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

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.