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