Toward the Complete Membrane Proteome

Abstract

To attain a comprehensive membrane proteome of two strains of Corynebacterium glutamicum (L-lysine-producing and the characterized model strains), both sample pretreatment and analysis methods were optimized. Isolated bacterial membranes were digested with trypsin/ cyanogen bromide or trypsin/chymotrypsin, and a complementary protein set was identified using the multidimensional protein identification technology (MudPIT). Besides a distinct number of cytosolic or membraneassociated proteins, the combined data analysis from both digests yielded 326 integral membrane proteins (ϳ50% of all predicted) covering membrane proteins both with small and large numbers of transmembrane helices. Also membrane proteins with a high GRAVY score (Kyte, J., and Doolittle, R. About 20 -30% of all genes in an organism code for integral membrane proteins. Integral membrane proteins are involved in central cellular processes and form the major protein class for drug targets. According to their structure they can be classified either as ␣-helical or as ␤-barrel proteins. Although the latter can be studied easily by proteomic techniques, ␣-helical membrane proteins still remain an analytical challenge. Due to their location in the lipid bilayer, membrane proteins are amphipathic, and the targets for tryptic cleavage, lysine and arginine, are mainly absent in transmembrane (TM) 1 helices and only found in the hydrophilic part of the protein. The size of exposed hydrophilic domains varies among integral membrane proteins from large (e.g. epidermal growth factor receptor) to small (e.g. rhodopsin). Because most integral membrane proteins are of low abundance and show a high GRAVY score (1), all proteomic protocols usually involve prefractionation steps for enrichment of the membrane fraction. Examples include organelle separation by freeflow electrophoresis (2) and removal of membrane-associated proteins by chaotropes (3) or alkaline pH washes (4). In the next step, membranes are either treated directly with protease or solubilized with detergents, and the intact proteins are further separated before digestion. However, even after enrichment, a high percentage of the identified proteins are usually not membrane proteins regardless of the applied proteomic approach. In addition, only a small fraction (usually less than 30%) of all predicted membrane proteins are detected. In conclusion, despite their high importance and consequentially a considerable effort in method development (5), current approaches aiming at obtaining a comprehensive coverage of the membrane proteome are far from satisfactory. The 2DE technique is unsuitable for the separation of integral membrane proteins mainly due to protein aggregation during the IEF step. For this reason only membrane proteins with a low GRAVY score and only one to two TM helices are detected. In comparison, the combination of SDS-PAGE and LC-ESI MS/MS has been applied with more success, but problems like protein insolubility and the loss of hydrophobic peptides, which prevent protein identificati...

Comparison of Proteins Expressed on Secretory Vesicle Membranes and Plasma Membranes of Human Neutrophils

“…Identifying transmembrane proteins is made difficult by the absence of sites for tryptic cleavage in transmembrane regions, variability in the size of exposed hydrophobic regions, low abundance of transmembrane proteins, poor separation by two-dimensional gel electrophoresis of integral membrane proteins, and poor solubility of hydrophobic peptides. Fischer et al (11) recently reported that tryptic digestion of bacterial membrane proteins extracted in 60% methanol increased identification of integral membrane proteins from 20 to 50% of the total proteins identified. Our results suggest that this approach is useful in membranes from mammalian cells, as one-third of the proteins identified in the present study were classified as integral membrane proteins by Gene Ontology.…”

“…To address this limitation, we used a recently described protein extraction method in which 60% methanol was added to 100 mM ammonium bicarbonate, as recently described by Fischer et al (11). To determine the effectiveness of methanol extraction in the recovery of membrane proteins, each protein was analyzed for known association with cellular membranes using the Gene Ontology database.…”

“…Percoll was removed from membranes by ultracentrifugation at 100,000 ϫ g for 90 min. Membrane pellets were resuspended in 50 mM ammonium bicarbonate, washed by centrifugation at 100,000 ϫ g, then resuspended in 60% methanol in 100 mM ammonium bicarbonate to extract membrane proteins (11). Proteins were digested by incubation with 3 g each of trypsin and chymotrypsin at 37°C overnight.…”

“…The ability to extract and solubilize membrane proteins is a major limitation to all proteomic approaches. To overcome this limitation, proteins were extracted from membranes using a recently described methanol extraction procedure, followed by two-dimensional HPLC and ESI-MS/MS (11). With this approach, we identified a number of membrane spanning and membrane associated proteins and uncovered significant differences between secretory vesicle-enriched and plasma membrane-enriched proteomes.…”

Keynotes on Membrane Proteomics

“…However, the use of chromatographic peptide fractionation tools, and especially those based on reverse-phase chromatography, will make the use of detergents more problematic, leading to specially-designed, detergent-free protocols (Wu et al 2003, Fischer et al 2006.…”