Simultaneous, Quantitative Characterization of Protein ADP-Ribosylation and Protein Phosphorylation in Macrophages.

The posttranslational modifications (PTMs) ADP-ribosylation and phosphorylation are important regulators of cellular pathways, and while mass spectrometry (MS)-based methods for the study of protein phosphorylation are well developed, protein ADP-ribosylation methodologies are still in a rapidly developing stage. The method described in this chapter uses immobilized metal affinity chromatography (IMAC), a phosphoenrichment matrix, to enrich ADP-ribosylated peptides which have been cleaved down to their phosphoribose attachment sites by a phosphodiesterase, thus isolating the ADP-ribosylated and phosphorylated proteomes simultaneously. To achieve the robust, relative quantification of PTM-level changes we have incorporated dimethyl labeling, a straightforward and economical choice which can be used on lysate from any cell type, including primary tissue. The entire pipeline has been optimized to work in ADP-ribosylation-compatible buffers and with protease-laden lysate from macrophage cells.