title: Strategies for the identification of arginine ADP-ribosylation sites.
abstract: Mono-ADP-ribosylation of arginine is a protein modification in eukaryotic cells regulating protein activity and thereby influencing signal transduction and metabolism. Due to the complexity of the modification and the fragmentation pattern in MS/MS CID experiments, the identification of ADP-ribosylation sites in complex mixtures is difficult. Here we describe a two-step strategy, in the first step enriching and identifying potentially ADP-ribosylated proteins and in the second step identifying the sites of modification by a combination of LC/MS-, LC/MS(E) (MS at elevated fragmentation energy)- and LC/MS/MS experiments. Using this technique we could identify two ADP-ribosylation sites in TNFα digested with trypsin, protease V8 and both proteases and thereby demonstrate the specific ADP-ribosylation of TNFα. In complex samples the detection of ADP-ribosylated peptides requires further enrichment of the modified peptides. We tested various materials routinely used for the isolation of phosphopeptides. IMAC as well as TiO(2) chromatography were successfully applied for the selective enrichment of ADP-ribosylated model peptides.
authors.affiliation: Institute of Immunology, University Medical Center Hamburg-Eppendorf, D-20246 Hamburg, Germany.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Laing
authors.forename: Friedrich
authors.initials: F
authors.lastname: Koch-Nolte
authors.forename: Friedrich
authors.initials: F
authors.lastname: Haag
authors.forename: Friedrich
authors.initials: F
authors.lastname: Buck
mesh_terms: D036002:ADP Ribose Transferases / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014025:Titanium / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D023084:alpha-Defensins / Q000378:metabolism
chemical_list: D010748:Phosphopeptides; D014409:Tumor Necrosis Factor-alpha; D023084:alpha-Defensins; C406413:human neutrophil peptide 1; C009495:titanium dioxide; D000246:Adenosine Diphosphate Ribose; D001120:Arginine; D014025:Titanium; D036002:ADP Ribose Transferases; D014357:Trypsin