Šlechtová Tereza, Gilar Martin, Kalíková Květa, Moore Stephanie M, Jorgenson James W, Tesařová Eva
Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Hlavova 8, 128 43, Prague, Czechia.
Waters Corporation, 34 Maple Street, Milford, MA 01757, USA.
J Chromatogr A. 2017 Mar 24;1490:126-132. doi: 10.1016/j.chroma.2017.02.024. Epub 2017 Feb 14.
Trypsin is the most widely used enzyme in proteomic research due to its high specificity. Although the in-solution digestion is predominantly used, it has several drawbacks, such as long digestion times, autolysis, and intolerance to high temperatures or organic solvents. To overcome these shortcomings trypsin was covalently immobilized on solid support and tested for its proteolytic activity. Trypsin was immobilized on bridge-ethyl hybrid silica sorbent with 300Å pores, packed in 2.1×30mm column and compared with Perfinity and Poroszyme trypsin columns. Catalytic efficiency of enzymatic reactors was tested using N-Benzoyl-l-arginine 4-nitroanilide hydrochloride as a substrate. The impact of buffer pH, mobile phase flow rate, and temperature on enzymatic activity was investigated. Digestion speed generally increased with the temperature from 20 to 37°C. Digestion speed also increased with pH from 7.0 to 9.0; the activity of prototype enzyme reactor was highest at pH 9.0, when it activity exceeded both commercial reactors. Preliminary data for fast protein digestion are presented.
由于胰蛋白酶具有高度特异性，它是蛋白质组学研究中使用最广泛的酶。尽管主要采用溶液内消化法，但该方法存在一些缺点，如消化时间长、自溶以及不耐高温或有机溶剂。为克服这些缺点，将胰蛋白酶共价固定在固体支持物上，并对其蛋白水解活性进行测试。将胰蛋白酶固定在孔径为300Å的桥乙基杂化硅胶吸附剂上，填充于2.1×30mm的色谱柱中，并与Perfinity和Poroszyme胰蛋白酶柱进行比较。以盐酸N-苯甲酰-L-精氨酸4-硝基苯胺为底物，测试酶反应器的催化效率。研究了缓冲液pH值、流动相流速和温度对酶活性的影响。消化速度一般随温度从20°C升高到37°C而加快。消化速度也随pH值从7.0升高到9.0而加快；原型酶反应器的活性在pH 9.0时最高，此时其活性超过了两种商业反应器。文中给出了快速蛋白质消化的初步数据。
