A Label-free Sirtuin 1 Assay based on Droplet-Electrospray Ionization Mass Spectrometry.
A Label-free Sirtuin 1 Assay based on Droplet-Electrospray Ionization Mass Spectrometry.
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DOI：
10.1039/c6ay00698a
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发表时间：
2016-05-07
期刊：
影响因子：
--
通讯作者：
Kennedy RT
中科院分区：
文献类型：
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作者：
Sun S;Buer BC;Marsh EN;Kennedy RT
Sirtuin 1 (SIRT1) 是一种 NAD+ 依赖性脱乙酰酶，与癌症、阿尔茨海默病、2 型糖尿病和血管疾病等年龄相关疾病有关。 SIRT1 调节剂因其潜在的治疗用途和作为研究 SIRT1 作用的化学探针的潜力而受到关注。用于识别 SIRT1 激活剂的基于荧光的测定已被证明具有与测定中使用的荧光团底物相关的伪影。这些问题凸显了无标记高通量筛选（HTS）策略的潜在效用。在这项工作中，我们描述了一种基于分段流电喷雾电离质谱 (ESI-MS) 的适用于 HTS 的无标记 SIRT1 测定。在该测定中，将 0.5 μM SIRT1 与 20 μM 乙酰化 21 个氨基酸肽一起孵育，后者充当蛋白质的底物。反应猝灭后，将稳定同位素标记的产物肽添加到测定混合物中作为内标。将所得样品格式化为由全氟萘烷分割的 100 nL 液滴，然后以 0.8 个样品/秒的速度注入 ESI-MS。为了实现直接 ESI-MS 分析，在用于测定之前，将 11 μM SIRT1 透析到 200 μM 甲酸铵 (pH 8.0) 缓冲液中。该缓冲液被证明对酶动力学的影响最小，并且与 ESI-MS 兼容。通过酶动力学研究优化测定条件，并通过筛选 80 种化合物库进行测试。测定Z因子为0.7。从文库中检测到四种抑制剂，没有检测到激活剂。
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 flow-electrospray 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 stable-isotope 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.