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. Author manuscript; available in PMC: 2021 Jul 19.
Published in final edited form as: Methods Mol Biol. 2021;2187:27–35. doi: 10.1007/978-1-0716-0814-2_2

A detergent-free method for preparation of lipid rafts for the shotgun lipidomics study

Chao Qin 1, Meixia Pan 1, Xianlin Han 1,2,*
PMCID: PMC8287891  NIHMSID: NIHMS1716737  PMID: 32770499

Abstract

Lipid rafts are microdomains on plasma membrane that contain high levels of cholesterol and sphingolipids. Because of the detergent-resistant property of lipid rafts, lipid rafts isolated by methods that use detergents frequently yield different results. Artifacts can also be introduced through the use of detergents. These limitations could be overcome with a detergent-free method which eliminates possible artificial influences. Importantly, lipid rafts prepared with a detergent-free method is more compatible to mass spectrometric analysis since the ion suppression effect is largely reduced.

This chapter describes a detergent-free two-step method for preparation of lipid rafts. Firstly, a purified plasma membrane fraction is prepared from cells by sedimentation of the post-nuclear supernatant (PNS) in a Percoll gradient. Secondly, the as-prepared plasma membranes are sonicated to release lipid rafts which are further isolated by flotation in a continuous gradient of Optiprep solution. Then, we introduce a typical shotgun lipidomics workflow that can be used as a cost-effective and relatively high throughput method to determine the lipidomes of lipid rafts.

The method also makes an easy start for lipidomics studies.

Keywords: Detergent-free preparation, lipid rafts, mass spectrometry, plasma membrane, shotgun lipidomics

1. Introduction

Lipid rafts are small plasma membrane domains that contain high levels of cholesterol and sphingolipids [1]. These lipids present in the lipid rafts tend to have highly saturated fatty-acid side chains, and tend to be closely packed and well-ordered [2]. This physical property gives the signature ability of their insolubility in nonionic detergents [3].

Caveolae are considered to be a subset of lipid rafts, which exist as small plasma membrane invaginations. Caveolae are distinguished from flat lipid rafts by their structure, which are achieved by the presence of cholesterol-binding protein (i.e., caveolin) [4, 5]. However, they are usually viewed equivalently as the subtypes of lipid rafts and are studied together, except under specific circumstances [6, 7].

Lipid rafts on plasma membrane are involved in many cellular processes, for example, trafficking [8] and cell signaling [9, 10]. Including various “-omics” studies upon lipid rafts, researchers have made significant progresses on discovering both structural and functional mechanisms. Many studies have been done to determine both proteins and lipids within these small plasma membrane domains, for example, glycosylphosphatidylinositol (GPI) anchored proteins [11, 12], some transmembrane proteins such as G protein-coupled receptors [13], flotillin [14], and receptor tyrosine kinases [15], as well as some lipidomics studies [16].

Along with the history of studying lipid rafts, various methods have been developed for isolation of lipid rafts. The 1% Triton X-100 extraction method [17] was commonly used and then a wide variety of other detergents were used and reported to isolate lipid rafts, including less than 1% Triton X-100, NP-40, octylglucoside, CHAPS, Lubrol, and Brij 98 [18–20]. Even though these methods result in a substantial overlap of proteins and lipid species, they give rise to obvious differences in regards to the lipid rafts that can be isolated due to the detergent-insoluble property [18–20].

Some detergent-free methods have also been developed to obtain a more comprehensive result of lipid rafts. For example, Song et al. reported a method for preparing detergent-free lipid rafts using the carbonate step gradient method [21]. This method involves a whole cell lysis process in a sodium carbonate buffer (pH 11.0) followed by sonication of the lysate. Lipid rafts are then centrifuged on a sucrose gradient and the bands located at the 5% and 35% sucrose interface. This method is relatively straightforward. However, there are two major concerns in regards to the process. Firstly, the flotation through 35% sucrose is not perfect for low-density membranes. Secondly, this method will also make the intracellular membranes in the fraction of lipid rafts.

