Sharma Divakar, Bisht Deepa
Department of Biochemistry, National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Tajganj, Agra, India.
Electrophoresis. 2016 May;37(9):1187-90. doi: 10.1002/elps.201600025. Epub 2016 Apr 5.
Lipophilic proteome profiling is crucial because they have an anticipated role in biological processes and pathogenesis of Mycobacterium tuberculosis. These lipophilic proteins might be used as potential targets for the development of newer diagnostic markers and drug targets due to their association with membranes and drugs. We developed an efficient and rapid method to enrich the lipophilic proteins extraction from M. tuberculosis H37Rv for 2DE. In the extraction of lipophilic proteins, nonionic detergent (Triton X-100) was added in sonication buffer that augmented the solubilization of the proteins at the time of sonication. Enriched whole cell lysate was subjected to direct phase separation using Triton X-114, without the need for preisolation of membranes. In this study, we report that our optimized extraction buffer increased the lipophilic proteins extraction and their improved resolution on 2D gel up to two- to threefolds (quantitatively and qualitatively) as compared to standard extraction buffer. Some proteins were identified by MALDI-TOF/MS.
亲脂蛋白质组分析至关重要，因为它们在结核分枝杆菌的生物学过程和发病机制中具有预期作用。由于这些亲脂性蛋白质与膜和药物相关，它们可能被用作开发新型诊断标志物和药物靶点的潜在目标。我们开发了一种高效快速的方法，用于从结核分枝杆菌H37Rv中富集亲脂性蛋白质以进行二维电泳。在亲脂性蛋白质的提取过程中，将非离子洗涤剂（Triton X-100）添加到超声缓冲液中，这在超声处理时增强了蛋白质的溶解。富集的全细胞裂解物使用Triton X-114进行直接相分离，无需预先分离膜。在本研究中，我们报告称，与标准提取缓冲液相比，我们优化的提取缓冲液使亲脂性蛋白质的提取量增加，并且在二维凝胶上的分辨率提高了两到三倍（在定量和定性方面）。一些蛋白质通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF/MS）进行了鉴定。
