Protein and lipid fingerprinting of native-like membrane complexes by combining TLC and protein electrophoresis
Journal of Lipid Research
This article is available online at http://www.jlr.org number of mammalian species (14-16) and comprises 90 wt.% lipids and 8-10 wt.% proteins. From the lipid fraction, phospholipids are the most abundant components. Among them, phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol, phosphatidylethanolamine (PE), phosphatidylserine, and small amounts of sphingolipids such as sphingomyelin (SM) have been identified by TLC. Neutral lipids, especially cholesterol, are also
... erol, are also present. Proteins coisolated together with LS complexes are typically in close contact with lipids, such as in the case of surfactant protein (SP) A (17), whereas other soluble, nonlipid interacting proteins present in the bronchoalveolar lavage (BAL) (18) are lost during LS purification (19). The so-called surfactant-associated proteins SP-A, SP-B, SP-C, and SP-D have been traditionally studied after their separation from their native lipid context by means of electrophoresis and Western blot. However, due to the high lipid content in BAL or isolated surfactant, electrophoresis and blotting often result in deformed protein bands and poor detection. Moreover, small fractions of strongly interacting lipids are usually coisolated with the hydrophobic proteins SP-B and SP-C, making their study by Western blot a difficult task. Here, we present a methodology that overcomes those difficulties by combining TLC and electrophoresis to qualitatively study the lipid and protein fingerprint of whole native surfactants (NSs) or other native membrane complexes. Lipid characterization by TLC typically includes the organic extraction of the lipid component from a given biological 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.-Lopez-Rodriguez, E., N. Roldan, B. Garcia-Alvarez, and J. Pérez-Gil. Protein and lipid fingerprinting of native-like membrane complexes by combining TLC and protein electrophoresis. J. Lipid Res. 2019. 60: 430-435.