Enhancement of phosphoprotein analysis using a fluorescent affinity tag and mass spectrometry

A fluorescent affinity tag (FAT) was synthesized and was utilized to selectively modify phosphorylated serine and threonine residues via beta‐elimination and Michael addition chemistries in a ‘one‐step’ reaction. This labeling technique was used for covalent modification of both phosphoproteins and phosphopeptides, allowing identification of these molecular species by fluorescence imaging after solution‐ or gel‐based separation methods. In addition to the strong fluorescence of the rhodamine tag, a commercially available antibody can be used to enrich low‐abundance post‐labeled phosphopeptides present in complex mixtures. Application of this methodology to phosphorylation‐site mapping has been evaluated for a phosphoprotein standard, bovine beta‐casein. Initial results demonstrated low femtomole detection limits after fluorescence image analysis of FAT‐labeled proteins or peptides.