Extraction of membrane proteins by differential solubilization for separation using two‐dimensional gel electrophoresis

Abstract

We describe the extraction and enrichment of membrane proteins for separation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) after differential solubilization of an Escherichia coli cell lysate. In a simple three-step sequential solubilization protocol applicable for whole cell lysates, membrane proteins are partitioned from other cellular proteins by their insolubility in solutions conventionally used for isoelectric focusing (IEF). As the first step, Tris-base was used to solubilize many cytosolic proteins. The resultant pellet was then subjected to conventional solubilizing solutions (urea, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, dithiothreitol, Tris, carrier ampholytes). Following the completion of this step, 89% of the initial E. coli sample mass was solubilized. Finally, the membrane protein rich pellet was partially solubilized using a combination of urea, thiourea, tributyl phosphine and multiple zwitterionic surfactants. Using N-terminal sequence tagging and peptide mass fingerprinting we have identified 11 membrane proteins from this pellet. Two of these outer membrane proteins (Omp), OmpW and OmpX, have previously been known only as an open reading frame in E. coli, while OmpC, OmpT and OmpTOLC have not previously been identified on a 2-D gel. The prefractionation of an entire cell lysate into multiple fractions, based on solubility, results in simplified protein patterns following 2-D PAGE using broad-range pH 3.5-10 immobilized pH gradients (IPGs). Additional advantages of sample prefractionation are that protein identification and gel matching, for database construction, is a more manageable task, the procedure requires no specialized apparatus, and the sequential extraction is conducted in a single centrifuge tube, minimizing protein loss.

Strategies for the enrichment and identification of basic proteins in proteome projects

“…These include subcellular location, pI and/or M r , as well as approaches aimed at separating a highly specific functional class [9,[23][24][25][26]. A major advantage of prefractionation is to enrich for selected proteins, for example the collection of a pure fraction of proteins within a particular pH range for separation on narrow or 'zoom' IPG 2-D gels [27][28].…”

Identification of proteins in activated human neutrophils susceptible to tyrosyl radical attack. A proteomic study using a tyrosylating fluorophore

“…Red cells contaminating neutrophils in the bottom layer were lysed with 15 vol of 0.15 M ammonium chloride solution for 30 min at 47C [19]. After washing three times in ice-cold HBSS 2 (Ca 21 -, Mg 21 -, and phenol red-free Hank's balanced salt solution, pH 7.4, containing 1 mg/mL glucose) neutrophils were resuspended to 10 7 /mL cells in the same buffer. Cell viability was .…”

“…After labeling soluble and membrane-associated proteins were isolated from neutrophils using a modified method [21]. Briefly, the cells maintained on ice were lysed in sterile Milli-Q water (0.1 mL) supplemented with 1 mM PMSF, 1 mM EGTA, 2 mg/mL leupeptin, and 1 mg/mL pepstatin for 20 min at 47C, with occasional vortexing.…”

Multiplex proteomic analysis by two‐dimensional differential in‐gel electrophoresis

“…Cerebral cortex tissues were subjected to a differential solubility extraction procedure essentially as described by Molloy et al [8]. Approximately 200 mg (wet wt.)…”