Cortese Marc S, Baird Jason P, Uversky Vladimir N, Dunker A Keith
Department of Biochemistry and Molecular Biology and Center for Computational Biology and Bioinformatics, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
J Proteome Res. 2005 Sep-Oct;4(5):1610-8. doi: 10.1021/pr050119c.
A method to enrich cell extracts in totally unfolded proteins was investigated. A literature search revealed that 14 of 29 proteins isolated by their failure to precipitate during perchloric acid (PCA) or trichloroacetic acid (TCA) treatment where also shown experimentally to be totally disordered. A near 100 000-fold reduction in yield was observed after 5% or 9% PCA treatment of total soluble E. coli protein. Despite this huge reduction, 158 and 142 spots were observed from the 5% and the 9% treated samples, respectively, on silver-stained 2-D SDS-PAGE gels loaded with 10 microg of protein. Treatment with 1% PCA was less selective with more visible spots and a greater than 3-fold higher yield. A substantial yield of unprecipitated protein was obtained after 3% TCA treatment, suggesting that the common use of TCA precipitation prior to 2-D gel analysis may result in loss of unstructured protein due to their failure to precipitate. Our preliminary analysis suggests that treating total protein extracts with 3-5% PCA and determining the identities of soluble proteins could be the starting point for uncovering unfoldomes (the complement of unstructured proteins in a given proteome). The 100 000-fold reduction in yield and concomitant reduction in number of proteins achieved by 5% PCA treatment produced a fraction suitable for analysis in its entirety using standard proteomic techniques. In this way, large numbers of totally unstructured proteins could be identified with minimal effort.
研究了一种在完全展开的蛋白质中富集细胞提取物的方法。文献检索表明，在高氯酸（PCA）或三氯乙酸（TCA）处理过程中未能沉淀而分离出的29种蛋白质中，有14种经实验证明也是完全无序的。用5%或9%的PCA处理总可溶性大肠杆菌蛋白后，产量下降了近100000倍。尽管产量大幅下降，但在加载10微克蛋白质的银染二维SDS-PAGE凝胶上，分别从5%和9%处理的样品中观察到158个和142个斑点。用1%的PCA处理选择性较低，斑点更明显，产量高出3倍以上。用3%的TCA处理后获得了大量未沉淀的蛋白质，这表明在二维凝胶分析之前常用的TCA沉淀可能会导致无结构蛋白质因未能沉淀而丢失。我们的初步分析表明，用3-5%的PCA处理总蛋白提取物并确定可溶性蛋白质的身份可能是揭示未折叠蛋白质组（给定蛋白质组中无结构蛋白质的补充）的起点。5%的PCA处理使产量降低了100000倍，同时蛋白质数量也相应减少，产生了一个适合使用标准蛋白质组学技术进行整体分析的组分。通过这种方式，可以轻松鉴定大量完全无结构的蛋白质。
