Man Tsz-Kwong, Li Yiting, Dang Tu Anh, Shen Jianhe, Perlaky Laszlo, Lau Ching C
Texas Children's Cancer Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA.
Proteome Sci. 2006 Mar 23;4:3. doi: 10.1186/1477-5956-4-3.
Research with clinical specimens is always hampered by the limited availability of relevant samples, necessitating the use of a single sample for multiple assays. TRIzol is a common reagent for RNA extraction, but DNA and protein fractions can also be used for other studies. However, little is known about using TRIzol-extracted proteins in proteomic research, partly because proteins extracted from TRIzol are very resistant to solubilization.
To facilitate the use of TRIzol-extracted proteins, we first compared the ability of four different common solubilizing reagents to solubilize the TRIzol-extracted proteins from an osteosarcoma cell line, U2-OS. Then we analyzed the solubilized proteins by Surface Enhanced Laser Desorption/Ionization technique (SELDI). The results showed that solubilization of TRIzol-extracted proteins with 9.5 M Urea and 2% CHAPS ([3-[(3-cholamidopropyl)-dimethylammonio]propanesulfonate]) (UREA-CHAPS) was significantly better than the standard 1% SDS in terms of solubilization efficiency and the number of detectable ion peaks. Using three different types of SELDI arrays (CM10, H50, and IMAC-Cu), we demonstrated that peak detection with proteins solubilized by UREA-CHAPS was reproducible (r > 0.9). Further SELDI analysis indicated that the number of ion peaks detected in TRIzol-extracted proteins was comparable to a direct extraction method, suggesting many proteins still remain in the TRIzol protein fraction.
Our results suggest that UREA-CHAPS performed very well in solubilizing TRIzol-extracted proteins for SELDI applications. Protein fractions left over after TRIzol RNA extraction could be a valuable but neglected source for proteomic or biochemical analysis when additional samples are not available.
临床样本研究总是受到相关样本有限可用性的阻碍，这使得有必要将单个样本用于多种检测。TRIzol是一种常用的RNA提取试剂，但DNA和蛋白质组分也可用于其他研究。然而，关于在蛋白质组学研究中使用TRIzol提取的蛋白质知之甚少，部分原因是从TRIzol中提取的蛋白质非常难以溶解。
为便于使用TRIzol提取的蛋白质，我们首先比较了四种不同常用溶解试剂溶解骨肉瘤细胞系U2-OS中TRIzol提取蛋白质的能力。然后我们通过表面增强激光解吸/电离技术（SELDI）分析溶解的蛋白质。结果表明，就溶解效率和可检测离子峰数量而言，用9.5 M尿素和2% CHAPS（[3-[(3-胆酰胺丙基)-二甲基铵]丙烷磺酸盐]）（UREA-CHAPS）溶解TRIzol提取的蛋白质明显优于标准的1% SDS。使用三种不同类型的SELDI芯片（CM10、H50和IMAC-Cu），我们证明用UREA-CHAPS溶解的蛋白质的峰检测具有可重复性（r > 0.9）。进一步的SELDI分析表明，在TRIzol提取的蛋白质中检测到的离子峰数量与直接提取方法相当，这表明许多蛋白质仍保留在TRIzol蛋白质组分中。
我们的结果表明，UREA-CHAPS在溶解TRIzol提取的蛋白质用于SELDI应用方面表现非常出色。当没有额外样本时，TRIzol RNA提取后剩余的蛋白质组分可能是蛋白质组学或生化分析中一个有价值但被忽视的来源。
