November 2009

A Little Digestive Aid for Analysis

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One problem with conventional shotgun proteomics strategies is that they generally underrepresent the membrane proteome because of inadequate solubilization and protease digestion. SDS-PAGE followed by in-gel digestion can partially solve this problem, but recovery can be low, and this approach is not suited for rapid and high-throughput systems. In this study, the researchers tried another trick, employing a digestion protocol that mimics the alimentary canal, in which bile salts such as cholate and deoxycholate are secreted together with trypsin, to increase solubility and digestion efficiency. Using this phase-transfer surfactant (PTS) strategy, the researchers estimated the copy numbers per cell of 1,453 Escherichia coli proteins, including 545 membrane proteins. They then applied their protocol to a quantitative analysis of guanosine tetra- and pentaphosphate-dependent signaling in E. coli wild-type and relA knockout strains. This study demonstrates, for the first time, that membrane proteins can be quantitatively extracted, digested and identified with similar robustness to soluble proteins.

relA KO E. coli
Quantitation and comparison of flagellum and chemotaxis proteins in wild-type and relA KO E. coli.  

Unbiased Quantitation of Escherichia coli Membrane Proteome Using Phase-transfer Surfactants

Takeshi Masuda, Natsumi Saito, Masaru Tomita, and Yasushi Ishihama

Mol. Cell. Proteomics, published online Sept. 18, 2009

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