Freije J Robert, Mulder Patty P M F A, Werkman Wendy, Rieux Laurent, Niederlander Harm A G, Verpoorte Elisabeth, Bischoff Rainer
Center for Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
J Proteome Res. 2005 Sep-Oct;4(5):1805-13. doi: 10.1021/pr050142y.
Tryptic digestion followed by identification using mass spectrometry is an important step in many proteomic studies. Here, we describe the preparation of immobilized, acetylated trypsin for enhanced digestion efficacy in integrated protein analysis platforms. Complete digestion of cytochrome c was obtained with two types of modified-trypsin beads with a contact time of only 4 s, while corresponding unmodified-trypsin beads gave only incomplete digestion. The digestion rate of myoglobin, a protein known to be rather resistant to proteolysis, was not altered by acetylating trypsin and required a buffer containing 35% acetonitrile to obtain complete digestion. The use of acetylated-trypsin beads led to fewer interfering tryptic autolysis products, indicating an increased stability of this modified enzyme. Importantly, the modification did not affect trypsin's substrate specificity, as the peptide map of myoglobin was not altered upon acetylation of immobilized trypsin. Kinetic digestion experiments in solution with low-molecular-weight substrates and cytochrome c confirmed the increased catalytic efficiency (lower K(M) and higher k(cat)) and increased resistance to autolysis of trypsin upon acetylation. Enhancement of catalytic efficiency was correlated with the number of acetylations per molecule. The favorable properties of the new chemically modified trypsin reactor should make it a valuable tool in automated protein analysis systems.
胰蛋白酶消化后通过质谱鉴定是许多蛋白质组学研究中的重要步骤。在此，我们描述了固定化乙酰化胰蛋白酶的制备方法，以提高集成蛋白质分析平台中的消化效率。使用两种修饰的胰蛋白酶珠，仅需4秒的接触时间就能实现细胞色素c的完全消化，而相应的未修饰胰蛋白酶珠只能实现不完全消化。肌红蛋白是一种已知对蛋白水解具有相当抗性的蛋白质，其消化率不受胰蛋白酶乙酰化的影响，需要含有35%乙腈的缓冲液才能实现完全消化。使用乙酰化胰蛋白酶珠会产生较少的干扰性胰蛋白酶自溶产物，表明这种修饰酶的稳定性有所提高。重要的是，这种修饰不会影响胰蛋白酶的底物特异性，因为固定化胰蛋白酶乙酰化后肌红蛋白的肽图没有改变。在溶液中使用低分子量底物和细胞色素c进行的动力学消化实验证实，乙酰化后胰蛋白酶的催化效率提高（较低的K(M)和较高的k(cat)），对自溶的抗性增强。催化效率的提高与每个分子的乙酰化数量相关。新型化学修饰胰蛋白酶反应器的良好特性使其成为自动化蛋白质分析系统中的一种有价值的工具。
