SIRCAMS, School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh, EH9 3JJ, UK.
Anal Chem. 2010 Mar 1;82(5):1897-904. doi: 10.1021/ac9026302.
We have developed an automated quench-flow microreactor which interfaces directly to an electrospray ionization (ESI) mass spectrometer. We have used this device in conjunction with ESI Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) to demonstrate the potential of this approach for studying the mechanistic details of enzyme reactions. For the model system chosen to test this device, namely, the pre-steady-state hydrolysis of p-nitrophenyl acetate by the enzyme chymotrypsin, the kinetic parameters obtained are in good agreement with those in the literature. To our knowledge, this is the first reported use of online quench-flow coupled with FTICR MS. Furthermore, we have exploited the power of FTICR MS to interrogate the quenched covalently bound enzyme intermediate using top-down fragmentation. The accurate mass capabilities of FTICR MS permitted the nature of the intermediate to be assigned with high confidence. Electron capture dissociation (ECD) fragmentation allowed us to locate the intermediate to a five amino acid section of the protein--which includes the known catalytic residue, Ser(195). This experimental approach, which uniquely can provide both kinetic and chemical details of enzyme mechanisms, is a potentially powerful tool for studies of enzyme catalysis.
我们开发了一种与电喷雾电离（ESI）质谱仪直接接口的自动淬灭流微反应器。我们使用该装置与 ESI 傅里叶变换离子回旋共振质谱（FTICR MS）结合，展示了这种方法在研究酶反应的机制细节方面的潜力。对于选择的模型系统，即酶糜蛋白酶对 p-硝基苯乙酸的预稳态水解，所获得的动力学参数与文献中的参数非常吻合。据我们所知，这是首次报道在线淬灭流与 FTICR MS 联用。此外，我们利用 FTICR MS 的强大功能，通过自上而下的片段化来检测淬灭的共价结合的酶中间产物。FTICR MS 的准确质量能力允许高度置信地确定中间产物的性质。电子俘获解离（ECD）片段化使我们能够将中间产物定位到蛋白质的五个氨基酸部分，其中包括已知的催化残基 Ser（195）。这种实验方法独特地提供了酶机制的动力学和化学细节，可以成为研究酶催化的有力工具。
