title: Strategy for the identification of sites of phosphorylation in proteins: neutral loss triggered electron capture dissociation.
abstract: We have previously demonstrated the suitability of data-dependent electron capture dissociation (ECD) for incorporation into proteomic strategies. The ability to directly determine sites of phosphorylation is a major advantage of electron capture dissociation; however, the low stoichiometry associated with phosphorylation means that phosphopeptides are often overlooked in data-dependent ECD analyses. In contrast, collision-induced dissociation (CID) tends to result in loss of the labile phosphate group, often at the expense of sequence fragments. Here, we demonstrate a novel strategy for the characterization of phosphoproteins which exploits the neutral loss feature of CID such that focused ECD of phosphopeptides is achieved. Peptides eluting from a liquid chromatograph are first subjected to CID, and if a neutral loss of 98 Da (corresponding to H3PO4) from the precursor is observed, ECD of that same precursor is performed; i.e., the method comprises neutral loss triggered ECD (NL-ECD-MS/MS). The method was applied to tryptic digests of beta-casein and alpha-casein. For alpha-casein, four sites of phosphorylation were identified with NL-ECD-MS/MS compared with a single site identified by ECD-MS/MS. The method also resulted in ECD of a doubly phosphorylated peptide. A further benefit of the method is that overall protein sequence coverage is improved. Sequence information from nonphosphorylated peptides is obtained as a result of the CID step.
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
authors.forename: Steve M M
authors.initials: SM
authors.lastname: Sweet
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Creese
authors.forename: Helen J
authors.initials: HJ
authors.lastname: Cooper
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D004583:Electrons; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared; D053719:Tandem Mass Spectrometry
chemical_list: D011506:Proteins