A more selective detergent-free procedure was developed by Smart et al. to prepare a better and closer to reality fraction of lipid rafts [22]. In this method, cells are homogenized in an isotonic sucrose buffer (pH 7.8), and a post-nuclear supernatant (PNS) is obtained. By combining the PNS with a self-forming Percoll gradient, a purified plasma membrane fraction is obtained through a sedimentation process. Then, the as-collected plasma membrane are sonicated to release lipid rafts (both flat rafts and caveolae). Finally, by flotation in a continuous Optiprep gradient in an isotonic solution, the lipid rafts were separated and collected.

This two-step method possesses several advantages. The first step has very high selectivity that plasma membrane is readily separated from the intracellular membrane fractions (such as Golgi, endoplasmic reticulum, and mitochondria), which eliminates the possible interference of plasma membrane from intracellular organelles [23]. The second step yields a close to reality lipid rafts for intact cells. Moreover, this fraction of lipid rafts can be further separated into flat rafts and caveolae [24]. This method produces a highly-purified result that closely reflects the actual composition of lipid rafts on plasma membrane of cells.

Since lipid rafts are rich in lipids, specifically cholesterol and sphingolipids, a lipidomics study is useful and informative for understanding the structure and function relationship. It is reasonable to take advantage of multidimensional mass spectrometry-based shotgun lipidomics (MDMS-SL), which possesses many advantages, to study lipidomics of lipid rafts [25–27].

This chapter describes the method for preparation of lipid rafts largely based on Smart’s method. The step-by-step workflow for isolation of lipid rafts is described and followed with a workflow from lipid extraction to data analysis of shotgun lipidomics for lipidomics analysis of lipid rafts.

2. Materials

Cells (~8 mg of total protein) are cultured in 100-mm dishes or T-75 flasks. All solutions are prepared using ultrapure water and analytical grade reagents. All solutions and materials should be ice-cold and/or stored at 4 °C before use.

2.1. Plasma Membrane

  1. Cell scrapers.

  2. Buffer A: 0.25 M Sucrose, 1 mM EDTA, and 20 mM Tris at pH 7.8.

  3. Conical tubes (1.5 mL) and a tabletop centrifuge.

  4. Dounce homogenizer (with a loose-fitting piston needle grinder).

  5. Needle (21g or higher).

  6. Percoll (Sigma-Aldrich). 30% Percoll diluted by buffer A is needed.

  7. Ultracentrifuge, 30 mL ultracentrifuge bottles, and a TI70 rotor (Beckman).

2.2. Lipid Rafts

  1. Optiprep (Sigma-Aldrich).

  2. Buffer A: 0.25 M Sucrose, 1 mM EDTA, and 20 mM Tris at pH 7.8.

  3. Buffer B: 0.25 M Sucrose, 6mM EDTA, and 120 mM Tris at pH 7.8.

  4. Buffer C: 50% Optiprep in Buffer B. Prepare the solution by diluting Optiprep with Buffer B.

  5. Sonicator with a 3-mm (diameter) sonication probe.

  6. Ultracentrifuge using a swinging bucket rotor SW41 (Beckman).

  7. A linear 20% to 10% Optiprep gradient (made by diluting Buffer C with Buffer A).

2.3. Shotgun Lipidomics

The total amount of sample should be around 0.1 mg protein. The reagents used should be HPLC or MS grade.

  1. Nano-electrospray ionization (ESI) source device and Chipsoft 8.3.1 software (TriVersa NanoMate, Advion Bioscience Ltd., Ithaca, NY, USA).

  2. Mass spectrometer (e.g., Thermo TSQ Quantiva™ Triple Quadrupole Mass Spectrometer, San Jose, CA, USA).

  3. 6 mL and 10 mL reusable culture tubes with PTFE lined cap.

  4. 5.75″ disposable borosilicate glass Pasteur pipets.

  5. Extraction solvent: chloroform/methanol (1/1, v/v) (Solvent A), 10 mM lithium chloride in H2O (Solvent B).

  6. Vortex mixer.

  7. Reagents: methanol (MeOH), chloroform (CHCl3), Millipore deionized water (H2O), isopropanol (IPA), and lithium hydroxide (LiOH).

