Thin-Layer Chromatography of Phospholipids

Abstract

Thin-layer chromatography (TLC) is a technique that has been routinely used for the separation and identification of lipids. Here we describe an optimized protocol for the steady state labeling, separation, and quantification of yeast phospholipids using 1-D TLC.

1 Introduction

Phospholipids constitute a major component of cell membranes, where they play essential roles in vital cellular processes. They are derived from the precursor phosphatidic acid (PA) and include phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylglycerol (PG), cardiolipin (CL), and phosphatidylinositol (PI), which is further phosphorylated to form a variety of phosphoinositides.

Increasingly, diseases are found to be associated with alterations in composition and levels of phospholipids. For example, Barth syndrome results from defects in cardiolipin levels and species [1, 2]; Charcot-Marie-Tooth disease is linked to altered levels of the phosphoinositide PI3,5P₂ [3]; and Alzheimer’s disease and cystic fibrosis are both associated with altered levels of PE and PC [4, 5]. The finding that altered phospholipids are implicated in many diseases necessitates quick and relatively easy methods for the detection and quantification of specific phospholipids.

Thin-layer chromatography (TLC) was first described in 1938 [6]. Since then, TLC has been used for the routine separation and identification of lipids. Its use offers several advantages in terms of cost, convenience, and simplicity. The method depends on the use of: (1) A stationary phase, most commonly silica gel, with particle size ranging from 10 to 50 μm for standard TLC, and 5 μm for high-performance TLC (HPTLC), and (2) a mobile phase, generally consisting of a combination of organic solvents. The composition varies in different protocols.

Phospholipids can be separated by 1-D or 2-D chromatography. 2-D TLC results in more sensitive separation; however, the number of samples is limited to one per plate. While 1-D TLC is somewhat less sensitive, multiple samples can be analyzed simultaneously on a single plate and in a shorter time than in 2-D TLC.

Here, we demonstrate the use of an optimized protocol for 1-D TLC for the detection and quantification of phospholipids from Saccharomyces cerevisiae.

2 Materials

2.1 Growth and Labeling of Phospholipids

1. Culture media: (a) Complex medium (yeast peptone dextrose, YPD): 1 % (w/v) yeast extract, 2 % (w/v) bactopeptone, 2 % (w/v) glucose, and (2 % (w/v) agar for plates only). (b) Synthetic medium (SM): To prepare 1 L of SM, use 0.69 g of vitamin-free yeast base mix, 2.01 g of ammonium sulfate, 20 g of glucose, vitamins, inositol, and amino acids as needed. The vitamin-free yeast base mix is based on the Difco recipe and contains the following: potassium phosphate (monobasic): 0.5 g; magnesium sulfate (anhydrous): 0.25 g; sodium chloride: 0.05 g; calcium chloride: 0.05 g; boric acid: 0.25 mg; copper sulfate (cupric sulfate anhydrous) 0.02 mg; potassium iodide: 0.05 mg; ferric chloride: 0.1 mg; manganese sulfate (monohydrate): 0.2 mg; sodium molybdate (molybdic acid, sodium salt): 0.1 mg; zinc sulfate: 0.2 mg.

2. Zymolyase 20-T (see ^{Note 1}).

3. Zymolyase buffer (50 mM Tris–SO₄ (pH 7.5), 1.2 M glycerol, 100 mM sodium thioglycolate): Weigh 0.6 g Tris base and transfer to a beaker. Add 60 mL dH₂O. Add 22.10 g of 50 % glycerol and 1.14 g of sodium thioglycolate. Mix and adjust pH to 7.5 using 1 N H₂SO₄. Make up to 100 mL with dH₂O. Store at 4 °C (see ^{Note 2}).

4. [³²P_i] orthophosphate: 10 μCi/mL for each sample used for steady state labeling (see ^{Note 3}).

2.2 Extraction

1. Extraction solvent: Chloroform/methanol 2:1 (v/v).

2. Microcentrifuge.

3. Agitator/Stirrer.

2.3 Chromatography

1. TLC plates (Whatman LK5 Silica gel 150 Å).

2. Prewash solution: Chloroform/methanol 1:1 (v/v). Prepare 150 mL of prewash solution by mixing 75 mL of chloroform with 75 mL of methanol.

3. Boric acid solution 1.8 % (w/v in 100 % ethanol): Weigh 9.0 g boric acid and add to 500 mL ethanol (see ^{Note 4}).

4. TLC tank lined with filter paper: Cut an appropriate size of filter paper to completely line all sides of the tank.

5. Developing solution (mobile phase): Chloroform/ethanol/water/tri-ethylamine 30:35:7:35 (v/v/v/v). Make a total of about 160 mL. Mix together 45 mL chloroform, 52.5 mL ethanol, 10.5 mL water, and 52.5 mL tri-ethylamine. Keep in an air-tight container until ready to use.

