Enhanced detergent extraction for analysis of membrane proteomes by two-dimensional gel electrophoresis

Abstract

Background: The analysis of hydrophobic membrane proteins by two-dimensional gel electrophoresis has long been hampered by the concept of inherent difficulty due to solubility issues. We have optimized extraction protocols by varying the detergent composition of the solubilization buffer with a variety of commercially available non-ionic and zwitterionic detergents and detergent-like phospholipids.

Results: After initial analyses by one-dimensional SDS-PAGE, quantitative two-dimensional analyses of human erythrocyte membranes, mouse liver membranes, and mouse brain membranes, extracted with buffers that included the zwitterionic detergent MEGA 10 (decanoyl-N-methylglucamide) and the zwitterionic lipid LPC (1-lauroyl lysophosphatidylcholine), showed selective improvement over extraction with the common 2-DE detergent CHAPS (3 [(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate). Mixtures of the three detergents showed additive improvements in spot number, density, and resolution. Substantial improvements in the analysis of a brain membrane proteome were observed.

Conclusion: This study demonstrates that an optimized detergent mix, coupled with rigorous sample handling and electrophoretic protocols, enables simple and effective analysis of membrane proteomes using two-dimensional electrophoresis.

Figure 1

Composite of 1D SDS-PAGE analyses of RBC ghost membranes extracted with A) 4% CHAPS, B) 2% SDS, C) 2% LPC, D) 2% lauric acid, E) 2% trans, trans-farnesol, F) 2% MEGA 8, G) 2% MEGA 9, H) 2% MEGA 10, I) 2% 1,2 dioleoyloxy -3-(dimethylamino)propane, J) 2% SB 3–10 (Sigma), K) 2% SB 3–10 (Calbiochem), L) C12E8, M)1-oleoyl-sn-glycerol, N) DL-α-O-benzylglycerol. Arrows indicate notable differences between extractions including 1, the multiple transmembrane spanning protein band III.

Figure 2

2-DE of RBC ghost membranes extracted with A) 4% CHAPS, B) 3% CHAPS : 1% LPC, C) 3% CHAPS : 1% MEGA 10, D) 3% CHAPS : 0.5% LPC : 0.5% MEGA 10. Extractions were carried out in buffer with 8 M urea, 2 M thiourea, protease inhibitor cocktail, and the indicated detergent for 1 hour on ice. Gels are representative of three independent experiments. Roman numerals indicate areas of improvement including i, the multiple transmembrane spanning protein band III.

Figure 3

2-DE of mouse brain membranes extracted with A) 4% CHAPS, B) 3% CHAPS : 1% LPC, C) 3% CHAPS : 1% MEGA 10, D) 3% CHAPS : 0.5% LPC : 0.5% MEGA 10. Extractions were carried out as for Fig. 2. Gels are representative of three independent experiments. Areas defined with Roman numerals are shown in Fig. 4.

Figure 5

2-DE gels of mouse liver membranes extracted with A) 4% CHAPS, B) 3% CHAPS : 1% LPC. Extractions were carried out as for Fig. 2. Gels are representative of three independent experiments. Arrow indicates specific differences between gels.

Figure 4

Enlargement and contrast of selected regions after 2-DE of mouse brain membranes (see areas defined in Fig 3). Areas i-vi show selective increases in spot number, resolution, and density. Samples were extracted with A) 4% CHAPS, B) 3% CHAPS : 1% LPC, C) 3% CHAPS : 1% MEGA 10, D) 3% CHAPS : 0.5% LPC : 0.5% MEGA 10. Results are representative of three independent experiments. Green arrows indicate spots showing increased volume and density, red arrows indicate decrease, blue arrows indicate novel spots.