Droplet electrospray ionization mass spectrometry for high throughput screening for enzyme inhibitors.
Droplet electrospray ionization mass spectrometry for high throughput screening for enzyme inhibitors.
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DOI：
10.1021/ac502542z
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发表时间：
2014-09-16
影响因子：
7.4
通讯作者：
Kennedy, Robert T.
中科院分区：
文献类型：
--
作者：
Sun, Shuwen;Kennedy, Robert T.
高通量筛选（HTS）对于鉴定具有所需性质的分子是重要的。质谱（MS）是潜在的强大的无标记HTS由于其高灵敏度，速度和分辨率。分段流是一种有趣的高通量MS格式，其中样本被不混溶流体分离成液滴，因为它可以用于可靠且精确地操作纳升体积，并且可以直接与电喷雾电离（ESI）MS耦合以进行快速分析。在这项研究中，我们描述了一种“MS读板器”，其将标准多孔板HTS工作流程与液滴ESI-MS相耦合。MS读板器可以将来自8个384孔板的3072个样品重新格式化为由不混溶油以4.5个样品/s分割的纳升液滴，并通过MS以2个样品/s顺序分析它们。使用该系统，针对1280种化学物质的组织蛋白酶B调节剂的无标记筛选在45分钟内完成，具有高Z因子（>0.72）并且没有假阳性（24个命中中的24个被确认）。该测定揭示了11种以前与组织蛋白酶抑制无关的结构。对于更大规模的筛选，可以同时进行重新格式化和分析，这将使超过145 000个样本能够在1天内进行分析。
High throughput screening (HTS) is important for identifying molecules with desired properties. Mass spectrometry (MS) is potentially powerful for label-free HTS due to its high sensitivity, speed, and resolution. Segmented flow, where samples are manipulated as droplets separated by an immiscible fluid, is an intriguing format for high throughput MS because it can be used to reliably and precisely manipulate nanoliter volumes and can be directly coupled to electrospray ionization (ESI) MS for rapid analysis. In this study, we describe a “MS Plate Reader” that couples standard multiwell plate HTS workflow to droplet ESI-MS. The MS plate reader can reformat 3072 samples from eight 384-well plates into nanoliter droplets segmented by an immiscible oil at 4.5 samples/s and sequentially analyze them by MS at 2 samples/s. Using the system, a label-free screen for cathepsin B modulators against 1280 chemicals was completed in 45 min with a high Z-factor (>0.72) and no false positives (24 of 24 hits confirmed). The assay revealed 11 structures not previously linked to cathepsin inhibition. For even larger scale screening, reformatting and analysis could be conducted simultaneously, which would enable more than 145 000 samples to be analyzed in 1 day.
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