Identification of an in vitro insulin receptor substrate-1 phosphorylation site by negative-ion muLC/ES-API-CID-MS hybrid scan technique.

Recently, we reported a fast on-line alkaline micro-liquid chromatography/electrospray-atmospheric pressure ionization/collision-induced dissociation/mass spectrometric approach for sensitive phosphopeptide screening of a tryptic digested protein and subsequent characterization of the identified phosphopeptide. Based on this study, we now applied an improved method for the identification of phosphorylation sites in insulin receptor substrate 1, an important mediator in insulin signal transduction which was phosphorylated in vitro by protein kinase C-zeta. The approach consists of an on-line alkaline negative-ion micro-liquid chromatography/electrospray-atmospheric pressure ionization/collision-induced dissociation/mass spectrometric hybrid scan experiment using a triple-quadrupole mass spectrometer with fractionation and subsequent off-line nanoES-MS (ion trap) analysis of the phosphopeptide-containing fractions. During the liquid chromatography (LC)/ES-MS experiment, the phosphopeptides of the enzymatic digest mixture of the studied insulin receptor substrate 1 fragment were detected under high skimmer potential (API-CID) using phosphorylation-specific m/z 79 marker ions as well as the intact m/z-values of the peptides which were recorded under low skimmer potential. Subsequently, the targeted fractions were analyzed by off-line nanoES-MS/MS and MS(3). Using this approach, serine 318 was clearly identified as a major in vitro protein kinase C-zeta phosphorylation site in the insulin receptor substrate -1 fragment. Together, our results indicate that the applied strategy is useful for unequivocal and fast analysis of phosphorylation sites in low abundant signaling proteins.