Determining PARP14 ADP‐Ribosylation Dynamics and Site‐Specificity Using MALDITOF

Poly (ADP‐ribose) polymerases, PARPs, are a family of 17 enzymes (in humans) that catalyze the transfer of ADP‐ribose onto target proteins from nicotinamide adenine dinucleotide (NAD + ). ADP‐ribosylation occurs in a highly conserved PARP catalytic domain found in all seventeen PARPs. Significant effort using modern proteomic methods has led to the identification of thousands of potential PARP targets in the cell. Unfortunately, this analysis has not yet uncovered which sites are preferentially targeted in vivo nor the determinants for PARP family‐member specific targeting. Further, current analytical methods require proteolysis of the target protein, limiting the study of dynamic ADP‐ribosylation. Herein, we present a matrix‐assisted laser desorption/ionization time of flight (MALDI‐ TOF) method using thin‐layer chromatography (TLC) that facilitates population‐wide analysis of ADP‐ribosylation. Using this method, we observe divergent ADP‐ribosylation dynamics for PARPs 14 and 15, with PARP15 modifying more sites on itself (+4–5 ADP‐ribose) than the closely related PARP14 protein (+1–2 ADP‐ribose) ‐ despite similar numbers of potential modification sites. Using this method, we also identify, for the first time, a minimal peptide fragment (18 amino‐acids) that is preferentially modified by PARP14. We have identified the specific glutamate (E) residue that is targeted on this peptide, and we demonstrate through mutagenesis and chemical treatment with hydroxylamine that PARP14 prefers acidic residues. The development of this technique will not only be useful in identifying the basic biochemistry governing ADP‐ribosylation but should find utility in the identification of PARP family‐member specific peptide targets and inhibitors.