Proteomic Sample Preparation through Extraction by Unspecific Adsorption on Silica Beads for ArgC-like Digestion.,Journal of Proteome Research

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Proteomic Sample Preparation through Extraction by Unspecific Adsorption on Silica Beads for ArgC-like Digestion.
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2019-02-13 , DOI: 10.1021/acs.jproteome.8b00882
Yannik Lewin ₁ , Moritz Neupärtl ₁ , Vahid Golghalyani _{1,

2} , Michael Karas ₁

Affiliation

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.

中文翻译：

通过非特异性吸附在硅胶微珠上提取蛋白质组学样品，以进行类似ArgC的消化。

用于基于质谱的蛋白质组学分析的样品前处理通常需要复杂的多步骤工作流程，这些工作流程通常容量有限或遭受样品损失。在这里，我们介绍了一种基于稀吸附的协议（ABP），用于从新鲜细胞裂解物中提取蛋白质，这使我们能够在苛刻的条件下（例如有机溶剂，高盐浓度或低pH值）对蛋白质样品进行修饰和标记。这提供了高通用性，同时还大大减少了制备过程中所需的步骤。与传统的丙酮沉淀，随后的溶液内消化（AP / IS）或助滤剂样品制备（FASP）相比，蛋白质的鉴定略有增加，并且证明了这两种方法在蛋白质组覆盖率方面均具有互补性。当与类似ArgC的消化法结合使用时，这种方法提供了5386个独特鉴定的蛋白质，比胰蛋白酶消化法（4554）显着增加了18.27％，同时由于每个蛋白质的肽与光谱匹配次数减少以及错过的裂解肽的数目减少，从而降低了光谱复杂性。此外，通过全面的数据分析，发现已鉴定的膜蛋白和组蛋白数量增加，碎片和强度覆盖率得到改善。

更新日期：2019-02-14

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