title: Identification and analysis of ADP-ribosylated proteins.
abstract: The analysis of ADP-ribosylated proteins is a challenging task, on the one hand because of the diversity of the target proteins and the modification sites, on the other hand because of the particular problems posed by the analysis of ADP-ribosylated peptides. ADP-ribosylated proteins can be detected in in vitro experiments after the incorporation of radioactively labeled or chemically modified ADP-ribose. Endogenously ADP-ribosylated proteins may be detected and enriched by antibodies directed against the ADP-ribosyl moiety or by ADP-ribosyl binding macro domains. The determination of the exact attachment site of the modification, which is a prerequisite for the understanding of the specificity of the various ADP-ribosyl transferases and the structural consequences of ADP-ribosylation, necessitates the proteolytic cleavage of the proteins. The resulting peptides can afterwards be enriched either by IMAC (using the affinity of the pyrophosphate group for heavy metal ions) or by immobilized boronic acid beads (using the affinity of the vicinal ribose hydroxy groups for boronic acid). The identification of the modified peptides usually requires tandem mass spectrometric measurements. Problems that hamper the mass spectrometric analysis by collision-induced decay (CID) can be circumvented either by the application of different fragmentation techniques (electron transfer or electron capture dissociation; ETD or ECD) or by enzymatic cleavage of the ADP-ribosyl group to ribosyl-phosphate.
authors.affiliation: Institute of Immunology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246, Hamburg, Germany.
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 / Q000378:metabolism*; D000818:Animals; D006801:Humans; D013058:Mass Spectrometry; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D010455:Peptides; D011506:Proteins; D000246:Adenosine Diphosphate Ribose; D036002:ADP Ribose Transferases