Dynamic ADP-Ribosylome, Phosphoproteome, and Interactome in LPS-Activated Macrophages

We
have used mass spectrometry (MS) to characterize protein signaling
in lipopolysaccharide (LPS)-stimulated macrophages from human blood,
human THP1 cells, mouse bone marrow, and mouse Raw264.7 cells. Protein
ADP-ribosylation was truncated down to phosphoribose, allowing for
enrichment and identification of the resulting phosphoribosylated
peptides alongside phosphopeptides. Size exclusion chromatography-MS
(SEC-MS) was used to separate proteoforms by size; protein complexes
were then identified by weighted correlation network analysis (WGCNA)
based on their correlated movement into or out of SEC fractions following
stimulation, presenting an analysis method for SEC-MS that does not
rely on established databases. We highlight two modules of interest:
one linked to the apoptosis signal-regulating kinase (ASK) signalosome
and the other containing poly­(ADP-ribose) polymerase 9 ­(PARP9). Finally, PARP inhibition was used
to perturb the characterized systems, demonstrating the importance
of ADP-ribosylation for the global interactome. All post-translational
modification (PTM) and interactome data have been aggregated into
a meta-database of 6729 proteins, with ADP-ribosylation characterized
on 2905 proteins and phosphorylation characterized on 2669 proteins.
This databasetitled MAPCD, for Macrophage ADP-ribosylation, Phosphorylation,
and Complex Dynamicsserves
as an invaluable resource for studying crosstalk between the ADP-ribosylome,
phosphoproteome, and interactome.