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.

Middle-Down Proteomics Reveals Dense Sites of Methylation and Phosphorylation in Arginine-Rich RNA-Binding Proteins.

Post-translational modifications (PTMs) within arginine (Arg)-rich RNA-binding proteins, such as phosphorylation and methylation, regulate multiple steps in RNA metabolism. However, the identification of PTMs within Arg-rich domains with complete trypsin digestion is extremely challenging due to the high density of Arg residues within these proteins. Here, we report a middle-down proteomic approach coupled with electron-transfer dissociation (ETD) mass spectrometry to map previously unknown sites of phosphorylation and methylation within the Arg-rich domains of U1-70K and structurally similar RNA-binding proteins from nuclear extracts of human embryonic kidney (HEK)-293T cells.

RNA-binding proteins with basic-acidic dipeptide (BAD) domains self-assemble and aggregate in Alzheimer's disease.

The U1 small nuclear ribonucleoprotein 70 kDa (U1-70K) and other RNA-binding proteins (RBPs) are mislocalized to cytoplasmic neurofibrillary Tau aggregates in Alzheimer's disease (AD), yet the co-aggregation mechanisms are incompletely understood. U1-70K harbors two disordered low-complexity domains (LC1 and LC2) that are necessary for aggregation in AD brain extracts. The LC1 domain contains highly repetitive basic (Arg/Lys) and acidic (Asp/Glu) residues, referred to as a basic-acidic dipeptide (BAD) domain.