2009年12月
Unbiased Quantitation of Escherichia coli Membrane Proteome Using Phase Transfer Surfactants
MOLECULAR & CELLULAR PROTEOMICS
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- ,
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- 巻
- 8
- 号
- 12
- 開始ページ
- 2770
- 終了ページ
- 2777
- 記述言語
- 英語
- 掲載種別
- 研究論文(学術雑誌)
- DOI
- 10.1074/mcp.M900240-MCP200
- 出版者・発行元
- AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
We developed a sample preparation protocol for rapid and unbiased analysis of the membrane proteome using an alimentary canal-mimicking system in which proteases are activated in the presence of bile salts. In this rapid and unbiased protocol, immobilized trypsin is used in the presence of deoxycholate and lauroylsarcosine to increase digestion efficiency as well as to increase the solubility of the membrane proteins. Using 22.5 mu g of Escherichia coli whole cell lysate, we quantitatively demonstrated that membrane proteins were extracted and digested at the same level as soluble proteins without any solubility-related bias. The recovery of membrane proteins was independent of the number of transmembrane domains per protein. In the analysis of the membrane-enriched fraction from 22.5 mu g of E. coli cell lysate, the abundance distribution of the membrane proteins was in agreement with that of the membrane protein-coding genes when this protocol, coupled with strong cation exchange prefractionation prior to nano-LC-MS/MS analysis, was used. Because this protocol allows unbiased sample preparation, protein abundance estimation based on the number of observed peptides per protein was applied to both soluble and membrane proteins simultaneously, and the copy numbers per cell for 1,453 E. coli proteins, including 545 membrane proteins, were successfully obtained. Finally, this protocol was applied to quantitative analysis of guanosine tetra-and pentaphosphate-dependent signaling in E. coli wildtype and relA knock-out strains. Molecular & Cellular Proteomics 8:2770-2777, 2009.
- リンク情報
- ID情報
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- DOI : 10.1074/mcp.M900240-MCP200
- ISSN : 1535-9476
- eISSN : 1535-9484
- PubMed ID : 19767571
- Web of Science ID : WOS:000272711800013