Crude-MS Strategy for in-Depth Proteome Analysis of the Methane-Oxidizing Methylocystis sp strain SC2

被引:11
作者
Hakobyan, Anna [1 ]
Liesack, Werner [1 ,3 ]
Glatter, Timo [2 ]
机构
[1] Max Planck Inst Terr Microbiol, Res Grp Methanotroph Bacteria & Environm Genom Tr, Karl von Frisch Str 10, D-35043 Marburg, Germany
[2] Max Planck Inst Terr Microbiol, Core Facil Mass Spectrometry & Prote, Karl von Frisch Str 10, D-35043 Marburg, Germany
[3] Philipps Univ Marburg, Ctr Synthet Microbiol SYNMIKRO, Karl von Frisch Str 16, D-35043 Marburg, Germany
关键词
methanotrophs; methane oxidation; pMMO; Methylocystis; Escherichia coli; proteomics; label-free quantification; LFQ; iBAQ; PRM; transmembrane proteins; METHYLOCOCCUS-CAPSULATUS BATH; SPECTROMETRY-BASED PROTEOMICS; II METHANOTROPHIC BACTERIA; SAMPLE PREPARATION METHOD; COMPLETE GENOME SEQUENCE; LANDFILL COVER SOIL; 16S RIBOSOMAL-RNA; AMMONIA ASSIMILATION; ESCHERICHIA-COLI; ABSOLUTE QUANTIFICATION;
D O I
10.1021/acs.jproteome.8b00216
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:3086 / 3103
页数:18
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