Quantification of cellular O-acetyl-ADP-ribose (OAADPr), a novel sirtuin metabolite, with HILIC LC-MS/MS

BIOL 151

Susan Lee, Lei Tong, and John M. Denu, jmdenu@wisc.edu. Department of Biomolecular Chemistry, University of Wisconsin-Madison, 551 MSC, 1300 University Ave, Madison, WI 53706
Sirtuins are NAD+-dependent deacetylases which have been implicated in chromatin silencing and life span extension. This deacetylation reaction leads to the formation of nicotinamide and the novel metabolite O-acetyl-ADP-ribose (OAADPr). Though OAADPr has been shown to have biological effects such as blocking oocyte maturation and in vitro binding to the yeast silencing complex, there is no information about cellular levels of OAADPr. We developed a new methodology to study cellular levels of OAADPr by comparing a YSA1 nudix hydrolase deletion strain to wild-type yeast using HILIC chromatography coupled with Multiple Reaction Monitoring (MRM) on a Q-trap mass spectrometer. We show that cellular levels of OAADPr can be quantitated and that deletion of the YSA1 hydrolase results in increased levels of both OAADPr and ADP-ribose. We plan to study how changes in cellular OAADPr and ADP-ribose correlate to biological phenotypes and hope to elucidate the functional role of these small molecules.