title: Combining Higher-Energy Collision Dissociation and Electron-Transfer/Higher-Energy Collision Dissociation Fragmentation in a Product-Dependent Manner Confidently Assigns Proteomewide ADP-Ribose Acceptor Sites.
abstract: Protein adenosine diphosphate (ADP)-ribosylation is a physiologically and pathologically important post-translational modification. Recent technological advances have improved analysis of this complex modification and have led to the discovery of hundreds of ADP-ribosylated proteins in both cultured cells and mouse tissues. Nevertheless, accurate assignment of the ADP-ribose acceptor site(s) within the modified proteins identified has remained a challenging task. This is mainly due to poor fragmentation of modified peptides. Here, using an Orbitrap Fusion Tribrid mass spectrometer, we present an optimized methodology that not only drastically improves the overall localization scores for ADP-ribosylation acceptor sites but also boosts ADP-ribosylated peptide identifications. First, we systematically compared the efficacy of higher-energy collision dissociation (HCD), electron-transfer dissociation with supplemental collisional activation (ETcaD), and electron-transfer/higher-energy collision dissociation (EThcD) fragmentation methods when determining ADP-ribose acceptor sites within complex cellular samples. We then tested the combination of HCD and EThcD fragmentation, which were employed in a product-dependent manner, and the unique fragmentation properties of the ADP-ribose moiety were used to trigger targeted fragmentation of only the modified peptides. The best results were obtained with a workflow that included initial fast, high-energy HCD (Orbitrap, FT) scans, which produced intense ADP-ribose fragmentation ions. These potentially ADP-ribosylated precursors were then selected and analyzed via subsequent high-resolution HCD and EThcD fragmentation. Using these resulting high-quality spectra, we identified a xxxxxxKSxxxxx modification motif where lysine can serve as an ADP-ribose acceptor site. Due to the appearance of serine within this motif and its close presence to the lysine, further analysis revealed that serine serves as a new ADP-ribose acceptor site across the proteome.
authors.affiliation: Department of Molecular Mechanisms of Disease, University of Zurich , 8057 Zurich, Switzerland.|Ph.D. Program in Molecular Life Sciences, University of Zurich/ETH Zurich , 8057 Zurich, Switzerland.
authors.forename: Vera
authors.initials: V
authors.lastname: Bilan
authors.affiliation: Department of Molecular Mechanisms of Disease, University of Zurich , 8057 Zurich, Switzerland.|Ph.D. Program in Molecular Life Sciences, University of Zurich/ETH Zurich , 8057 Zurich, Switzerland.
authors.forename: Mario
authors.initials: M
authors.lastname: Leutert
authors.affiliation: Functional Genomics Center Zurich, University of Zurich/ETH Zurich , 8057 Zurich, Switzerland.
authors.forename: Paolo
authors.identifier: 0000-0001-8429-3557
authors.initials: P
authors.lastname: Nanni
authors.affiliation: Functional Genomics Center Zurich, University of Zurich/ETH Zurich , 8057 Zurich, Switzerland.
authors.forename: Christian
authors.initials: C
authors.lastname: Panse
authors.affiliation: Department of Molecular Mechanisms of Disease, University of Zurich , 8057 Zurich, Switzerland.
authors.forename: Michael O
authors.identifier: 0000-0002-7323-2270
authors.initials: MO
authors.lastname: Hottiger
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004579:Electron Transport; D006367:HeLa Cells; D006801:Humans; D010455:Peptides / Q000032:analysis*; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D010455:Peptides; D011506:Proteins; D000246:Adenosine Diphosphate Ribose