Methylocystis sp. strain SC2 is a representative of the alphaproteobacterial methane oxidizers or type IIa methanotrophs. These microorganisms play a crucial role in methane cycling. Here, we developed an efficient analytical proteomics workflow for strain SC2. It tackles the major challenges related to the high amount of integral membrane proteins that need to be efficiently solubilized and digested for downstream analysis. Each step of the workflow, including cell lysis, protein solubilization and digestion, and MS peptide quantification, was assessed and optimized. Our new crude-lysate-MS approach proved to increase protein quantification accuracy and proteome coverage of strain SC2. It captured 62% of the predicted SC2 proteome, with up to 10-fold increase in membrane-associated proteins relative to less effective conditions. The use of crude cell lysate for downstream analysis showed to be highly efficient for SC2 and other members of the family Methylocystaceae. Using two contrasting nitrogen conditions, we further validated our workflow efficiency by analyzing the SC2 proteome for differentially expressed proteins involved in methane and nitrogen metabolism. Our crude-MS approach may be applied to a variety of proteomic workflows incorporating cell types with challenging solubilization properties. Data are available via ProteomeXchange with identifier PXD009027.

中文翻译：

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对甲烷氧化的甲基囊藻进行深度蛋白质组分析的Crude-MS策略。菌株SC2

甲基囊肿sp。SC2菌株是α-proteobacterial甲烷氧化剂或IIa型甲烷营养生物的代表。这些微生物在甲烷循环中起着至关重要的作用。在这里，我们为菌株SC2开发了有效的分析蛋白质组学工作流程。它解决了与大量整合膜蛋白有关的重大挑战，需要有效地溶解和消化这些整合膜蛋白以进行下游分析。评估和优化了工作流程的每个步骤，包括细胞裂解，蛋白质溶解和消化以及MS肽定量。我们的新粗裂解物-MS方法证明可以提高菌株SC2的蛋白质定量准确性和蛋白质组覆盖率。它捕获了62％的预测SC2蛋白质组，与膜条件相关的蛋白质相对于无效条件最多增加了10倍。甲基囊藻科。使用两个相反的氮条件，我们通过分析SC2蛋白质组中参与甲烷和氮代谢的差异表达蛋白，进一步验证了我们的工作流程效率。我们的粗质谱方法可应用于结合了具有挑战性增溶特性的细胞类型的各种蛋白质组学工作流程。数据可通过ProteomeXchange获得，其标识符为PXD009027。