Enabling Biocatalysis by High-Throughput Protein Engineering Using Droplet Microfluidics Coupled to Mass Spectrometry.
Enabling Biocatalysis by High-Throughput Protein Engineering Using Droplet Microfluidics Coupled to Mass Spectrometry.
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DOI：
10.1021/acsomega.7b01973
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发表时间：
2018-02-28
期刊：
影响因子：
4.1
通讯作者：
Moore JC
中科院分区：
文献类型：
--
作者：
Diefenbach XW;Farasat I;Guetschow ED;Welch CJ;Kennedy RT;Sun S;Moore JC
定向进化是推动生物催化在药物合成中应用的关键技术。传统的定向进化方法是使用在微孔板中表达酶的细菌细胞进行，并通过 HPLC、高效液相色谱-质谱 (HPLC-MS) 或光学检测器测量催化反应，这需要较长的循环时间或定制的底物。为了更好地适应现代快节奏的工艺化学开发（新基质快速需要解决方案），与电喷雾电离 (ESI)-MS 连接的液滴微流体提供了一个无标记的高通量筛选平台。为了将该方法应用于工业酶筛选并探索可能进一步提高总体通量的潜在方法，我们优化了现有的液滴质谱方法。通过在毛细管电泳 (CE)-MS 源中用聚四氟乙烯针替换不锈钢 ESI 针，液滴之间的残留传统上是一个重要问题，已降至不可检测的水平。通过调节 CE-MS 源内的鞘流，吞吐量可提高至 3 Hz，液滴尺寸范围广泛 (10-50 nL)。通过使用两个不同的转氨酶库筛选反应来证明优化的方法。液滴-MS 和 LC-MS 方法之间发现良好的相关性 (r2 ∼ 0.95)，命中变体 100% 匹配。我们通过在液滴内进行体外转录翻译并通过 MS 直接分析完整的反应混合物液滴，进一步探索了该系统的功能。合成的蛋白质达到了与蛋白质标准相当的活性，并且 MS 似乎对复杂样品具有良好的耐受性。上述应用的成功表明，微流体液滴的质谱分析是显着加速酶进化库筛选的可用选择。
Directed Evolution is a key technology driving the utility of biocatalysis in pharmaceutical synthesis. Conventional approaches to Directed Evolution are conducted using bacterial cells expressing enzymes in microplates, with catalyzed reactions measured by HPLC, high-performance liquid chromatography-mass spectrometry (HPLC-MS), or optical detectors, which require either long cycle times or tailor-made substrates. To better fit modern, fast-paced process chemistry development where solutions are rapidly needed for new substrates, droplet microfluidics interfaced with electrospray ionization (ESI)-MS provides a label-free high-throughput screening platform. To apply this method to industrial enzyme screening and to explore potential approaches that may further improve the overall throughput, we optimized the existing droplet–MS methods. Carryover between droplets, traditionally a significant issue, was reduced to undetectable level by replacing the stainless steel ESI needle with a Teflon needle within a capillary electrophoresis (CE)–MS source. Throughput was improved to 3 Hz with a wide range of droplet sizes (10–50 nL) by tuning the sheath flow within the CE–MS source. The optimized method was demonstrated by screening reactions using two different transaminase libraries. Good correlations (r2 ∼ 0.95) were found between the droplet–MS and LC–MS methods, with 100% match on hit variants. We further explored the capability of the system by performing in vitro transcription–translation inside the droplets and directly analyzing the intact reaction mixture droplets by MS. The synthesized protein attained comparable activity to the protein standard, and the complex samples appeared well tolerated by the MS. The success of the above applications indicates that the MS analysis of the microfluidic droplets is an available option for considerably accelerating the screening of enzyme evolution libraries.
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DOI：
10.1177/2472555217717473
发表时间：
2017-12
期刊：
SLAS discovery : advancing life sciences R & D
影响因子：
--
作者：
Heap RE;Hope AG;Pearson LA;Reyskens KMSE;McElroy SP;Hastie CJ;Porter DW;Arthur JSC;Gray DW;Trost M
通讯作者：
Trost M
影响因子：
7.4
作者：
Sun, Shuwen;Slaney, Thomas R.;Kennedy, Robert T.
通讯作者：
Kennedy, Robert T.
影响因子：
16.6
作者：
Sun, Huihua;Yeo, Wan Lin;Ang, Ee Lui
通讯作者：
Ang, Ee Lui
影响因子：
15
作者：
Si T;Li B;Comi TJ;Wu Y;Hu P;Wu Y;Min Y;Mitchell DA;Zhao H;Sweedler JV
通讯作者：
Sweedler JV
影响因子：
6.4
作者：
Hibbert, EG;Dalby, PA
通讯作者：
Dalby, PA