Optimizing of a protein extraction method for Mycobacterium tuberculosis proteome analysis using mass spectrometry
Optimizing of a protein extraction method for Mycobacterium tuberculosis proteome analysis using mass spectrometry
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DOI：
10.1016/j.mimet.2016.10.021
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发表时间：
2016-12-01
影响因子：
2.2
通讯作者：
Martin, Anandi
中科院分区：
文献类型：
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作者：
Rabodoarivelo, Marie Sylvianne;Aerts, Maarten;Martin, Anandi
蛋白质组分析的关键步骤包括实施可靠的细胞裂解方法，并获得高产量的定性蛋白质。在分枝杆菌中，蛋白质提取步骤常常受到富含分枝菌酸的厚蜡状细胞壁的阻碍。因此需要严格的破坏技术来从细胞中释放蛋白质。在这里，我们展示了结核分枝杆菌 (Mbt) 的优化蛋白质提取程序，该程序产生的蛋白质提取物可用于所有当前使用的蛋白质组学平台，包括基于凝胶和 LC-MS 的策略。我们比较了在增溶缓冲液中同时使用硫脲和尿素和/或 SDS 和 DTT、结合或不结合超声处理和/或珠敲打的效果。在对快速生长的类 Mbt 生物（即耻垢分枝杆菌和偶然分枝杆菌）进行一些初步优化步骤后，在结核分枝杆菌上测试了最终的蛋白质提取方案。根据从每种测试方法中回收的蛋白质浓度以及通过 SDS PAGE 评估的提取蛋白质的质量，我们提出了一种含有硫脲和尿素的裂解缓冲液，并结合两种机械细胞破碎方法：超声处理和珠敲打。优化的方案产生的蛋白质提取物可用于基于任何蛋白质组学策略或平台的结核分枝杆菌蛋白质组学研究。 (C) 2016 Elsevier B.V. 保留所有权利。
A critical step in proteomic analyses comprises the implementation of a reliable cell lysis method with high yields of qualitative proteins. In Mycobacteria, the protein extraction step is often hampered by the thick waxy cell wall which is rich in mycolic acids. Harsh disruption techniques to release proteins from the cells are thus required. Here, we demonstrate an optimized protein extraction procedure for Mycobacterium tuberculosis (Mbt) that results in protein extracts that are useful for all currently used proteomics platforms, including gel and LC-MS based strategies. We compared the effectiveness of using both thiourea and urea and/or SDS and DTT in the solubilization buffer, in combination or not with sonication and/or bead beating. After some preliminary optimization steps on fast-growing Mbt-like organisms, namely Mycobacterium smegmatis and Mycobacterium fortuitum, the final protein extraction protocol was tested on M. tuberculosis. Based on the concentrations of the proteins recovered from each of the tested methods and on the quality of the extracted proteins as evaluated by SDS PAGE, we propose a lysis buffer that contains both thiourea and urea, in combination with two mechanical cell disruption methods: sonication and bead beating. The optimized protocol results in protein extracts that are useful in M. tuberculosis proteomics studies based on any proteomics strategy or platform. (C) 2016 Elsevier B.V. All rights reserved.