Mapping the Subcellular Proteome of Shewanella oneidensis MR-1 using Sarkosyl-Based Fractionation and LC-MS/MS Protein Identification

被引:39
作者
Brown, Roslyn N. [1 ]
Romine, Margaret F. [1 ]
Schepmoes, Athena A. [1 ]
Smith, Richard D. [1 ]
Lipton, Mary S. [1 ]
机构
[1] Pacific NW Natl Lab, Div Biol Sci, Richland, WA 99352 USA
关键词
subcellular; proteomics; Sarkosyl; fractionation; Shewanella; SIGNAL RECOGNITION PARTICLE; OUTER-MEMBRANE CYTOCHROMES; TANDEM MASS-SPECTROMETRY; SALMONELLA-TYPHIMURIUM PROTEOME; GRAM-NEGATIVE BACTERIA; TIME TAG APPROACH; ESCHERICHIA-COLI; PUTREFACIENS MR-1; HYPOTHETICAL PROTEINS; CYTOPLASMIC MEMBRANE;
D O I
10.1021/pr100215h
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
A simple and effective subcellular proteomic method for fractionation based on osmotic lysis, differential centrifugation, and Sarkosyl solubilization was applied to the Gram-negative bacterium Shewanella oneidensis to gain insight into its subcellular architecture. Global differences in bacterial cytoplasm, inner membrane, periplasm, and outer membrane protein fractions were observed by SDS PAGE and heme staining, and tryptic peptides were analyzed using high-resolution liquid chromatography tandem mass spectrometry. Proteins predicted to be localized to each subcellular fraction were enriched similar to 2-fold (on average) in each fraction compared to crude cell lysates. In addition, the Sarkosyl solubilization method facilitated separation of the inner and outer membranes, making the procedure amenable for effective probing of the subcellular proteome of Gram-negative bacteria via liquid chromatography tandem mass spectrometry. With 40% of the observable proteome represented, this study provides extensive information on both subcellular architecture and relative abundance of proteins in S. oneidensis and provides a foundation for future work on subcellular organization and protein membrane interactions in other Gram-negative bacteria.
引用
收藏
页码:4454 / 4463
页数:10
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