ABSTRACT
The sirtuin enzymes, a class of NAD(+)-dependent histone deacetylases, are a focal point of epigenetic research because of their roles in regulating gene expression and cellular differentiation by deacetylating histones and a host of transcription factors, including p53. Here, the authors present two label-free screening methodologies to study sirtuin activity using high-throughput mass spectrometry. The first method involves the detection of native peptides and provides a platform for more detailed mechanistic studies by enabling the concurrent and direct measurement of multiple modification states. The second method obviates the need for substrate-specific assay development by measuring the O-acetyl-ADP-ribose co-product formed by sirtuin-dependent deacetylation. Both methodologies were applied to investigating the deacetylation of multiple-peptide substrates by multiple-sirtuin enzymes. Kinetic data, including binding constants, inhibition, and, in some cases, activation, are demonstrated to correlate well, both between the methodologies and with previous literature precedent. In addition, the ability to monitor sirtuin activity via O-acetyl-ADP-ribose production permits experimentation on whole-protein substrates. The deacetylation of whole-histone proteins by SIRT3, and inhibition thereof, is presented and demonstrates the feasibility of screening sirtuins using more biologically relevant molecules.
