Journal
JOURNAL OF PROTEOME RESEARCH
Volume 18, Issue 4, Pages 1607-1622Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.jproteome.8b00895
Keywords
higher-energy collision dissociation (HCD); electron transfer higher-energy collision dissociation (EThcD); Orbitrap Fusion Lumos; gas phase segmentation; parallel reaction monitoring (PRM); proteomics; post-translational modification (PTM)
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Funding
- Kowa Company, Ltd., Nagoya, Japan
- National Institutes of Health [R01HL126901]
- University of Zurich
- Swiss National Science Foundation [31003A_176177]
- Swiss National Science Foundation (SNF) [31003A_176177] Funding Source: Swiss National Science Foundation (SNF)
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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-gamma-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 ADPribosylated 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-gamma 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-gamma and ADP-ribosylation signaling pathways.
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