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. 2018:1813:271-283.
doi: 10.1007/978-1-4939-8588-3_19.

Quantitative Determination of MAR Hydrolase Residue Specificity In Vitro by Tandem Mass Spectrometry

Robert Lyle McPherson  1 , Shao-En Ong  2 , Anthony K L Leung  3   4   5
Affiliations

Quantitative Determination of MAR Hydrolase Residue Specificity In Vitro by Tandem Mass Spectrometry

Robert Lyle McPherson et al. Methods Mol Biol. 2018.

Erratum in

Abstract

ADP-ribosylation is a posttranslational modification that involves the conjugation of monomers and polymers of the small molecule ADP-ribose onto amino acid side chains. A family of ADP-ribosyltransferases catalyzes the transfer of the ADP-ribose moiety of nicotinamide adenine dinucleotide (NAD+) onto a variety of amino acid side chains including aspartate, glutamate, lysine, arginine, cysteine, and serine. The monomeric form of the modification mono(ADP-ribosyl)ation (MARylation) is reversed by a number of enzymes including a family of MacroD-type macrodomain-containing mono(ADP-ribose) (MAR) hydrolases. Though it has been inferred from various chemical tests that these enzymes have specificity for MARylated aspartate and glutamate residues in vitro, the amino acid and site specificity of different family members are often not unambiguously defined. Here we describe a mass spectrometry-based assay to determine the site specificity of MAR hydrolases in vitro.

Keywords: ADP-ribosylation; ADP-ribosylhydrolase; Macrodomain; Mass spectrometry.

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Figures

Figure 1.
Figure 1.. Determining the site specificity of ADP-ribosylhydrolases.
Workflow describing the steps necessary for (1) identifying sites of MARylation on in vitro modified substrates by LC-MS/MS and (2) quantifying their intensities under different conditions (e.g., incubation with MAR hydrolases).
Figure 2.
Figure 2.. Generating and desalting MARylated PARP10CD.
SDS-PAGE analysis of PARP10CD incubated with or without 1 mM NAD+ for 2h at 37 oC analyzed by (A) total protein stain or (B) western blotting with anti-PAN-ADP-ribose. (C) Gel filtration chromatography of PARP10CD automodification reaction, blue trace is the absorbance of the sample at 280 nm over the course of the run.
Figure 3.
Figure 3.. Identification and quantification of phosphoribosylated sites on PARP10CD.
(A) MS/MS spectra of a PARP10CD peptide containing a high-confidence phosphoribose modification at D839. (B) Total ion chromatogram (TIC) of LC-MS/MS run where peptide from (A) was identified (C) Extracted ion chromatogram (XIC) of peptide from (A). (Data from ref. 15)
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