Wang Jigang, Lee Yew Mun, Li Caixia, Li Ping, Li Zhen, Lim Teck Kwang, Gong Zhiyuan, Lin Qingsong
Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543.
Int J Anal Chem. 2015;2015:763969. doi: 10.1155/2015/763969. Epub 2015 Mar 19.
Majority of the proteomic studies on tissue samples involve the use of gel-based approach for profiling and digestion. The laborious gel-based approach is slowly being replaced by the advancing in-solution digestion approach. However, there are still several difficulties such as difficult-to-solubilize proteins, poor proteomic analysis in complex tissue samples, and the presence of sample impurities. Henceforth, there is a great demand to formulate a highly efficient protein extraction buffer with high protein extraction efficiency from tissue samples, high compatibility with in-solution digestion, reduced number of sample handling steps to reduce sample loss, low time consumption, low cost, and ease of usage. Here, we evaluated various existing protein extraction buffers with zebrafish liver tumor samples and found that sodium deoxycholate- (DOC-) based extraction buffer with heat denaturation was the most effective approach for highly efficient extraction of proteins from complex tissues such as the zebrafish liver tumor. A total of 4,790 proteins have been identified using shotgun proteomics approach with 2D LC, which to our knowledge is the most comprehensive study for zebrafish liver tumor proteome.
大多数针对组织样本的蛋白质组学研究都采用基于凝胶的方法进行分析和消化。这种耗时费力的基于凝胶的方法正逐渐被不断发展的溶液内消化方法所取代。然而，仍然存在一些困难，比如蛋白质难以溶解、复杂组织样本中的蛋白质组分析效果不佳以及样本存在杂质等问题。因此，迫切需要配制一种高效的蛋白质提取缓冲液，该缓冲液要具备从组织样本中高效提取蛋白质、与溶液内消化具有高度兼容性、减少样本处理步骤以减少样本损失、耗时短、成本低且易于使用等特点。在此，我们用斑马鱼肝脏肿瘤样本评估了各种现有的蛋白质提取缓冲液，发现基于脱氧胆酸钠（DOC）并进行热变性处理的提取缓冲液是从斑马鱼肝脏肿瘤等复杂组织中高效提取蛋白质的最有效方法。使用二维液相色谱的鸟枪法蛋白质组学方法共鉴定出了4790种蛋白质，据我们所知，这是对斑马鱼肝脏肿瘤蛋白质组最全面的研究。
