Assessment of the Proteolytic Activity of α-Chymotrypsin Immobilized on Colloidal Particles by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry
Assessment of the Proteolytic Activity of α-Chymotrypsin Immobilized on Colloidal Particles by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry
复制标题
通过基质辅助激光解吸电离飞行时间质谱法评估固定在胶体颗粒上的 α-胰凝乳蛋白酶的蛋白水解活性
DOI：
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发表时间：
2015
期刊：
影响因子：
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通讯作者：
K. Rezwan
中科院分区：
文献类型：
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作者：
Ludmilla Derr;Sascha Steckbeck;R. Dringen;L. Colombi Ciacchi;L. Treccani;K. Rezwan
将酶固定在固体材料上是生物技术应用和蛋白质组学研究中的一个有前途的策略。它可以提高酶的稳定性，并且使得能够更方便地处理，容易地从反应溶液中分离，以及酶的循环再利用。为了研究颗粒结合蛋白酶的蛋白水解性质，糜蛋白酶共价固定在二氧化硅和氧化铝胶体颗粒。结合糜蛋白酶在不同时间，在连续的蛋白水解周期，并在存储长达数周后的酶活性进行了研究，通过基质辅助激光解吸/电离飞行时间质谱（MALDI-ToF MS）。使用这种方法，在不使用人工蛋白酶底物或中间化学品的情况下鉴定蛋白水解产物。以溶菌酶为模型蛋白，采用固定化胰凝乳蛋白酶进行酶解，并对酶解产物进行了测定。与用于固定化反应的胰凝乳蛋白酶的活性相比，更多的活性胰凝乳蛋白酶与氧化铝（初始浓度的1 - 10%）结合，而不是与二氧化硅（低于1%）胶体颗粒结合。与过量的未结合的胰凝乳蛋白酶相比，溶菌酶的消化较慢，胰凝乳蛋白酶固定在胶体颗粒上，只有60%的最大量的溶菌酶肽检测。固定在胶体颗粒上的胰凝乳蛋白酶的蛋白水解活性在室温下储存期间保持长达至少7周，而在连续的消化期间降低。
The immobilization of enzymes on solid materials is a promising strategy in biotechnological applications and proteomics. It can improve the enzymes’ stability, and enables a more convenient handling, easy separation from the reaction solution, and cyclic reuse of the enzymes. In order to investigate the proteolytic properties of a particle-bound protease, chymotrypsin was covalently immobilized on silica and alumina colloidal particles. The enzymatic activity of the bound chymotrypsin at different times, in consecutive proteolytic cycles, and after storage up to several weeks was investigated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS). Using this approach, the proteolysis products were identified without using artificial protease substrates or intermediate chemicals. Lysozyme was used as a model protein to perform enzymatic digestion using immobilized chymotrypsin and the peptides generated from the proteolytic digestion were determined. Compared to the activity of chymotrypsin applied for the immobilization reactions, more active chymotrypsin was bound to alumina (between 1 and 10% of the initial concentration) than to silica (below 1%) colloidal particles. Compared to an excess of unbound chymotrypsin, the digestion of lysozyme was slower with chymotrypsin immobilized on colloidal particles and only 60% of the maximal amounts of lysozyme peptides were detected. The proteolytic activity of chymotrypsin immobilized on colloidal particles was maintained during storage at room temperature for up to at least seven weeks, while it was lowered during consecutive digestions.
通过 MALDI-ToF MS 耗竭测定指导鉴定材料的结合肽序列
影响因子：
3.1
作者：
S. Steckbeck;J. Schneider;L. Wittig;K. Rischka;I. Grunwald;L. Colombi Ciacchi
通讯作者：
L. Colombi Ciacchi
影响因子：
3.1
作者：
Rivera, Jose G.;Messersmith, Phillip B.
通讯作者：
Messersmith, Phillip B.