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Rapid Analysis of ADP-Ribosylation Dynamics and Site-Specificity Using TLC-MALDI.

Sean R WallaceLeila Y ChihabMiles YamasakiBraden T YoshinagaYazmin M TorresDamon RideauxZeeshan JavedSoumya TurumellaMichelle ZhangDylan R LawtonAmelia A FullerIan Carter-O'Connell
Published in: ACS chemical biology (2021)
Poly(ADP-ribose) polymerases, PARPs, transfer ADP-ribose onto target proteins from nicotinamide adenine dinucleotide (NAD+). Current mass spectrometric analytical methods require proteolysis of target proteins, limiting the study of dynamic ADP-ribosylation on contiguous proteins. Herein, we present a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) method that facilitates multisite analysis of ADP-ribosylation. We observe divergent ADP-ribosylation dynamics for the catalytic domains of PARPs 14 and 15, with PARP15 modifying more sites on itself (+3-4 ADP-ribose) than the closely related PARP14 protein (+1-2 ADP-ribose)─despite similar numbers of potential modification sites. We identify, for the first time, a minimal peptide fragment (18 amino-acids) that is preferentially modified by PARP14. Finally, we demonstrate through mutagenesis and chemical treatment with hydroxylamine that PARPs 14/15 prefer acidic residues. Our results highlight the utility of MALDI-TOF in the analysis of PARP target modifications and in elucidating the biochemical mechanism governing PARP target selection.
Keyphrases
  • mass spectrometry
  • dna damage
  • dna repair
  • amino acid
  • ms ms
  • liquid chromatography
  • crispr cas
  • oxidative stress
  • ionic liquid