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

• DOI: 10.1021/acschembio.1c00542
• Publication Date: 2021-10-15T22:41:30Z
• Authors: Sean R. Wallace; Leila Y. Chihab; Miles Yamasaki; Braden T. Yoshinaga; Yazmin M. Torres; Damon Rideaux; Zeeshan Javed; Soumya Turumella; Michelle Zhang; Dylan R. Lawton; Amelia A. Fuller; Ian Carter-O’Connell
• Keywords: 0301 basic medicine; 18 amino; Chemical Sciences not elsewhere classified; parp15 modifying; results highlight; chemical treatment; Immunology; Biophysics; potential modification sites; Biochemistry; Inorganic Chemistry; dynamic adp; 03 medical and health sciences; ADP-Ribosylation; Protein Domains; 616; Genetics; target proteins; ionization time; nicotinamide adenine dinucleotide; catalytic domains; rapid analysis; first time; specificity using tlc; Cell Biology; contiguous proteins; observe divergent adp; parp target modifications; Astronomical and Space Sciences not elsewhere classified; Infectious Diseases; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; assisted laser desorption; facilitates multisite analysis; transfer adp; minimal peptide fragment; Chromatography, Thin Layer; Poly(ADP-ribose) Polymerases; Biotechnology; Biological Sciences not elsewhere classified; preferentially modified

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.