomics has been largely limited by the lack of technologies for specific detection of phosphoproteins in gels. Here for the first time we report a strategy for simultaneous visualization and identification of phosphoproteome in gels (VIPing) through coupling specific detection of phosphoproteins with protein identification and phosphorylation site mapping by tandem mass spectrometry. The core of the strategy is a novel compound multifunctionalized with a titanium ion(IV) for outstanding selectivity toward phosphorylated residues, a fluorophore for visualization, and a biotin group for phosphopeptide enrichment. The sensitivity and specificity of the VIPing strategy was demonstrated using standard protein mixtures and complex cell extracts, and the method was applied to study the phosphorylation changes of an essential tyrosine kinase Syk and interacting proteins upon B-cell stimulation. The novel technique provides a powerful platform for gel-based phosphoproteomic studies.