title: Phosphopeptide/phosphoprotein mapping by electron capture dissociation mass spectrometry.
abstract: Of methods for dissociation of multiply charged peptide and protein ions, electron capture dissociation (ECD) has the advantages of cleaving between a high proportion of amino acids, without loss of such posttranslational modifications as glycosylation and carboxylation. Here this capability is successfully extended to phosphorylation, for which collisionally activated dissociation (CAD) can cause extensive loss of H3PO4 and HPO3. As shown here, these losses are minimal in ECD spectra, an advantage for measuring the degree of phosphorylation. For phosphorylated peptides, ECD and CAD spectra give 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-specific characterization of this key post-translational modification on a protein without its prior degradation, such as proteolysis.
authors.affiliation: Department of Physical and Structural Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA.
authors.forename: S D
authors.initials: SD
authors.lastname: Shi
authors.forename: M E
authors.initials: ME
authors.lastname: Hemling
authors.forename: S A
authors.initials: SA
authors.lastname: Carr
authors.forename: D M
authors.initials: DM
authors.lastname: Horn
authors.forename: I
authors.initials: I
authors.lastname: Lindh
authors.forename: F W
authors.initials: FW
authors.lastname: McLafferty
mesh_terms: D000595:Amino Acid Sequence; D002364:Caseins / Q000737:chemistry; D013058:Mass Spectrometry; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry*; D010750:Phosphoproteins / Q000737:chemistry*
chemical_list: D002364:Caseins; D010748:Phosphopeptides; D010750:Phosphoproteins