Phosphopeptide/phosphoprotein mapping by electron capture dissociation mass spectrometry

Of methods for dissociation of multiply charged peptide and protein ions, e lectron capture dissociation (ECD) has the advantages of cleaving between a high proportion of amino acids, without loss of such posttranslational mod ifications as glycosylation and carboxylation, Here this capability is succ essfully extended to phosphorylation, for which collisionally activated dis sociation (CAD) can cause extensive loss of H3PO4 and HPO3, As shown here, these losses are minimal in ECD spectra, an advantage for measuring the deg ree of phosphorylation. For phosphorylated peptides, ECD and CAD spectra gi ve complementary backbone cleavages for identifying modification sites. For a 24-kDa heterogeneous phosphoprotein, bovine beta -casein, activated ion ECD cleaved 87 of 208 backbone bonds that identified a phosphorylation site at Ser-15, and localized three more among Ser-17,-18, -19, and -22 and Thr -24, and the last among four other sites: This is the first direct site-spe cific characterization of this key post-translational modification on a pro tein without its prior degradation, such as proteolysis.