Porto Conte Ricerche, Tramariglio, Alghero, Italy; Dipartimento di Scienze Biomediche, Università di Sassari, Sassari, Italy.
Proteomics. 2013 Sep;13(17):2597-607. doi: 10.1002/pmic.201200478. Epub 2013 Aug 7.
This work presents a comparative evaluation of several detergent-based sample preparation workflows for the MS-based analysis of bacterial proteomes, performed using the model organism Escherichia coli. Initially, RapiGest- and SDS-based buffers were compared for their protein extraction efficiency and quality of the MS data generated. As a result, SDS performed best in terms of total protein yields and overall number of MS identifications, mainly due to a higher efficiency in extracting high molecular weight (MW) and membrane proteins, while RapiGest led to an enrichment in periplasmic and fimbrial proteins. Then, SDS extracts underwent five different MS sample preparation workflows, including: detergent removal by spin columns followed by in-solution digestion (SC), protein precipitation followed by in-solution digestion in ammonium bicarbonate or urea buffer, filter-aided sample preparation (FASP), and 1DE separation followed by in-gel digestion. On the whole, about 1000 proteins were identified upon LC-MS/MS analysis of all preparations (>1100 with the SC workflow), with FASP producing more identified peptides and a higher mean sequence coverage. Each protocol exhibited specific behaviors in terms of MW, hydrophobicity, and subcellular localization distribution of the identified proteins; a comparative assessment of the different outputs is presented.
这项工作对几种基于去污剂的样品制备工作流程进行了比较评估，用于基于 MS 的细菌蛋白质组分析，使用模型生物大肠杆菌进行。最初，比较了 RapiGest 和 SDS 基缓冲液在蛋白质提取效率和生成的 MS 数据质量方面的性能。结果表明，SDS 在总蛋白质产量和 MS 鉴定总数方面表现最佳，主要是因为其提取高分子量（MW）和膜蛋白的效率更高，而 RapiGest 则导致周质和菌毛蛋白富集。然后，SDS 提取物经历了五种不同的 MS 样品制备工作流程，包括：通过旋转柱去除去污剂，然后进行溶液内消化（SC）；用碳酸氢铵或尿素缓冲液进行蛋白质沉淀，然后进行溶液内消化；滤过辅助样品制备（FASP）；以及 1DE 分离后进行胶内消化。总的来说，所有制备物的 LC-MS/MS 分析鉴定了约 1000 种蛋白质（SC 工作流程超过 1100 种），FASP 产生了更多的鉴定肽和更高的平均序列覆盖率。每个方案在鉴定蛋白质的 MW、疏水性和亚细胞定位分布方面都表现出特定的行为；对不同输出结果进行了比较评估。
