Clean Energy Research Center, University of Yamanashi, 4-3-11 Takeda, Kofu Yamanashi, 400-8511, Japan.
J Am Soc Mass Spectrom. 2012 Apr;23(4):728-35. doi: 10.1007/s13361-011-0323-5.
Although there are a lot of well established methods for monitoring enzyme-catalyzed reactions, most of them are based on changes in spectroscopic properties during the conversion of substrates to products. However, reactions without optical changes are common, which are inapplicable to these spectroscopic methods. As an alternative technique for enzymologic research, mass spectrometry (MS) is favored due to its specificity, sensitivity, and the ability to obtain stoichiometric information. In this work, probe electrospray ionization (PESI) source coupled with a time of flight mass spectrometer was employed to monitor some typical protease-catalyzed reactions, including pepsinolysis and trypsinolysis of cytochrome c in real time. Due to the high electrical conductivity of each reaction system, corona discharges are likely to occur, which would decrease intensities of mass spectrometric signals. An ultra-fine sampling probe and an auxiliary vapor spray were adopted to prevent corona discharges. Experimental results from peptic and tryptic digestions of cytochrome c showed different and characteristic catalytic pathways. With the data presented in this study, PESI-MS can be considered as a potential tool for real-time monitoring of enzymatic reactions because of its simplicity in instrumental configuration, wide applicability under harsh conditions, and flexibility in combination with other techniques.
虽然有很多成熟的方法可以监测酶催化反应，但大多数方法都是基于底物转化为产物过程中光谱性质的变化。然而，没有光学变化的反应很常见，这些方法不适用于这些光谱方法。作为酶学研究的替代技术，质谱（MS）因其特异性、灵敏度以及获得化学计量信息的能力而受到青睐。在这项工作中，采用探针电喷雾（PESI）源与飞行时间质谱仪相结合，实时监测一些典型的蛋白酶催化反应，包括细胞色素 c 的胃蛋白酶解和胰蛋白酶解。由于每个反应体系的高导电性，很可能会发生电晕放电，从而降低质谱信号的强度。采用超细采样探针和辅助蒸汽喷雾来防止电晕放电。细胞色素 c 的胃蛋白酶解和胰蛋白酶解实验结果显示出不同的特征性催化途径。有了本研究提供的数据，由于其仪器配置简单、在恶劣条件下具有广泛的适用性以及与其他技术结合的灵活性，PESI-MS 可以被视为实时监测酶反应的一种潜在工具。
