Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Martin-Luther-Universität Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, D-06120 Halle (Saale), Germany.
Anal Chem. 2010 Feb 15;82(4):1434-43. doi: 10.1021/ac9025362.
We describe the preparation of a capillary trypsin immobilized monolithic enzyme reactor (IMER) for a rapid and efficient digestion of proteins down to the femtomole level. Trypsin was immobilized on a poly(glycidyl methacrylate-co-acrylamide-co-ethylene glycol dimethycrylate) monolith using the glutaraldehyde technique. Digestion efficiencies of the IMER were evaluated using model proteins and protein mixtures as well as chemically cross-linked lysozyme regarding the addition of denaturants and increasing digestion temperature. The trypsin IMER described herein is applicable for the digestion of protein mixtures. Even at a 1000-fold molar excess of one protein, low-abundance proteins are readily identified, in combination with MS/MS analysis. An online setup of the IMER with reversed phase nano-HPLC separation and nano-ESI-MS/MS analysis was established. The great potential of the trypsin IMER for proteomics applications comprise short digestion times in the range of seconds to minutes, in addition to improved digestion efficiencies, compared to in-solution digestion.
我们描述了一种毛细管胰蛋白酶固定化整体酶反应器（IMER）的制备方法，用于快速有效地将蛋白质消化至飞摩尔水平。胰蛋白酶通过戊二醛技术固定在聚（甲基丙烯酸缩水甘油酯-丙烯酰胺-乙二醇二甲基丙烯酸酯）整体柱上。使用模型蛋白质和蛋白质混合物以及化学交联溶菌酶评估了 IMER 的消化效率，考察了添加变性剂和提高消化温度对其的影响。本文所述的胰蛋白酶 IMER 适用于蛋白质混合物的消化。即使在一种蛋白质的摩尔过量 1000 倍的情况下，结合 MS/MS 分析，低丰度蛋白质也很容易被鉴定。建立了 IMER 与反相纳米 HPLC 分离和纳升电喷雾-MS/MS 分析的在线装置。与溶液内消化相比，胰蛋白酶 IMER 在蛋白质组学应用方面具有很大的潜力，包括在几秒钟到几分钟的短消化时间内，以及提高的消化效率。
