A label-free Sirtuin 1 assay based on droplet-electrospray ionization mass spectrometry

Abstract

Sirtuin 1(SIRT1) is a NAD + -dependent deacetylase which has been implicated in age-related diseases such as cancer, Alzheimer's disease, type 2 diabetes, and vascular diseases. SIRT1 modulators are of interest for their potential therapeutic use and potential as chemical probes to study the role of SIRT1. Fluorescence-based assays used to identify SIRT1 activators have been shown to have artifacts related to the fluorophore substrates used in the assays. Such problems highlight the potential utility of a label-free high throughput screening (HTS) strategy. In this work, we describe a label-free SIRT1 assay suitable for HTS based on segmented flowelectrospray ionization-mass spectrometry (ESI-MS). In the assay, 0.5 μM SIRT1 was incubated with 20 μM acetylated 21-amino acid peptide, which acts as substrate for the protein. A stableisotope labeled product peptide was added to the assay mixture as an internal standard after reaction quenching. The resulting samples are formatted into 100 nL droplets segmented by perfluorodecalin and then infused at 0.8 samples/s into an ESI-MS. To enable direct ESI-MS analysis, 11 μM SIRT1 was dialyzed into a 200 μM ammonium formate (pH 8.0) buffer prior to use in the assay. This buffer was demonstrated to minimally affect enzyme kinetics and yet be compatible with ESI-MS. The assay conditions were optimized through enzyme kinetic study, and tested by screening an 80-compound library. The assay Z-factor was 0.7. Four inhibitors and no activators were detected from the library.

Epigenetic assays for chemical biology and drug discovery

“…This ambiguity can be reduced significantly with the cellular thermal shift assay (CETSA) that enables target engagement by compounds to be studied [ 207 ]. Other assay techniques that are now being used more commonly include advanced mass spectrometry [ 208 – 210 ] and when applied in conjunction with advanced image analysis of clinical samples, exquisite detail of cellular processes can be deciphered as shown in the case of the in situ detection of topoisomerase [ 211 ].…”

How electrospray potentials can disrupt droplet microfluidics and how to prevent this

“…In droplet microfluidics, usually only the disperse polar, mostly aqueous phase is electrically conductive, so it is necessary to consider how the electrical potential can be transferred from the emitter to the droplets. The easiest way to realize this is to implement an electrically conductive capillary emitter which is compatible with capillarybased droplet systems [11][12][13][14][15][16][17] as well as with polymer chip devices.…”

Enabling Biocatalysis by High-Throughput Protein Engineering Using Droplet Microfluidics Coupled to Mass Spectrometry

“…This allows for taking advantage of the tiny volume of droplets to save considerable reagent cost. Additionally, the identification of the appropriate oil–surfactant system broadens the scope of droplet–MS screening setup, as samples no longer need to be confined to relatively clean, prepared sample plates, 24 or buffer-switched reactions 16 but can be employed as routine reaction vessels in a protein engineering process.…”