Identification by electrospray ionization mass spectrometry of the sites of tyrosine phosphorylation induced in activated Jurkat T cells on the protein tyrosine kinase ZAP-70.

摘要: We have developed a rapid and sensitive two capillary-column chromatography mass spectrometry-based method for the determination of protein phosphorylation sites following recovery individual phosphopeptides from two-dimensional phosphopeptide maps. With standard phosphopeptide, we demonstrate detection sensitivity at least 250 fmol this system. applied technique to analysis in vitro tyrosine induced on T cell-specific kinase ZAP-70 absence presence p56lck. show that has primary autophosphorylation site Tyr-292, with secondary Tyr-126. also additional Tyr-69, Tyr-178, Tyr-492, Tyr-493 upon addition kinase, By comparative mapping, isolated Jurkat cells autophosphorylates Tyr-292 Similar 32P-labeled stimulated anti-T cell receptor antibodies reveals Tyr-492 as principal antigen receptor-induced phosphorylation, but not Tyr-126 site. The high degree achieved technology should greatly facilitate direct biochemical inducible events, an experimental strategy until now been both time consuming difficult.