Institute of Pharmaceutical Chemistry , Goethe-University , Frankfurt am Main 60438 , Germany.
Biopharmaceutical Development, Analytical Sciences , MedImmune, Ltd. , Granta Park, Great Abington CB21 6GH , United Kingdom.
J Proteome Res. 2019 Mar 1;18(3):1289-1298. doi: 10.1021/acs.jproteome.8b00882. Epub 2019 Feb 13.
Sample preparation for mass-spectrometry-based proteomic analyses usually requires intricate, multistep workflows that are often limited in capacity or suffer from sample loss. Here, we introduce a lean adsorption-based protocol (ABP) for the extraction of proteins from fresh cell lysates that enables us to modify and tag protein samples under harsh conditions, such as organic solvents, high salt concentrations, or low pH values. This offers high versatility while also reducing the required steps in the preparation process significantly. Protein identifications are slightly increased compared to traditional acetone precipitation followed by an in-solution digestion (AP/IS) or filter aided sample preparation (FASP) and proved complementary to both methods regarding proteome coverage. When combined with ArgC-like digestion, this approach delivered 5386 uniquely identified proteins, a substantial increase of 18.27% over tryptic digestion (4554), while decreasing spectra complexity due to a lower number of peptide to spectra matches per protein and the number of missed cleaved peptides. In addition, an increased number of identified membrane proteins and histones as well as improved fragmentation and intensity coverage were observed through comprehensive data analysis.
基于质谱的蛋白质组学分析的样品制备通常需要复杂的、多步骤的工作流程，这些流程通常受到容量限制或存在样品损失的问题。在这里，我们介绍了一种精简的基于吸附的方案（ABP），用于从新鲜的细胞裂解物中提取蛋白质，使我们能够在苛刻的条件下对蛋白质样品进行修饰和标记，例如有机溶剂、高盐浓度或低 pH 值。这提供了高度的多功能性，同时也大大减少了制备过程中的步骤。与传统的丙酮沉淀后进行溶液消化（AP/IS）或过滤辅助样品制备（FASP）相比，蛋白质鉴定略有增加，并且在蛋白质组覆盖范围方面与这两种方法互补。当与 ArgC 样消化结合使用时，该方法可鉴定 5386 个独特的蛋白质，与胰蛋白酶消化（4554）相比，增加了 18.27%，而由于每个蛋白质的肽与谱匹配的数量以及缺失的切割肽的数量减少，谱复杂性降低。此外，通过全面数据分析，观察到鉴定的膜蛋白和组蛋白数量增加，片段化和强度覆盖度提高。
