HIGH-SENSITIVITY DETERMINATION OF TYROSINE-PHOSPHORYLATED PEPTIDES BYONLINE ENZYME REACTOR AND ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY

We describe a simple, fast, sensitive, and nonisotopic bioanalytical t echnique for the detection of tyrosine-phosphorylated peptides and the determination of sites of protein tyrosine phosphorylation. The techn ique employs a protein tyrosine phosphatase micro enzyme reactor coupl ed on-line to either capillary electrophoresis or liquid chromatograph y and electrospray ionization mass spectrometry instruments. The micro enzyme reactor was constructed by immobilizing genetically engineered , metabolically biotinylated human protein tyrosine phosphatase beta o nto the inner surface of a small piece of a 50-mu m inner diameter, 36 0-mu m outer diameter fused silica capillary or by immobilization of t he phosphatase onto 40-90-mu m avidin-activated resins. By coupling th ese reactors directly to either a capillary electrophoresis column or a liquid chromatography column, we were able to rapidly perform enzyma tic dephosphorylation and separation of the reaction products. Detecti on and identification of the components of the reaction mixture exitin g these reactors were done by mass analysis with an on-line electrospr ay ionization mass spectrometer. Tyrosine-phosphorylated peptides, eve n if present in a complex peptide mixture, were identified by subtract ive analysis of peptide patterns generated with or without phosphatase treatment. Two criteria, namely a phosphatase-induced change in hydro pathy and charge, respectively, and a change in molecular mass by 80 D a, were used jointly to identify phosphopeptides. We demonstrate that, with this technique, low picomole amounts of a tyrosine-phosphorylate d peptide can be detected in a complex peptide mixture generated by pr oteolysis of a protein and that even higher sensitivities can be reali zed if more sensitive detection systems are applied.