Protein and lipid fingerprinting of native-like membrane complexes by combining TLC and protein electrophoresis

Abstract

TLC has traditionally been used to analyze lipids isolated from membrane complexes. Here, we describe a method based on the combination of TLC and SDS-PAGE to qualitatively analyze the protein/lipid profile of membrane complexes such as those of lung surfactant. For this purpose, native lung surfactant was applied onto a silica TLC plate in the form of an aqueous suspension, preserving not only hydrophilic proteins associated with lipids but also native protein-lipid interactions. Using native membrane complexes in TLC allows the differential migration of lipids and their separation from the protein components. As a result, (partly) delipidated protein-enriched bands can be visualized and analyzed by SDS-PAGE to identify proteins originally associated with lipids. Interestingly, the hydrophobic surfactant protein C, which interacts tightly with lipids in native membrane complexes, migrates through the TLC plate, configuring specific bands that differ from those corresponding to lipids or proteins. This method therefore allows the detection and analysis of strong native-like protein-lipid interactions.

Keywords: diagnostic tools; lipoprotein; lung surfactant; phospholipids; proteomics; thin-layer chromatography.

Fig. 1.

TLC of surfactant in aqueous solution (NS) and organic solvent (OE). Samples applied include aqueous suspensions of NS purified from porcine bronchoalveolar lavage and its OE applied as a chloroform/methanol solution. Applied lipid standards include PC, PE, PG, PI, PS, SM, LPC, and Chol. Bands stained with iodine revealed SM, PC, PG, PE, NLs (including Chol), and the AP band where water-soluble proteins remain. Chol, cholesterol; LPC, lysophosphatidylcholine; NL, neutral lipid; PI, phosphatidylinositol; PS, phosphatidylserine.

Fig. 2.

Electrophoretic analysis of proteins associated with surfactant samples as extracted from TLC plates. A: Silver staining of an SDS-PAGE performed with the material scrapped from the AP and the PC TLC bands from NS or OE. B: Silver-stained gel of the AP of BAL and NS after TLC. Equivalent amounts of phospholipid were applied (100 µg). C: Immunodetection of surfactant-associated proteins in the AP and PC lanes by Western blot. D: Iodine-stained (top) and fluorescamine-stained (bottom) TLC of lipid/protein samples incorporating different amounts of SP-C expressed in percentage by weight and applied as aqueous suspensions onto the TLC plate. Arrows indicate the position of the band corresponding to SP-C.

Fig. 3.

TLC analysis of different lipid/protein complexes applied as aqueous suspensions. A: Iodine-developed TLC plate (left) of lipid or lipid/protein membrane complexes applied as aqueous suspensions of PC, PC/eNOS N-terminal segment, and PC/BR and fluorescamine-developed TLC plate (right) revealing the corresponding protein bands. B: Iodine-developed TLC plate (left) of lipid/protein membrane complexes applied as aqueous suspensions of DPPC:POPC:POPG membranes bearing the peptides KL4, KL4PQ, or KL2A2 and fluorescamine-developed TLC plate (right) revealing the corresponding peptide bands. C: Iodine-developed TLC plate (left) of different proteins and peptides (SP-C, eNOS, BR, KL4, KL4PQ, KL2A2) or lipids (POPC) applied in organic solvent and fluorescamine-developed TLC plate (right) revealing the corresponding protein bands at the AP. BR, bacteriorhodopsin; DPPC, dipalmitoylphosphatidylcholine; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol.