Online Electrospray Ionization Mass Spectrometric Monitoring of Protease-Catalyzed Reactions in Real Time
Online Electrospray Ionization Mass Spectrometric Monitoring of Protease-Catalyzed Reactions in Real Time
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DOI：
10.1007/s13361-011-0323-5
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发表时间：
2012-02
影响因子：
3.2
通讯作者：
Zhan Yu;L. Chen;M. K. Mandal;H. Nonami;R. Erra-Balsells;K. Hiraoka
中科院分区：
文献类型：
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作者：
Zhan Yu;L. Chen;M. K. Mandal;H. Nonami;R. Erra-Balsells;K. Hiraoka
虽然有许多成熟的方法来监测酶催化反应，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是常见的，这是不适用于这些光谱方法。质谱法作为酶学研究的一种替代技术，因其特异性、敏感性和获得化学计量信息的能力而受到青睐。本研究采用探针电喷雾电离（PESI）源与飞行时间质谱联用，实时监测了典型的蛋白酶催化反应，包括胃蛋白酶溶解和胰蛋白酶溶解细胞色素。由于每个反应体系的高导电性，很可能发生电晕放电，这将降低质谱信号的强度。采用超细取样探针和辅助蒸汽喷雾防止电晕放电。细胞色素的消化性消化和色氨酸消化的实验结果显示出不同的、有特点的催化途径。根据本研究提供的数据，PESI-MS可以被认为是实时监测酶促反应的潜在工具，因为它的仪器配置简单，在恶劣条件下的广泛适用性，以及与其他技术结合的灵活性。
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 cytochromecin 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 cytochromecshowed 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.