Biomedical Proteomics Research Group, Department of Structural Biology and Bioinformatics, Geneva University, 1 Michel Servet, Geneva, Switzerland.
J Microbiol Methods. 2010 Jan;80(1):56-62. doi: 10.1016/j.mimet.2009.10.019. Epub 2009 Nov 12.
In bottom-up proteomics, rapid and efficient protein digestion is crucial for data reliability. However, sample preparation remains one of the rate-limiting steps in proteomics workflows. In this study, we compared the conventional trypsin digestion procedure with two accelerated digestion protocols based on shorter reaction times and microwave-assisted digestion for the preparation of membrane-enriched protein fractions of the human pathogenic bacterium Staphylococcus aureus. Produced peptides were analyzed by Shotgun IPG-IEF, a methodology relying on separation of peptides by IPG-IEF before the conventional LC-MS/MS steps of shotgun proteomics. Data obtained on two LC-MS/MS platforms showed that accelerated digestion protocols, especially the one relying on microwave irradiation, enhanced the cleavage specificity of trypsin and thus improved the digestion efficiency especially for hydrophobic and membrane proteins. The combination of high-throughput proteomics with accelerated and efficient sample preparation should enhance the practicability of proteomics by reducing the time from sample collection to obtaining the results.
在自下而上的蛋白质组学中，快速有效的蛋白质消化对于数据可靠性至关重要。然而，样品制备仍然是蛋白质组学工作流程中的限速步骤之一。在这项研究中，我们比较了传统的胰蛋白酶消化程序与两种基于更短反应时间的加速消化方案，以及微波辅助消化，用于制备人类致病菌金黄色葡萄球菌的膜富集蛋白级分。产生的肽通过 Shotgun IPG-IEF 进行分析，该方法依赖于在传统的 LC-MS/MS 步骤之前通过 IPG-IEF 分离肽。在两个 LC-MS/MS 平台上获得的数据表明，加速消化方案，特别是依赖于微波辐射的方案，增强了胰蛋白酶的切割特异性，从而提高了消化效率，特别是对疏水性和膜蛋白。高通量蛋白质组学与加速和高效样品制备的结合，通过减少从样品采集到获得结果的时间，应该增强蛋白质组学的实用性。
