Using TLC-MALDI-TOF to Interrogate In Vitro Peptidyl Proximal Preferences of PARP14 and Glycohydrolase Specificity

The transfer of ADP–ribose (ADPr) from nicotinamide adenine dinucleotide (NAD+) to target proteins is mediated by a class human diphtheria toxin-like ADP-ribosyltransferases (ARTDs; previously referred as poly-ADP–ribose polymerases or PARPs) and the removal ADPr catalyzed family glycohydrolases. Although thousands potential modification sites have been identified using high-throughput mass-spectrometry, relatively little known about sequence specificity encoded near site. Herein, we present matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) method that facilitates in vitro analysis proximal factors guide ARTD selection. We identify minimal 5-mer peptide necessary sufficient drive glutamate/aspartate targeting PARP14 while highlighting importance adjacent residues targeting. measure stability resultant ester bond show non-enzymatic pH temperature dependent, independent, occurs within hours. Finally, use ADPr–peptides highlight differential activities glycohydrolase their preferences. Our results (1) utility MALDI-TOF analyzing ARTD–substrate interactions (2) sequences governing removal.