title: Covalent perturbation as a tool for validation of identifications and PTM mapping applied to bovine alpha-crystallin.
abstract: Proteomic identifications hinge on the measurement of both parent and fragment masses and matching these to amino acid sequences via database search engines. The correctness of the identifications is assessed by statistical means. Here we present an experimental approach to test identifications. Chemical modification of all peptides in a sample leads to shifts in masses depending on the chemical properties of each peptide. The identification of a native peptide sequence and its perturbed version with a different parent mass and fragment ion masses provides valuable information. Labeling all peptides using reductive alkylation with formaldehyde is one such perturbation where the ensemble of peptides shifts mass depending on the number of reactive amine groups. Matching covalently perturbed fragmentation patterns from the same underlying peptide sequence increases confidence in the assignments and can salvage low scoring post-translationally modified peptides. Applying this strategy to bovine alpha-crystallin, we identify 9 lysine acetylation sites, 4 O-GlcNAc sites and 13 phosphorylation sites.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark.|Department of Clinical Biochemistry, Copenhagen University Hospital Hvidovre, Hvidovre, Denmark.
authors.forename: Jakob
authors.initials: J
authors.lastname: Bunkenborg
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark.
authors.forename: Lasse Gaarde
authors.initials: LG
authors.lastname: Falkenby
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark.
authors.forename: Lea Mørch
authors.initials: LM
authors.lastname: Harder
authors.affiliation: The Rockefeller University, New York, NY, USA.
authors.forename: Henrik
authors.initials: H
authors.lastname: Molina
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D006031:Glycosylation; D010455:Peptides / Q000032:analysis; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D053719:Tandem Mass Spectrometry; D038201:alpha-Crystallins / Q000032:analysis*
chemical_list: D010455:Peptides; D038201:alpha-Crystallins
keywords: Alpha-crystallin; O-glycosylation; PTM; Technology; Validation