Label Free Screening of Enzyme Inhibitors at Femtomole Scale Using Segmented Flow Electrospray Ionization Mass Spectrometry
Label Free Screening of Enzyme Inhibitors at Femtomole Scale Using Segmented Flow Electrospray Ionization Mass Spectrometry
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DOI：
10.1021/ac3011389
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发表时间：
2012-07-03
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;Slaney, Thomas R.;Kennedy, Robert T.
基于液滴的微流体是筛选和优化化学反应的一个有吸引力的平台。使用这种方法，可以可靠地操作纳升体积的样品，并以高精度、自动化和吞吐量执行诸如试剂添加等操作。大多数使用液滴微流体的研究都依赖于光学技术来检测反应；然而，这需要在被研究的反应中改变工程颜色或荧光。在这项工作中，我们将电喷雾电离质谱仪(ESI-MS)与纳升尺度的分段流动反应相结合，以实现对反应产物的直接(无标记)分析。该系统被应用于组织蛋白酶B抑制剂的筛选。在这种方法中，测试化合物(包括三种已知的抑制剂)的溶液在由全氟十氢萘分割的试管中排列成纳升液滴阵列。样品通过一系列三通泵送，以添加酶、底物(肽)和猝灭剂。然后，将生成的反应混合物注入涂有金属涂层的熔融二氧化硅ESI发射器中进行MS分析。该系统具有高通量的潜力，因为试剂添加步骤是在每个样品0.7S的条件下进行的，而电喷雾质谱联用技术在每个样品高达1.2S的条件下进行。在ESI发射器中，结转是无关紧要的，根据所用TEE的不同，每个试剂添加的结转在2%到9%之间。该分析方法可靠，Z因子接近0.8。该方法每次反应需要0.8pmol的受试化合物、1.6pmoL的底物和5fmoL的酶。分段流动ESI-MS允许以良好的吞吐量和超低的样品消耗直接、无标记地筛选反应。
Droplet-based microfluidics is an attractive platform for screening and optimizing chemical reactions. Using this approach, it is possible to reliably manipulate nanoliter volume samples and perform operations such as reagent addition with high precision, automation, and throughput. Most studies using droplet microfluidics have relied on optical techniques to detect the reaction; however, this requires engineering color or fluorescence change into the reaction being studied. In this work, we couple electrospray ionization mass spectrometry (ESI-MS) to nanoliter scale segmented flow reactions to enable direct (label-free) analysis of reaction products. The system is applied to a screen of inhibitors for cathepsin B. In this approach, solutions of test compounds (including three known inhibitors) are arranged as an array of nanoliter droplets in a tube segmented by perfluorodecalin. The samples are pumped through a series of tees to add enzyme, substrate (peptides), and quenchant. The resulting reaction mixtures are then infused into a metal-coated, fused silica ESI emitter for MS analysis. The system has potential for high-throughput as reagent addition steps are performed at 0.7 s per sample and ESI-MS at up to 1.2 s per sample. Carryover is inconsequential in the ESI emitter and between 2 and 9% per reagent addition depending on the tee utilized. The assay was reliable with a Z-factor of similar to 0.8. The method required 0.8 pmol of test compound, 1.6 pmol of substrate, and 5 fmol of enzyme per reaction. Segmented flow ESI-MS allows direct, label free screening of reactions at good throughput and ultralow sample consumption.