title: Optimization of LTQ-Orbitrap Mass Spectrometer Parameters for the Identification of ADP-Ribosylation Sites.
abstract: ADP-ribosylation of proteins alters their function or provides a scaffold for the recruitment of other proteins, thereby regulating several important cellular processes. Mono- or poly-ADP-ribosylation is catalyzed by different ADP-ribosyltransferases (ARTs) that have different subcellular localizations and modify different amino acid acceptor sites. However, our knowledge of ADP-ribosylated proteins and their acceptor amino acids is still limited due to the lack of suitable mass spectrometry (MS) tools. Here, we describe an MS approach for the detection of ADP-ribosylated peptides and identification of the ADP-ribose acceptor sites, combining higher-energy collisional dissociation (HCD) and electron-transfer dissociation (ETD) on an LTQ-Orbitrap mass spectrometer. The presence of diagnostic ions of ADP-ribose in the HCD spectra allowed us to detect putative ADP-ribosylated peptides to target in a second LC-MS/MS analysis. The combination of HCD with ETD fragmentation gave a more comprehensive coverage of ADP-ribosylation sites than that with HCD alone. We successfully identified different ADP-ribose acceptor sites on several in vitro modified proteins. The combination of optimized HCD and ETD methods may be applied to complex samples, allowing comprehensive identification of ADP-ribosylation acceptor sites.
authors.affiliation: Institute of Veterinary Biochemistry and Molecular Biology, ‡Functional Genomics Center Zurich, University of Zurich/ETH Zurich, §Life Science Zurich Graduate School, University of Zurich , CH-8057 Zurich, Switzerland.
authors.forename: Florian
authors.initials: F
authors.lastname: Rosenthal
authors.affiliation: Institute of Veterinary Biochemistry and Molecular Biology, ‡Functional Genomics Center Zurich, University of Zurich/ETH Zurich, §Life Science Zurich Graduate School, University of Zurich , CH-8057 Zurich, Switzerland.
authors.forename: Paolo
authors.initials: P
authors.lastname: Nanni
authors.affiliation: Institute of Veterinary Biochemistry and Molecular Biology, ‡Functional Genomics Center Zurich, University of Zurich/ETH Zurich, §Life Science Zurich Graduate School, University of Zurich , CH-8057 Zurich, Switzerland.
authors.forename: Simon
authors.initials: S
authors.lastname: Barkow-Oesterreicher
authors.affiliation: Institute of Veterinary Biochemistry and Molecular Biology, ‡Functional Genomics Center Zurich, University of Zurich/ETH Zurich, §Life Science Zurich Graduate School, University of Zurich , CH-8057 Zurich, Switzerland.
authors.forename: Michael O
authors.initials: MO
authors.lastname: Hottiger
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D013058:Mass Spectrometry / Q000379:methods*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011506:Proteins; D000246:Adenosine Diphosphate Ribose
keywords: ADP-ribosylation; ETD; HCD; LTQ-Orbitrap; PARP; electron-transfer dissociation; higher-energy collisional dissociation; histone; mass spectrometry