February 2020 Meeting

Speaker: Casey Daniels, NIH NIAID

Topic: The dynamic ADP-ribosylome, phosphoproteome, and interactome in LPS-activated macrophages

Date: Monday, February 10th, 2020

Time: 6:15 pm Dinner, 7:15 pm Presentation

Location: Shimadzu Scientific Instruments, Inc. Training Center 7100 Riverwood Drive, Columbia, MD 21046 (Directions)

Dinner: Please RSVP to Meghan Burke (meghan.burke@nist.gov) by Friday, February 7th if you will be attending the dinner.

Abstract: The innate immune response relies on efficient, robust, and controlled protein signaling networks to relay information related to pathogen or viral detection. This communication is mediated primarily through protein-protein interactions and post-translational modifications (PTMs), events which are best characterized by mass spectrometry (MS)-based proteomics. This in-depth study uses MS to identify changes in protein signaling networks of lipopolysaccharide (LPS)-stimulated human and mouse macrophages, at the level of single PTMs and protein complexes. Protein ADP-ribosylation is truncated down to its phosphoribose attachment structure, allowing for enrichment of the resulting phosphoribosylated peptides along with co-occurring phoshopeptides by immobilized metal affinity chromatography. Additionally, size exclusion chromatography-MS (SEC-MS) is used to separate protein complexes and proteoforms based on size; known and novel protein complexes are then identified by weighted correlation network analysis (WGCNA) – a machine learning algorithm for unsupervised clustering into modules – based on their correlated movement into or out of SEC fractions following LPS stimulation. It is only after unsupervised clustering has been performed that established databases are used to characterize the protein modules, which prove to be highly interactive based on known protein-protein interactions, and of similar biological processes, molecular functions, and/or cellular compartments based on gene ontology. Two modules of interest – one linked to the ASK complex, the other containing PARP9 as a hub protein – are studied further through immunoprecipitation and PTM analysis. Finally, PARP inhibition is used to perturb the characterized systems, demonstrating the importance of protein ADP-ribosylation for the global protein interactome. All PTM and interactome data has been aggregated into a meta-database of 6,729 proteins, with ADP-ribosylation characterized on 2,905 proteins, and phosphorylation characterized on 2,669 proteins; expanding the number of proteins with endogenous ADP-ribosylation sites characterized, identifying ADP-ribosylation in primary human cells for the first time, and surveying protein phosphorylation in human macrophages for the first time. This database serves as an invaluable resource for studying crosstalk between the ADP-ribosylome, phosphoproteome, and interactome.