title: Studying the fragmentation behavior of peptides with arginine phosphorylation and its influence on phospho-site localization.
abstract: Phospho-proteomic studies opened a broad view onto the main mechanisms of regulating cellular processes. Our recent discovery of a protein arginine kinase and its target in bacteria added a previously undescribed type of phosphorylation to control protein activity. Several challenges arise from large in vivo studies of this and other types of phosphorylations. The main factors impeding correct localization are low spectral quality, neutral loss of phosphoric acid, and gas-phase rearrangements, which have recently been described for phospho-serine, -threonine, and -tyrosine. Studies on histidine-phosphorylated peptides, a nitrogen-bound phosphorylation, also reported loss of phosphoric acid upon collision-induced dissociation. We were interested in studying the behaviour of arginine phosphorylation under different fragmentation conditions and its influence on site localization. First, we determined the percentage of false localizations obtained by three different search engines and a software tool dedicated for phospho-site determination. Next, we demonstrate that application of collisional activation for analysis of arginine-phosphorylated peptides leads to extensive elimination of phosphoric acid and increases the numbers of false localizations, while the modification is maintained on the arginine side chain upon electron-transfer dissociation. Furthermore, we also observed a rearrangement of the phosphorylation onto serine and glutamic acid side chains upon collisional activation.
authors.affiliation: Christian-Doppler-Laboratory for Proteome Analysis, Department of Biochemistry and Microbiology, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.
authors.forename: Andreas
authors.initials: A
authors.lastname: Schmidt
authors.forename: Gustav
authors.initials: G
authors.lastname: Ammerer
authors.forename: Karl
authors.initials: K
authors.lastname: Mechtler
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry* / Q000378:metabolism; D016477:Artifacts; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002918:Chymotrypsin / Q000737:chemistry; D006801:Humans; D008666:Metalloendopeptidases / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D010446:Peptide Fragments; D010748:Phosphopeptides; D001120:Arginine; D002918:Chymotrypsin; D014357:Trypsin; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase