Background

Lipid research has gained great momentum in recent years, stimulated in part by the interest in lipid-associated disorders in humans, in part by aspirations of biofuel and biopharmaceutical production. Lipids play vital roles in many biological processes, including the formation of bio-membranes, the storage of energy, and numerous signal transduction processes. The budding yeast, Saccharomyces cerevisiae, has been an excellent model organism for studying the physiological and pathophysiological roles of lipids at the cellular level (Popa et al., 2016). Primary lipid metabolism is highly conserved between yeast and humans in both the basic biochemical pathways and the regulatory circuitries that link lipid metabolism to energy homeostasis, cell growth, and development. One advantage of using yeast in lipid-related research is that the pathways and molecular compositions are often less complicated than those in mammals, facilitating their scientific investigation. In yeast, PA (phosphatidate acid), PC (phosphatidylcholine), PE (phosphatidylethanolamine), PI (phosphatidylinositol), PS (phosphatidylserine), DAG (diacylycerols), and their respective lyso-derivatives are the most abundant membrane lipid classes, while PG (phosphatidylglycerol) and CL (cardiolipin) are less abundant. TAG (triacylglycerol) and SE (sterol ester) act as storage lipids. The most abundant yeast sphingolipids consist of IPC (inositolphosphoceramide), MIPC (mannose inositolphosphoceramide), and M(IP)₂C (mannose diinositolphosphoceramide) (Klug and Daum, 2014).

Lipid extraction procedures described in the literature generally share common principles but vary in processing steps. Among these, the most cited Folch method uses CHCl₃/CH₃OH (2:1 v/v) as the solvent system (Folch et al., 1957). Alternate proportions of CHCl₃/CH₃OH or replacement of CHCl₃ with 2-propanol or hexane have also been utilized (Fuchs et al., 2015; Knittelfelder and Kohlwein, 2017a and 2017b). In this protocol, we use CHCl₃/CH₃OH (2:1 v/v) as the solvent for lipid extraction and break yeast cells with glass beads in a mechanical bead mill. We repeat the extraction five times to ensure efficient lipid extraction.

Silica gel is the most wildly used stationary phase for chromatographic lipid separation. Impregnated with different substances, modified silica gel can be used to separate various lipid classes. Among the most popular modifications, silver nitrate impregnation has better potential for separating glycerolipids containing unsaturated fatty acyl chains (Dobson et al., 1995); boric acid is primarily used to separate the various isomers of DAG or PL (Ando et al., 2000). Like in other chromatographic techniques, TLC can be classified as “normal” or “reversed” phase based on the polarities of their mobile and stationary phases. Normal phase (polar stationary phase and non-polar mobile phase) chromatography is the standard method to separate lipids of interest that may have different polarities caused by a variation in their head groups (Fuchs et al., 2011). Typically, the size of gel particles for TLC is 10-50 μm. In contrast, the size of the high-performance thin-layer chromatography (HPTLC) gel particles is about 5 μm with narrow distributions (Fuchs et al., 2011), which results in higher separation quality and smaller sample quantity. It is noteworthy that bio-lipids are very complex molecules, and no single separation method is sufficient to separate all lipid species. With the stationary phase chosen, the separating efficiency of TLC or HPTLC mainly relies on the mobile phase. A 0.4% (NH₄)SO₄-impregnated silica gel plate together with chloroform-methanol-acetic acid-acetone-water (40:25:7:4:2 v/v/v/v/v) as the mobile phase can be used to separate phospholipids, lysophospholipids, and SM (sphingomyelin) (Wang and Gustafson, 1992). A chloroform-methanol-water mixture (35:15:2 or 50:40:10 v/v/v) is generally used for the separation of SM; a different proportion, chloroform-methanol-water mixture (65:25:4 v/v/v), can be used to separate phospholipids (Knittelfelder and Kohlwein, 2017a and 2017b). A mobile phase consisting of hexane-diethyl ether-acetic acid (70/30/1 v/v/v) is initially used to separate cholesterol isomers; it can also be used to separate neutral lipids such as TAGs, DAGs, and MAGs (monoacylglycerols). Alternatively, neutral lipids can be separated by TLC silica gel 60 plates and petroleum ether-diethyl ether-acetic acid (32:8:0.8 v/v/v/) mixture as the mobile phase (Knittelfelder and Kohlwein, 2017a and 2017b). Overall, it is a trial-and-error process to choose a proper stationary phase and mobile phase based on the characteristics of individual lipid species. In the present protocol, a silica gel 60 plate is used as the stationary phase, and a hexane-diethyl ether-acetic acid (70/30/1 v/v/v) mixture and a chloroform-methanol-water mixture (65:25:4 v/v/v) are used as the mobile phase to separate neutral lipids and phospholipids, respectively. Note that the mobile phase for chromatographic lipid separation is not to be confused with the solvent system for lipid extraction, which is discussed in the preceding paragraph.

After separation, the resulting positions of different lipid species on TLC plates need to be visualized, usually by color reactions with chemical spray reagents. An iodine vapor bath is one of the most widely used methods. The brown iodine color will disappear spontaneously once the plates are removed from the iodine vapor (Palumbo and Zullo, 1987). It has been reported that iodine can be difficult to remove from highly unsaturated lipids, but in our experience, the brown color fades away too quickly before follow-up steps can be properly performed. Alternative spray reagents include 2,7-dichlorofluorescein, rhodamine 6 G, and primuline. Together with iodine, they belong to non-destructive reagents, with the modification of lipid structures being reversible (Fuchs et al., 2011). 0.2% amino black 10 B in 1 M NaCl and 3.2% H₂SO₄, 0.5% MnCl₂ in 50% ethanol are two destructive reagents with high sensitivity (Fuchs et al., 2011 and 2015; Knittelfelder and Kohlwein, 2017a and 2017b). In this protocol, we use Coomassie Brilliant Blue R-250, a non-destructive reagent first used to stain lipids in 1984 (Nakamura and Handa, 1984). In our experience, the detection sensitivity and durability of Coomassie Brilliant Blue R-250 are better than those of iodine vapor. Conveniently, the staining steps only take 30 min.