title: Pinpointing phosphorylation sites: Quantitative filtering and a novel site-specific x-ion fragment.
abstract: Phosphoproteomics deals with the identification and quantification of thousands of phosphopeptides. Localizing the phosphorylation site is however much more difficult than establishing the identity of a phosphorylated peptide. Further, recent findings have raised doubts of the validity of the site assignments in large-scale phosphoproteomics data sets. To improve methods for site localization, we made use of a synthetic phosphopeptide library and SILAC-labeled peptides from whole cell lysates and analyzed these with high-resolution tandem mass spectrometry on an LTQ Orbitrap Velos. We validated gas-phase phosphate rearrangement reactions during collision-induced dissociation (CID) and used these spectra to devise a quantitative filter that by comparing signal intensities of putative phosphorylated fragment ions with their nonphosphorylated counterparts allowed us to accurately pinpoint which fragment ions contain a phosphorylated residue and which ones do not. We also evaluated higher-energy collisional dissociation (HCD) and found this to be an accurate method for correct phosphorylation site localization with no gas-phase rearrangements observed above noise level. Analyzing a large set of HCD spectra of SILAC-labeled phosphopeptides, we identified a novel fragmentation mechanism that generates a phosphorylation site-specific neutral loss derived x-ion, which directly pinpoints the phosphorylated residue. Together, these findings significantly improve phosphorylation site localization confidence.
authors.affiliation: Department for Proteomics, NNF Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3b, DK-2200 Copenhagen, Denmark.
authors.forename: Christian D
authors.initials: CD
authors.lastname: Kelstrup
authors.forename: Omid
authors.initials: O
authors.lastname: Hekmat
authors.forename: Chiara
authors.initials: C
authors.lastname: Francavilla
authors.forename: Jesper V
authors.initials: JV
authors.lastname: Olsen
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D002457:Cell Extracts / Q000032:analysis* / Q000737:chemistry; D004735:Energy Transfer; D005740:Gases / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D007477:Ions / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D010748:Phosphopeptides / Q000032:analysis / Q000737:chemistry; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D012984:Software; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
chemical_list: D002457:Cell Extracts; D005740:Gases; D007477:Ions; D010446:Peptide Fragments; D010710:Phosphates; D010748:Phosphopeptides; D014357:Trypsin