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 microg 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 microg 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.