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

  • Robert Lyle McPherson
  • Shao-En Ong
  • Anthony K. L. LeungEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1813)


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.

Key words

ADP-ribosylation Mass spectrometry Macrodomain ADP-ribosylhydrolase 



This work was supported by a Johns Hopkins Catalyst Award (AKLL) and research grants from the Johns Hopkins University School of Medicine Sherrilyn and Ken Fisher Center for Environmental Infectious Disease (AKLL). The proteomics work was also in part supported by R01GM104135S1 (AKLL and RLM), S10OD021502 (SEO), and T32CA009110 (RLM) from the U.S. National Institutes of Health, and the American Cancer Society Research Scholar Award 129539-RSG-16-062-01-RMC (AKLL).


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Robert Lyle McPherson
    • 1
  • Shao-En Ong
    • 2
  • Anthony K. L. Leung
    • 1
    • 3
    • 4
    Email author
  1. 1.Department of Biochemistry and Molecular Biology, Bloomberg School of Public HealthJohns Hopkins UniversityBaltimoreUSA
  2. 2.Department of PharmacologyUniversity of WashingtonSeattleUSA
  3. 3.Department of Molecular Biology and GeneticsJohns Hopkins UniversityBaltimoreUSA
  4. 4.Department of Oncology, School of MedicineJohns Hopkins UniversityBaltimoreUSA

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