Institute of Organic Chemistry and Center for Molecular Biosciences (CMBI), Leopold-Franzens University Innsbruck, 6020 Innsbruck, Austria.
J Am Soc Mass Spectrom. 2021 Jul 7;32(7):1841-1845. doi: 10.1021/jasms.1c00126. Epub 2021 Jun 8.
In microdroplets, rates of chemical or biomolecular reactions can exceed those in the bulk phase by more than a million times. As electrospray ionization-based mass spectrometry (MS) involves the formation of charged microdroplets, reaction acceleration and online MS monitoring of reaction products can readily be performed at the same time. We investigated accelerated enzymatic reactions in microdroplets and focused on the proteolytic enzyme pepsin. Electrosonic spray ionization (ESSI) was utilized for developing the ultrarapid pepsin in-spray digestion of two different proteins, cytochrome and RocC, at low pH values. The optimization of the protein digestion aimed at achieving maximum sequence coverage for the analyzed proteins. Furthermore, carefully designed control experiments allowed us to unambiguously prove that enzymatic protein cleavage almost exclusively occurs within the spray at a millisecond time scale and not prior to microdroplet generation.
在微滴中，化学或生物分子反应的速率可以超过本体相中的速率一百万倍以上。由于基于电喷雾电离的质谱（MS）涉及带电荷的微滴的形成，因此可以在同一时间进行反应加速和在线 MS 监测反应产物。我们研究了微滴中的加速酶反应，并专注于蛋白水解酶胃蛋白酶。利用电声喷雾电离（ESSI）来发展超低速胃蛋白酶在喷雾中的消化两种不同的蛋白质，细胞色素和 RocC，在低 pH 值下进行。蛋白质消化的优化旨在实现分析蛋白质的最大序列覆盖。此外，精心设计的对照实验使我们能够明确证明酶蛋白裂解几乎仅在毫秒时间尺度内在喷雾内发生，而不在微滴生成之前发生。
