Wang Haixing, Qian Wei-Jun, Mottaz Heather M, Clauss Therese R W, Anderson David J, Moore Ronald J, Camp David G, Khan Arshad H, Sforza Daniel M, Pallavicini Maria, Smith Desmond J, Smith Richard D
Biological Sciences Division, Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
J Proteome Res. 2005 Nov-Dec;4(6):2397-403. doi: 10.1021/pr050160f.
Challenges associated with the efficient and effective preparation of micro- and nanoscale (micro- and nanogram) clinical specimens for proteomic applications include the unmitigated sample losses that occur during the processing steps. Herein, we describe a simple "single-tube" preparation protocol appropriate for small proteomic samples using the organic cosolvent, trifluoroethanol (TFE) that circumvents the loss of sample by facilitating both protein extraction and protein denaturation without requiring a separate cleanup step. The performance of the TFE-based method was initially evaluated by comparisons to traditional detergent-based methods on relatively large scale sample processing using human breast cancer cells and mouse brain tissue. The results demonstrated that the TFE-based protocol provided comparable results to the traditional detergent-based protocols for larger, conventionally sized proteomic samples (>100 microg protein content), based on both sample recovery and numbers of peptide/protein identifications. The effectiveness of this protocol for micro- and nanoscale sample processing was then evaluated for the extraction of proteins/peptides and shown effective for small mouse brain tissue samples (approximately 30 microg total protein content) and also for samples of approximately 5000 MCF-7 human breast cancer cells (approximately 500 ng total protein content), where the detergent-based methods were ineffective due to losses during cleanup and transfer steps.
为蛋白质组学应用高效且有效地制备微米和纳米级（微克和纳克级）临床样本所面临的挑战包括在处理步骤中出现的不可避免的样本损失。在此，我们描述了一种简单的“单管”制备方案，该方案适用于使用有机助溶剂三氟乙醇（TFE）的小蛋白质组样本，通过促进蛋白质提取和蛋白质变性来避免样本损失，而无需单独的净化步骤。基于TFE的方法的性能最初是通过在使用人乳腺癌细胞和小鼠脑组织进行相对大规模样本处理时与传统的基于去污剂的方法进行比较来评估的。结果表明，基于TFE的方案在样本回收率和肽/蛋白质鉴定数量方面，对于更大的、常规大小的蛋白质组样本（蛋白质含量>100微克），能提供与传统的基于去污剂的方案相当的结果。然后评估了该方案在微米和纳米级样本处理中提取蛋白质/肽的有效性，结果表明其对小鼠脑组织小样本（总蛋白质含量约30微克）以及约5000个MCF - 7人乳腺癌细胞样本（总蛋白质含量约500纳克）有效，而基于去污剂的方法由于在净化和转移步骤中的损失在这些样本处理中无效。
