Advances in Label-Free Screening Approaches for Studying Histone Acetyltransferases

Abstract

Histone acetyltransferases (HATs) catalyze the transfer of an acetyl group from an acetyl-coenzyme A donor molecule to specific lysine residues within proteins. The acetylation state of proteins, particularly histones, is known to modulate their intermolecular binding properties and control various cellular processes, most notably transcriptional activation. In addition, deregulation of HAT activity has been linked to the development of a number of cancers; therefore, compounds that affect these enzymes have strong potential as therapeutic agents. The research presented here demonstrates three label-free HAT screening approaches, all based on the fast and direct measurement of one or more substrate-product pairs by high-throughput mass spectrometry techniques. The first approach involves monitoring all possible acetylation states of a peptide concurrently to measure HAT activity. The second approach measures acetylation reactions, on both peptides and whole protein substrates, via direct detection of the acetyl-coenzyme A cosubstrate and coenzyme A coproduct. Lastly, the authors demonstrate the ability to monitor directly the acetylation state of whole histone proteins in the same high-throughput manner using time-of-flight mass spectrometry. The generation of compound-mediated inhibition data using each of these techniques establishes mass spectrometry as a versatile, label-free, and biologically relevant screening approach to this challenging target class.

Sirtuin 1 (SIRT1): The Misunderstood HDAC

“…Enzymatic assays there are now many assay formats to screen sirt1 enzyme for both activators and inhibitors. these include fluorescence, [136][137][138][139] time-resolved fluorescence, chemiluminescence, 140 microfluidic mobility shift, 140 fluorescence polarization, 141 mass spectrometry, 141,142 capillary electrophoresis, 143 high-performance liquid chromatography (hPlC), 7,144 and radioactive [145][146][147] formats. these are described in more detail within this issue.…”

Measurement of glycolysis reactants by high-throughput solid phase extraction with tandem mass spectrometry: Characterization of pyrophosphate-dependent phosphofructokinase as a case study

“…The MS-based analysis of multienzyme glycolytic systems has been described previously [28], further suggesting that high-throughput SPE-MS/MS could be instrumental in simultaneously studying multiple enzymes within a complex network. Because SPE-MS/MS can accommodate a variety of packing materials, can be run in reverse of normal phase, and can be connected to any mass spectrometer, this detection strategy facilitates the measurement of a wide breadth of analytes including small molecules [29][30][31][32][33][34], peptides [35][36][37], and whole proteins [38]. This work, therefore, also contributes to a growing list of applications for high-throughput SPE-MS/MS analysis.…”

Sirtuins as Regulators of the Yeast Metabolic Network

“…Recently, Sir2 was the basis for several genome-scale screens to identify genetic and physical interactors. These include a genome-wide screen to identify anti-silencer factors (Raisner and Madhani, 2008), and proteomic analyses that use methods of label-free protein quantification (Rye et al, 2011). Together with other genes identified in earlier targeted and untargeted screens, they form a large network which is indicative of the global function of Sir2.…”