Rapid and efficient protein enzymatic digestion: An experimental comparison
Rapid and efficient protein enzymatic digestion: An experimental comparison
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DOI：
10.1002/elps.201100123
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发表时间：
2012-01-01
期刊：
影响因子：
2.9
通讯作者：
Bobalova, Janette
中科院分区：
文献类型：
--
作者：
Dycka, Filip;Bobal, Pavel;Bobalova, Janette
蛋白质组学的主要目标是识别和检查从复杂生物系统中提取的大量蛋白质。这通常通过将各种蛋白质分离技术与酶消化蛋白质的质谱分析相结合来实现。最近，已经开发了该标准方案的几种替代方案，用于有效和快速的蛋白质消化。一种选择是使用修饰的胰蛋白酶代替天然胰蛋白酶用于蛋白质的凝胶内消化。微波、超声波辅助的蛋白质酶促消化和红外辐射加速的蛋白质水解是其他合适的替代方法。本文报道了利用不同技术对蛋白质进行快速酶消化的可变性能的应用。这些方法的优点是能够在较短的时间内检测蛋白质。例如，使用替代的蛋白质消化仅需要几分钟，而传统方法需要几个小时。为了证明这种快速方法的适用性，使用碳酸酐酶、牛血清白蛋白、溶菌酶和从植物（大麦、拟南芥）中提取的蛋白质的消化。考虑到常规方法所需的反应时间要长得多，这些应用的方法倾向于给予凝胶内消化高得多的效率等级。本研究探讨了快速，高效和低成本的蛋白水解策略的消化过程中，蛋白质鉴定的基础上使用超声波和红外技术。此外，还对应用技术进行了比较研究。发现了几个差异，表明红外辐射加速蛋白水解的潜在用途。
The major objective of proteomics is to identify and examine the large numbers of proteins extracted from complex biological systems. This is generally achieved by combining various techniques of protein separation with a mass spectrometric analysis of proteins that are digested enzymatically. Recently, several alternatives to this standard protocol have been developed for efficient and fast protein digestion. One option is the use of modified trypsin instead of native trypsin for the in-gel digestion of proteins. Microwave, ultrasonic-assisted protein enzymatic digestion and proteolysis accelerated by infrared radiation are other suitable alternatives. The application of the variable performance of the fast enzymatic digestion of proteins by using different techniques is reported here. The advantage of these methods is to have the ability to detect proteins in a shorter span of time. For example, using alternative protein digestion takes only minutes, in contrast to the several hours required by conventional methods. To demonstrate the suitability of this fast procedure, the digestion of carbonic anhydrase, bovine serum albumin, lysozyme and proteins extracted from plants (Hordeum vulgare, Arabidopsis thaliana) were used. Considering that the required reaction time for the conventional method is much longer, these applied methodic approaches tend to give in-gel digestion a much higher efficiency rating. This study examines the fast, efficient and low-cost proteolytic strategies for the digestion process, and for protein identification based on the use of ultrasound and infrared technology. In addition, comparisons of the applied techniques were studied. Several differences were found, suggesting the potential use of proteolysis accelerated by infrared radiation.