Detection and Quantification of Sirtuin Metabolite O-acetyl-ADP-ribose (OAADPr) in Mouse Liver Extracts Using ~(13)C-OAADPr and LC-MS/MS

摘要

Sirtuins are NAD~(+)-dependent protein deacetylases that are involved in diverse cellular processes such as lifespan extension and metabolic regulation. Interestingly, sirtuin deacetylation generates a novel metabolite, O-acetyl-ADP-ribose (OAADPr). Though this metabolite has been shown to have a variety of biological effects, such as blocking oocyte maturation and binding to the histone macroH2A1.1, SIR complex, and cation channel TRPM2, its cellular levels have not been determined and its possible roles as a secondary messenger are still unclear. Here we report the first method to detect and quantify OAADPr in biological samples using an isotopic internal standard, ~(13)C-OAADPr, and LC-MS/MS. We show the utility of this method by quantifying OAADPr in control (SIRT3~(+)/~(+)) and sirtuin SIRT3 knockout mouse liver extracts.