Fast and efficient proteolysis by microwave-assisted protein digestion using trypsin-immobilized magnetic silica microspheres
Fast and efficient proteolysis by microwave-assisted protein digestion using trypsin-immobilized magnetic silica microspheres
复制标题
使用胰蛋白酶固定的磁性二氧化硅微球通过微波辅助蛋白质消化进行快速高效的蛋白水解
DOI：
10.1021/ac800023r
复制
发表时间：
2008-05-15
影响因子：
7.4
通讯作者：
Zhang, Xiangmin
中科院分区：
文献类型：
--
作者：
Lin, Shuang;Yao, Guoping;Zhang, Xiangmin
利用固定化胰蛋白酶的磁性二氧化硅微球，建立了一种快速、高效的微波辅助蛋白质酶解方法。在这项工作中，固定化的酶到MS微球是非常简单的，只有通过一步反应与3-缩水甘油氧基丙基三甲氧基硅烷（GLYMO），它提供了环氧基团作为反应性间隔。考虑到磁性微球是一种优良的微波吸收剂，我们开发了一种新的微波辅助消化方法的基础上，易于制备的胰蛋白酶固定化MS微球。该方法结合了固定化胰蛋白酶的优点和微波快速消化的优点，具有较高的消化效率。以牛血清白蛋白和肌红蛋白为模型蛋白，对该方法的条件进行了优化。在15秒内产生的肽片段可以通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）分析确信地鉴定。与当前的溶液中消化相比，观察到等同或更好的消化效率。此外，由于固定化胰蛋白酶具有独特的磁响应性，因此可以在外部磁体的帮助下容易地分离固定化胰蛋白酶，从而可以重复使用。即使在胰蛋白酶固定化MS微球运行七次后也获得了高活性。为了进一步验证其在蛋白质组分析中的效率，应用了大鼠肝脏提取物的一个反相液相色谱（RPLC）级分。孵育15 s后，鉴定了对应于两种蛋白质的16种完全独特的肽。最后，以大鼠肝脏为例，对其应用价值进行了评价。通过液相色谱-电喷雾-串联质谱（LC-ESI-MS/MS）分析，观察到与典型的溶液中消化相当的消化效率，但孵育时间大大缩短。这种新的微波辅助消化方法将加速蛋白质组技术在生物医学和临床研究中的应用。
A fast and efficient proteolysis approach of microwave-assisted protein digestion was developed by using trypsin-immobilized magnetic silica (MS) microspheres. In the work, immobilization of the enzyme onto MS microspheres was very simple and only through a one-step reaction with 3-glycidoxypropyltrimethoxysilane (GLYMO) which provides the epoxy group as a reactive spacer. Considering that the magnetic particles are excellent microwave absorbers, we developed a novel microwave-assisted digestion method based on the easily prepared trypsin-immobilized MS microspheres. This novel digestion method combined the advantages of immobilized trypsin and the rapid-fashion of microwave-assisted digestion, which resulted in high digestion efficiency. BSA and myoglobin were used as model proteins to optimize the conditions of this method. Peptide fragments produced in 15 s could be confidently identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Equivalent or better digestion efficiency was observed comparing to current in-solution digestion. Besides, because of the unique magnetic responsivity, the immobilized trypsin can be isolated easily with the help of an external magnet and thus used repeatedly. High activity was obtained even after seven runs of the trypsin-immobilized MS microspheres. To further verify its efficiency in proteome analysis, one reversed-phase liquid chromatography (RPLC) fraction of rat liver extract was applied. After 15 s incubation, 16 totally unique peptides corresponding to two proteins were identified. Finally, the rat liver sample was used to evaluate its worth for the application. With analysis by liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS/MS), comparable digestion efficiency was observed with typical in-solution digestion but the incubation time was largely shortened. This new microwave-assisted digestion method will hasten the application of the proteome technique to biomedical and clinical research.