Two-dimensional electrophoresis of membrane proteins

Abstract

One third of all genes of various organisms encode membrane proteins, emphasizing their crucial cellular role. However, due to their high hydrophobicity, membrane proteins demonstrate low solubility and a high tendency for aggregation. Indeed, conventional two-dimensional gel electrophoresis (2-DE), a powerful electrophoretic method for the separation of complex protein samples that applies isoelectric focusing (IEF) in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension, has a strong bias against membrane proteins. This review describes two-dimensional electrophoretic techniques that can be used to separate membrane proteins. Alternative methods for performing conventional 2-DE are highlighted; these involve replacing the IEF with electrophoresis using cationic detergents, namely 16-benzyldimethyl-n-hexadecylammonium chloride (16-BAC) and cetyl trimethyl ammonium bromide (CTAB), or the anionic detergent SDS. Finally, the separation of native membrane protein complexes through the application of blue and clear native gel electrophoresis (BN/CN-PAGE) is reviewed, as well as the free-flow electrophoresis (FFE) of membranes.

Two‐dimensional gel electrophoresis and mass spectrometry for biomarker discovery

“…One-third of the human genome codes for membrane proteins [144,145]. Besides providing a physical barrier between the cell and its environment and the various intracellular compartments, membrane proteins also regulate cell maintenance, cell-cell and organelle-cytosol interaction and communication via signalling mechanisms and the mediation of transport of ions, metabolites and peptides/proteins.…”

“…Nevertheless, several improvements in the technique have been developed to overcome this, and apparently successful analyses of purified or enriched plasma membrane proteins by 2-DE have been published (for recent review [144]). The 2-DE analysis of human erythrocyte membrane proteins [147][148][149][150], human liver plasma membrane [151] and cell envelope or outer membrane proteins from bacteria [152,153] are recent examples.…”

“…To increase the number of membrane proteins separated on the 2-D gel, approaches involve differential fractionation with the use of organic solvents and/or detergents such as Triton X-100, Triton X-114, dodecylmaltoside or ASB14 in the lysis buffer to enrich and improve the solublization of membrane proteins. Other approaches include delipidation that has shown to be beneficial for 2-DE separation and the use of soft IPG strips (,4% of polyacrylamide pores) to reduce protein aggregation, among others (for recent review [80,95,96,144,154]). Biotinylation of proteins at the cell surface, followed by highly specific purification of membrane proteins using avidin, is another strategy that has been used successfully for the enrichment and separation of surface membrane proteins by 2-DE [155].…”

Ionic liquid 1-butyl-3-methyl imidazolium tetrafluoroborate for shotgun membrane proteomics

“…As illustrated in Fig. 3, the identified TMDs could be divided into four sections: [1,3), [3,5), [5,8), and [8, ∞), and IMPs identified by BMIM BF 4 method are distributed in all sections, with number higher than those identified by other methods.…”

“…Integral membrane proteins (IMPs) lie at the critical junctions between intracellular compartments, cells, and their environments, which play unique roles in mediating various cellular processes, including intercellular communication, vesicle trafficking, ion transport, protein translocation/ integration, and the propagation of signaling cascades [1][2][3]. However, the identification of IMPs meets great challenges due to their hydrophobicity [4][5][6][7].…”