基于探针电喷雾电离质谱的酶促反应实时监测新方法

It is well accepted that the real-time reaction monitoring methodologies are more favored than the non-real-time methodologies because the real-time reaction monitoring can provide more useful information describing the reaction progress, which are crucial for the research of reaction mechanisms and optimization. Due to the reason that enzymes play important roles in both biological systems and modern industries, 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, time-resolved electrospray ionization mass spectrometry is a powerful tool for kinetic studies in this regime due to its high selectivity and sensitivity. Herein, we propose a new method for monitoring enzymatic conversions in real time by employing probe electrospray ionization mass spectrometry (PESI-MS), a newly developed ESI-MS based technique. With its advantageous properties like minimal requirement for sample pretreatment, direct analysis, high tolerance to acid and buffer, PESI-MS can be considered as a potentially universal method for real-time reaction monitoring. In this work, we plan to apply PESI-MS for monitoring of various interesting enzymatic processes to obtain qualitative and quantitative information of every reactant and product in real time. The established method of PESI-MS can be considered as a strong candidate for real-time monitoring of enzymatic reactions due to its simplicity in instrumental configuration, wide applicability under harsh conditions, and flexibility in combination with other techniques. The real-time MS data obtained by PESI-MS can be profoundly helpful for the research of enzymatic kinetics and enzymatic mechanisms.

实时反应监测与非实时反应监测比较更受青睐，这是由于实时反应监测可以提供更多的反映反应进程的信息，有助于反应机理研究与提高反应产率。由于酶促反应在生命体系以及工业上的重要性，人们建立了许多基于光谱学的酶促反应的实时反应监测方法。但是实际中很多酶促反应过程中并无光学变化，因此这些方法适用范围不大。本课题将建立一个以探针电喷雾电离质谱（PESI-MS）技术为分析手段的新型酶促反应实时监测方法。这种方法具有实时、无需样品制备、直接分析、耐高缓冲盐高酸度等特点。本课题以监测某些典型酶促反应为例，通过获取酶促反应过程中反应物与产物种类与含量的实时信息，研究酶催化底物反应的动力学过程。本课题拟建立的PESI-MS方法具有装置简单、应用范围广、方便灵活等特点，是一个潜在的酶学研究新方法。通过PESI-MS所获得的酶促反应实时质谱数据将有助于深刻认识酶促反应动力学过程以及酶的催化机理。

与非实时分析相比，人们更青睐实时分析方法，这是由于实时分析方法可以提供更多的反映化学/生物反应过程中的信息，有助于人们研究反应机理以及提高反应产率。探针电喷雾电离质谱（PESI-MS）法是一种采用实体探针作为电离源的新型质谱分析方法，具有灵敏度高、盐耐受能力强、分析速度快、方便灵活的特点，在原位分析、活体分析、实时在线分析等领域具有一定应用。受本项目资助，我们以PESI-MS技术为基础，并针对酶促反应特点进行离子源关键部件的改造与性能提高，建立了一个区别于其他分析手段的新型酶促反应实时监测方法，并成功地应用于蛋白酶促反应的实时在线分析中。与其他基于质谱技术或非质谱技术的酶促反应在线分析方法比较，PESI-MS方法除了可提供反应产物的分子量信息之外，还可提供大量的反应过程信息，这对于研究酶与底物间相互作用、酶促反应动力学特点等都是非常重要的。在上述研究结果基础上，我们进一步简化PESI-MS的设计，建立了直接采样探针质谱（DSP-MS）法，研究了DSP-MS在原位分析中的应用，并在此基础上采用电化学刻蚀法制备更加纤细强韧的探针以及流量更低的辅助溶剂喷雾，成功地将DSP-MS方法应用于植物、动物单细胞质谱分析中。