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J Proteome Res. 2019 Apr 5;18(4):1607-1622. doi: 10.1021/acs.jproteome.8b00895. Epub 2019 Mar 21.

A Study into the ADP-Ribosylome of IFN-γ-Stimulated THP-1 Human Macrophage-like Cells Identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-Ribosylation.

Author information

1
Center for Interdisciplinary Cardiovascular Sciences, Division of Cardiovascular Medicine, Department of Medicine , Brigham Women's Hospital, Harvard Medical School , Boston , Massachusetts 02115 , United States.
2
Department of Molecular Mechanisms of Disease , University of Zurich , 8057 Zurich , Switzerland.
3
Center for Excellence in Vascular Biology, Cardiovascular Division , Brigham Women's Hospital, Harvard Medical School , Boston , Massachusetts 02115 , United States.
4
Channing Division of Network Medicine, Department of Medicine , Brigham Women's Hospital, Harvard Medical School , Boston , Massachusetts 02115 , United States.

Abstract

ADP-ribosylation is a post-translational modification that, until recently, has remained elusive to study at the cellular level. Previously dependent on radioactive tracers to identify ADP-ribosylation targets, several advances in mass spectrometric workflows now permit global identification of ADP-ribosylated substrates. In this study, we capitalized on two ADP-ribosylation enrichment strategies, and multiple activation methods performed on the Orbitrap Fusion Lumos, to identify IFN-γ-induced ADP-ribosylation substrates in macrophages. The ADP-ribosyl binding protein, Af1521, was used to enrich ADP-ribosylated peptides, and the antipoly-ADP-ribosyl antibody, 10H, was used to enrich ADP-ribosylated proteins. ADP-ribosyl-specific mass spectra were further enriched by an ADP-ribose product ion triggered EThcD and HCD activation strategy, in combination with multiple acquisitions that segmented the survey scan into smaller ranges. HCD and EThcD resulted in overlapping and unique ADP-ribosyl peptide identifications, with HCD providing more peptide identifications but EThcD providing more reliable ADP-ribosyl acceptor sites. Our acquisition strategies also resulted in the first ever characterization of ADP-ribosyl on three poly-ADP-ribose polymerases, ARTD9/PARP9, ARTD10/PARP10, and ARTD8/PARP14. IFN-γ increased the ADP-ribosylation status of ARTD9/PARP9, ARTD8/PARP14, and proteins involved in RNA processes. This study therefore summarizes specific molecular pathways at the intersection of IFN-γ and ADP-ribosylation signaling pathways.

KEYWORDS:

Orbitrap Fusion Lumos; electron transfer higher-energy collision dissociation (EThcD); gas phase segmentation; higher-energy collision dissociation (HCD); parallel reaction monitoring (PRM); post-translational modification (PTM); proteomics

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