title: Mapping PARP-1 auto-ADP-ribosylation sites by liquid chromatography-tandem mass spectrometry.
abstract: We demonstrate a novel method for the identification of poly(ADP-ribose) polymerase-1 (PARP-1) autopoly(ADP-ribosyl)ation sites that is suited to collision induced dissociation (CID) tandem mass spectrometry. By employing phosphodiesterase to remove the majority of the poly(ADP-ribose) (pADPr) modification, we reduce the complexity of tandem mass spectrometric analysis of pADPr-modified tryptic peptides. The simplified ribose-5'-phosphate form of the peptides produce tandem mass spectra by CID that are readily interpreted and enable effective localization of the exact sites of PARP-1-catalyzed poly(ADP-ribosyl)ation. In conjunction with a phosphopeptide-like enrichment strategy that captures the ribose-5'-phosphate peptides, we identified eight novel sites of PARP-1 automodification, confirmed the localization of two sites previously reported, and provided evidence for two additional targeted peptides with ambiguous modification site assignments. Given the simplicity of the approach, the method is readily applicable to analysis of complex samples.
authors: Department of Medicinal Chemistry, University of Washington , Seattle, Washington, United States.
authors: John D
authors: JD
authors: Chapman
authors: Jean-Philippe
authors: JP
authors: Gagné
authors: Guy G
authors: GG
authors: Poirier
authors: David R
authors: DR
authors: Goodlett
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism*; D002853:Chromatography, Liquid; D006801:Humans; D010446:Peptide Fragments / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000378:metabolism; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D010446:Peptide Fragments; D000244:Adenosine Diphosphate; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D010727:Phosphoric Diester Hydrolases