3 Methods

3.1 Steady State Labeling of Phospholipids

1. Streak the desired yeast strain on a fresh YPD plate and grow until colonies are about 2 mm in diameter.

2. Prepare an overnight culture by picking one colony and transferring it to 5 mL of the desired medium (YPD or SM).

3. Inoculate each of three tubes containing 2 mL of the desired media with cells from the overnight culture to a starting A₅₅₀ of 0.05 (see ^{Note 5}).

4. To two tubes only, add 20 μCi ³²P_i; (10 μCi/mL culture). Use the third tube (with no ³²Pi added) to monitor the absorbance.

5. To achieve steady state labeling, incubate all three tubes in a rotary shaker at the desired temperature for 5–6 generations (see ^{Note 6}).

6. Harvest the cells by centrifuging at 5,500 × g for 5 min, and wash once with 1 mL distilled water. Remove the supernatant. In this and all subsequent steps, discard radioactive materials properly.

7. To facilitate the extraction of phospholipids, digest the cell wall by adding 1 mL zymolyase solution to the cell pellets (see ^{Note 7}). Mix and keep on stirrer for 15 min at room temperature.

8. Harvest the spheroplasts (the cells without the cell wall) by centrifuging at 5,500 × g for 5 min. Discard the supernatant, which contains the zymolyase and all debris.

3.2 Extraction of the Phospholipids

1. To lyse the spheroplasts and extract the phospholipids, add 250 μL of chloroform/methanol 2:1 (v/v), resuspend and incubate on stirrer at room temperature for 45 min to 1 h.

2. To separate the organic phase containing the phospholipids from the aqueous phase containing non-lipid cellular material, add 50 μL of water, vortex, and centrifuge at 1,000 × g for 1 min.

3. Using a pipette with a fine tip, or a Hamilton syringe, carefully remove the lower (organic) phase and transfer to a microcentrifuge tube. The sample can be loaded directly onto the TLC plate or concentrated under a gentle stream of nitrogen at room temperature (see ^{Note 8}).

3.3 Preparation of the TLC Plates (See Fig. 1)

Fig. 1.

TLC plate preparation for loading several samples onto the same plate. (a) The pre-adsorbent zone is divided into several zones using a fine tip pencil. (b) The silica from a width of about 0.5 cm is gently scraped off. (c) The sample is applied as a fine streak at about 1.5 cm from the base of the plate

1. At least 2 h before starting, line the TLC tank with filter paper and equilibrate for at least 2 h with chloroform/ethanol/water/tri-ethylamine 30:35:7:35 (v/v/v/v), keeping the tank covered with a lid.

2. To remove impurities from the TLC plates, pre-develop plates with chloroform/methanol 1:1 (v/v) in a clean tank and air-dry in a fume hood.

3. Prepare a 1.8 % boric acid solution in 100 % ethanol. Pour into a glass tray that can easily accommodate the TLC plate. Fill the glass tray to a depth of about 2 cm. Dip the TLC plate in the solution for 2 min, making sure that it is uniformly impregnated with boric acid (see ^{Note 9}). Air-dry the plate for 15 min, then activate it at 100 °C for 15 min. Let it cool.

4. Loading lanes: (a) Using a fine point pencil, divide the pre-adsorbent zone (the lower thick area of the plate) into 2 cm fractions. (b) Scrape a width of ~0.5 cm using a flat spatula or a razor blade. (c) Using a fine tip, spot or streak 20–30 μL of the phospholipid sample onto the pre-adsorbent area at about 1.5 cm from the base. Allow sample to dry (see ^{Note 10}).

5. Gently lower the loaded TLC plate into the pre-equilibrated chromatography tank, making sure the samples are above the surface of the developing solvent (see ^{Note 11}).

6. Place the lid on the tank and allow the solvent (the mobile phase) to ascend to about 1 cm from the top of the plate (see ^{Note 12}).

7. Remove the plate from the tank, mark the level of the solvent front by making a small etch on both sides of the plate, and air-dry completely in a fume hood.

8. Cover the TLC plate with plastic wrap, avoiding creases, and place the plate in an imaging cassette, face up. Place the phosphorimaging film on top, lock the cassette, and expose for 15–60 min.

9. Remove the film from the cassette and scan it using a Storm/Typhoon phosphorimager system. Separation of phospholipids from wild-type and crd1 mutant cells, which lack CL, and accumulate PG, is shown in Fig. 2.

10. Quantify intensity of the bands using available software (e.g., ImageQuant).

11. To determine the rate of synthesis of phospholipids, a slightly modified procedure is followed (see ^{Note 13}).

Fig. 2.

Separation of phospholipids by 1-D TLC. Cells were grown in YPD, labeled with ³²P_i, washed and digested with zymolyase. Lipids were extracted from spheroplasts with chloroform/methanol 2:1 (v/v), and then analyzed by 1-D TLC. Phospholipids from wild-type cells (A), and from crd1 mutant cells (B), which lack CL are depicted. Individual lipid species were identified by the comigration of standards. PDME phosphatidyldimethylethanolamine, CL cardiolipin, PA phosphatidic acid, PE phosphatidylethanolamine, PG phosphatidylglycerine, PS phosphatidylserine, PI phosphatidylinositol, PC phosphatidylcholine