About 20-30% of all genes in an organism encode integral membrane proteins, which are involved in numerous cellular processes [27] . Membrane proteins constitute 30% of the typical proteome, yet their propensity to aggregate and precipitate in solution confounds their analysis [28] . The target residues for tryptic cleavage (i.e., lysine and arginine) are mainly absent in transmembrane helices and preferentially found in the hydrophilic part of these lipid bilayer-incorporated proteins. Because of the protein aggregation step of IEF, 2DE is unsuitable for the separation of integral membrane proteins and is limited to detection of membraneassociated proteins and membrane proteins with a low hydrophobicity [29] . Membrane solubilization methods have been deployed to analyze enriched membrane fractions and address the solubility issue by using detergents [30] , organic solvents [31] , and organic acids [32] compatible with subsequent proteolytic digestion/chemical cleavage, separation and analysis by LC/MS. In this approach, (1) an enriched yeast membrane fraction is solubilized with 90% formic acid in the presence of cyanogens bromide. The concentrated organic acid provides the solubilization agent, and cyanogen bromide, functional under acidic conditions, allows many embedded membrane proteins to be cleaved, (2) a membrane-enriched microsomal fraction is solubilized by boiling in 0.5% SDS and, following isotope-coded affinity tag (ICAT) labeling, is diluted to reduce the concentration of SDS, and (3) by using an enriched membrane sample, the proteins are thermally denatured and sonicated in 60% organic solvent (methanol) in the presence of trypsin. The resultant peptide mixture is then analyzed by LC/MS. All three of these methods are effective and optimize the identifications of membrane proteins. Another method using high pH and protenase K is optimized specifically for the global analysis of both membrane and soluble proteins [33] . High pH favors the formation of membrane sheets, while proteinase K cleaves exposed hydrophilic domains of membrane proteins. Commercially available nonionic detergents, dodecyl maltoside, and decaethylene glycol mono hexadecyl are proved most efficient membrane protein solubilizers [34] . Another more successful approach to isolate membrane proteins relies on cell surface labeling in combination with high resolution two-dimensional (2D) LC-MS/MS [35] . In addition, improved analytical tools should be developed, that is, multidimensional liquid chromatography of peptide mixtures generated from membrane proteins, nanoflow chromatographic techniques for hydrophobic transmembrane peptides, and native electrophoresis of membrane protein complexes, which, in combination with mass spectrometry, should lead to the identification of the majority of proteins in the membrane proteome of simple microorganisms. It is important to quantify not only the identified membrane proteins but also to determine the levels of interacting partners. Subcellular fractionation techniques that employ a combination of centrifugation steps are a common choice for preparing plasma membrane-(PM-) enriched fractions including detergent-resistant membrane fractions, commonly known as lipid rafts. These methods can offer a significant improvement in specificity for PM proteins over approaches that do not perform any subcellular fractionation, but rather use whole-cell or tissue preparations [36] . Chemical-tagging methods [37] have been a more applied technique used to enrich for PM proteins and are often used in conjunction with physical separation strategies. This method allows for a specific class of protein or modification of interest to be physically separated from other nontagged proteins. Importantly, when chemical tags are attached to the extracellular domain of PM proteins on intact cells, they offer an unrivaled specificity for PM proteins, because they offer a manner to distinguish true PM proteins from intracellular contaminants. Cell-surface biotinylation, the covalent attachment of a biotin tag to the extracellular domain of PM proteins, is also a popular choice [38] [39] [40] .