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.

Comprehensive ADP‐ribosylome analysis identifies tyrosine as an ADP‐ribose acceptor site

“…Moreover, our ability to confidently assign only S-ADPr, R-ADPr, and Y-ADPr modifications in vivo raises concerns as to why E-ADPr and D-ADPr were not robustly detected using the methodologies applied here; especially given that other studies provide strong evidence that these modifications are written in vivo and in vitro [8,9,12]. It could simply be that the peptide fragmentation (EThcD versus CID [32,33]) methods used here limit D-ADPr and E-ADPr modification identifications. T-ADPribosylation modifications were not identified, suggesting that T is not at all ADP-ribosylated by a mammalian ART or to an undetectable extent under the conditions tested.…”

“…It will also be important to explore whether other MS search engines and data analysis pipelines identify more ADPr-peptides and assign ADPr-sites with greater confidence [29][30][31]. It could simply be that the peptide fragmentation (EThcD versus CID [32,33]) methods used here limit D-ADPr and E-ADPr modification identifications. It could simply be that the peptide fragmentation (EThcD versus CID [32,33]) methods used here limit D-ADPr and E-ADPr modification identifications.…”

Intracellular Mono-ADP-Ribosylation in Signaling and Disease

“…It is important for the reader to realize these obstacles when discussing the functionality of intracellular MARylation. We refer to excellent recent reviews that discuss the detection and analysis of MARylation in detail [72][73][74].…”

Update: Post-translational protein modifications in plant metabolism

“…Whereas collision-induced dissociation is the most common peptide fragmentation technique used for protein identifications, electron-transfer dissociation and electron-capture dissociation are valuable complementary techniques to identify PTMs and/or improve the localization of PTMs (e.g. phosphorylation [Jünger and Aebersold, 2014] and ADP ribosylation [Haag and Buck, 2015]). Major improvements of modern mass spectrometers, in combination with novel enrichment strategies, have allowed for large-scale identification studies for numerous PTMs.…”