A review of tandem mass spectrometry characterization of adenosine diphosphate-ribosylated peptides

Hengel, Shawna M.; Goodlett, David R.

doi:10.1016/j.ijms.2011.06.003

Cited by 35 publications

(55 citation statements)

References 27 publications

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“…Amino acid assignment of ADP-ribosylation sites by MS is challenging due to chemical lability of the protein-ribose linkage and inadvertent fragmentation of ADPr (Hengel and Goodlett, 2012). Fortuitously, existing Ub antibodies (Abs) were useful for deducing the site of ADPr modification.…”

Section: Resultsmentioning

confidence: 99%

Ubiquitin Modification by the E3 Ligase/ADP-Ribosyltransferase Dtx3L/Parp9

et al. 2017

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SUMMARY ADP-ribosylation of proteins is emerging as an important regulatory mechanism. Depending on the family member, ADP-ribosyltransferases conjugate either a single ADP-ribose to a target, or generate ADP-ribose chains. Here we characterize Parp9, a mono-ADP-ribosyltransferase reported to be enzymatically inactive. Parp9 undergoes heterodimerization with Dtx3L, a histone E3 ligase involved in DNA damage repair. We show that the Dtx3L/Parp9 heterodimer mediates NAD+- dependent mono-ADP-ribosylation of ubiquitin, exclusively in the context of ubiquitin processing by E1 and E2 enzymes. Dtx3L/Parp9 ADP-ribosylates the carboxyl group of Ub Gly76. Because Gly76 is normally used for Ub conjugation to substrates, ADP-ribosylation of the Ub carboxyl terminus precludes ubiquitylation. Parp9 ADP-ribosylation activity therefore restrains the E3 function of Dtx3L. Mutation of the NAD+ binding site in Parp9 increases the DNA repair activity of the heterodimer. Moreover, poly-ADP-ribose binding to the Parp9 macrodomains increases E3 activity. Dtx3L heterodimerization with Parp9 enables NAD+ and poly- ADP-ribose regulation of E3 activity.

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Section: Resultsmentioning

confidence: 99%

Ubiquitin Modification by the E3 Ligase/ADP-Ribosyltransferase Dtx3L/Parp9

et al. 2017

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“…At the moment, this is difficult to study because no direct read-outs for ARTD10 activity in cells exist. Furthermore, mono-ADP-ribosylation cannot be measured as easily by mass spectrometry as for example phosphorylation [46,47], as also illustrated by the fact that publications that describe automodification sites in ARTD1 contradict each other [48,49]. To be able to fully understand the role of mono-ADP-ribosylation in cell physiology, reliable methods have to be developed to study this modification.…”

Section: Discussionmentioning

confidence: 99%

ARTD10 substrate identification on protein microarrays: regulation of GSK3β by mono-ADP-ribosylation

et al. 2013

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BackgroundAlthough ADP-ribosylation has been described five decades ago, only recently a distinction has been made between eukaryotic intracellular poly- and mono-ADP-ribosylating enzymes. Poly-ADP-ribosylation by ARTD1 (formerly PARP1) is best known for its role in DNA damage repair. Other polymer forming enzymes are ARTD2 (formerly PARP2), ARTD3 (formerly PARP3) and ARTD5/6 (formerly Tankyrase 1/2), the latter being involved in Wnt signaling and regulation of 3BP2. Thus several different functions of poly-ADP-ribosylation have been well described whereas intracellular mono-ADP-ribosylation is currently largely undefined. It is for example not known which proteins function as substrate for the different mono-ARTDs. This is partially due to lack of suitable reagents to study mono-ADP-ribosylation, which limits the current understanding of this post-translational modification.ResultsWe have optimized a novel screening method employing protein microarrays, ProtoArrays®, applied here for the identification of substrates of ARTD10 (formerly PARP10) and ARTD8 (formerly PARP14). The results of this substrate screen were validated using in vitro ADP-ribosylation assays with recombinant proteins. Further analysis of the novel ARTD10 substrate GSK3β revealed mono-ADP-ribosylation as a regulatory mechanism of kinase activity by non-competitive inhibition in vitro. Additionally, manipulation of the ARTD10 levels in cells accordingly influenced GSK3β activity. Together these data provide the first evidence for a role of endogenous mono-ADP-ribosylation in intracellular signaling.ConclusionsOur findings indicate that substrates of ADP-ribosyltransferases can be identified using protein microarrays. The discovered substrates of ARTD10 and ARTD8 provide the first sets of proteins that are modified by mono-ADP-ribosyltransferases in vitro. By studying one of the ARTD10 substrates more closely, the kinase GSK3β, we identified mono-ADP-ribosylation as a negative regulator of kinase activity.

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“…For this reason, these fragmentation techniques have been considered to be the best choice for the identification of ADP-ribosylation sites 17,21 .…”

Section: Introductionmentioning

confidence: 99%

“…In a first LC-MS/MS run, putative ADP-ribosylated peptides are detected (based on the presence of marker ions in the CID spectra), which are then targeted for ETD fragmentation and subsequent peptide identification in a second LC-MS/MS analysis 17,21 .…”

Section: Introductionmentioning

confidence: 99%

Optimization of LTQ-Orbitrap Mass Spectrometer Parameters for the Identification of ADP-Ribosylation Sites

et al. 2015

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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.

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A review of tandem mass spectrometry characterization of adenosine diphosphate-ribosylated peptides

Cited by 35 publications

References 27 publications

Ubiquitin Modification by the E3 Ligase/ADP-Ribosyltransferase Dtx3L/Parp9

Ubiquitin Modification by the E3 Ligase/ADP-Ribosyltransferase Dtx3L/Parp9

ARTD10 substrate identification on protein microarrays: regulation of GSK3β by mono-ADP-ribosylation

Optimization of LTQ-Orbitrap Mass Spectrometer Parameters for the Identification of ADP-Ribosylation Sites

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