Unbiased quantitation of Escherichia coli membrane proteome using phase transfer surfactants

Takeshi Masuda, Natsumi Saito, Masaru Tomita, Yasushi Ishihama

Research output: Contribution to journalArticle

76 Citations (Scopus)

Abstract

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 μg 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 μg 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 wild-type and relA knock-out strains.

Original languageEnglish
Pages (from-to)2770-2777
Number of pages8
JournalMolecular and Cellular Proteomics
Volume8
Issue number12
DOIs
Publication statusPublished - 2009 Dec

Fingerprint

Proteome
Surface-Active Agents
Escherichia coli
Membrane Proteins
Membranes
Solubility
Guanosine Pentaphosphate
Proteins
Guanosine Tetraphosphate
Deoxycholic Acid
Escherichia coli Proteins
Canals
Bile Acids and Salts
Trypsin
Cations
Digestion
Peptide Hydrolases
Cell Count
Genes
Recovery

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Analytical Chemistry

Cite this

Unbiased quantitation of Escherichia coli membrane proteome using phase transfer surfactants. / Masuda, Takeshi; Saito, Natsumi; Tomita, Masaru; Ishihama, Yasushi.

In: Molecular and Cellular Proteomics, Vol. 8, No. 12, 12.2009, p. 2770-2777.

Research output: Contribution to journalArticle

Masuda, Takeshi ; Saito, Natsumi ; Tomita, Masaru ; Ishihama, Yasushi. / Unbiased quantitation of Escherichia coli membrane proteome using phase transfer surfactants. In: Molecular and Cellular Proteomics. 2009 ; Vol. 8, No. 12. pp. 2770-2777.
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