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A study into the ADP-ribosylome of IFN-γ-stimulated THP-1 human macrophage-like cells identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-ribosylation


Higashi, Hideyuki; Maejima, Takashi; Lee, Lang Ho; Yamazaki, Yukiyoshi; Hottiger, Michael O; Singh, Sasha A; Aikawa, Masanori (2019). A study into the ADP-ribosylome of IFN-γ-stimulated THP-1 human macrophage-like cells identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-ribosylation. Journal of Proteome Research, 18(4):1607-1622.

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.

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.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:05 Vetsuisse Faculty > Department of Molecular Mechanisms of Disease
07 Faculty of Science > Department of Molecular Mechanisms of Disease
Dewey Decimal Classification:570 Life sciences; biology
Scopus Subject Areas:Life Sciences > Biochemistry
Physical Sciences > General Chemistry
Uncontrolled Keywords:Biochemistry, General Chemistry, 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
Language:English
Date:5 April 2019
Deposited On:22 Mar 2019 07:30
Last Modified:29 Jul 2020 10:30
Publisher:American Chemical Society (ACS)
ISSN:1535-3893
OA Status:Closed
Publisher DOI:https://doi.org/10.1021/acs.jproteome.8b00895
PubMed ID:30848916
Project Information:
  • : FunderSNSF
  • : Grant ID31003A_176177
  • : Project TitleUncovering the function of cell stress-associated ADP-ribosylomes

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