Rapid Analysis of ADP-Ribosylation Dynamics and Site-Specificity Using TLC-MALDI.
ACS Chem Biol
; 16(11): 2137-2143, 2021 11 19.
Article
em En
| MEDLINE
| ID: mdl-34647721
ABSTRACT
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)âdespite 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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Cromatografia em Camada Fina
/
Poli(ADP-Ribose) Polimerases
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Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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ADP-Ribosilação
Idioma:
En
Revista:
ACS Chem Biol
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Estados Unidos