  8. Lipid internal standards (see Note 7):
    1. 1,2-Dimyristoleoyl-sn-glycero-3-phosphocholine (di14:1 PC).
    2. Cholesterol-d7 (d7-CHL).
    3. 1,2-Dipentadecanoyl-sn-glycero-3-phosphoglycerol (sodium salt) (di15:0 PG).
    4. 1,2-Dimyristoyl-sn-glycero-3-phospho-L-serine (sodium salt) (di14:0 PS).
    5. 1,2-Dimyristoyl-sn-glycero-3-phosphate (sodium salt) (di14:0 PA).
    6. N-Lauroryl sphingomyelin (N12:0 SM).
    7. N-Heptadecanoyl ceramide (N17:0 Cer).
    8. 1-Heptadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (17:0 lysoPC).
    9. 1-Myristoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (17:0 lysoPE).

3. Methods

All procedures should be carried out at 4 °C.

3.1. Plasma Membrane

  1. Wash cells twice with 5 mL of Buffer A (100-mm dishes or T-75 flasks).

  2. Collect the cells by scraping with 3 mL Buffer A and pipette to a conical tube (see Note 1).

  3. Pellet the as-collected cells (7-8 mg of total protein) by centrifuging in a tabletop centrifuge for 5 min at 1000 × g (2200 rpm).

  4. Resuspend the cells in 1 mL of Buffer A. Use the Dounce homogenizer to homogenize the cells with 20 strokes, and pass through a 21 g (or higher) needle 20 times. (see Note 2).

  5. Transfer the homogenate into a 1.5 mL conical tube and centrifuge for 10 min at 1000 × g (2200 rpm).

  6. Remove the supernatant and store it on ice as the post-nuclear supernatant (PNS).

  7. Resuspend the cell pellet in 1 mL of Buffer A and homogenize then centrifuge as aforementioned.

  8. Combine the supernatant with the former and form a final PNS (about 4 mg of total protein).

  9. Carefully add 2 mL of PNS on the top of 23 mL of 30% Percoll in Buffer A (see Note 3).

  10. Centrifuge for 30 min at 84,000 × g in the TI70 rotor.

  11. Collect the plasma membrane fraction with a pipette. The plasma membrane fraction should be a visible band at around 5.7 cm from the bottom of the centrifuge bottle (see Note 4).

3.2. Lipid Rafts

  1. Adjust the volume of the as-prepared plasma membrane fraction to 2 mL by adding Buffer A in a centrifuge tube.

  2. Sonicate the solution for 15 seconds by placing the 3-mm sonication probe in the middle of the solution. Repeat this process 6 times with a 1-minute rest in between two rounds (see Note 5).

  3. Mix the 2 mL sonicate with 1.84 mL of Buffer C and 0.16 mL of Buffer A (Optiprep concentration 23%) at the bottom of a 12-mL centrifuge tube.

  4. Pour 8 mL of a 20% to 10% Optiprep gradient on top of the sample (in the same 12 mL tube)

  5. Centrifuge at 52,000 × g for 90 minutes at 4 °C in ultracentrifuge using a swinging bucket rotor SW41 (21,000 rpm, Beckman)

  6. Collect the top 5 mL of the gradient (fractions 1-7) and mix it with 4 mL of Buffer C in a centrifuge tube.

  7. Overlay 2 mL of 5% Optiprep (made by diluting Buffer C with Buffer A) on top of the collected gradient.

  8. Centrifuge at 52,000 × g for 90 minutes at 4°C in ultracentrifuge using a swinging bucket rotor SW41 (21,000 rpm, Beckman).

  9. Collect the opaque band that present in the 5% Optiprep about 4-5 mm above the interface (see Note 6).

3.3. Lipid Extraction

  1. Make each internal standard into a stock solution and then select the internal standards needed to make a pre-mixture (e.g., di14:1 PC, d7 CHL, and N12:0 SM) (see Note 8). The amount of each single lipid species in the pre-mixture should be based on the abundance of the corresponding lipid class in the samples. The molecular species of internal standards are selected because they represent <0.1% of the endogenous cellular lipid mass levels as predetermined by ESI-MS analysis.

  2. In a 6-mL glass tube, add the isolated lipid rafts (~0.1 mg protein or higher), internal standard pre-mixture, 4 mL of Solvent A, and 2 mL of Solvent B. (see Note 9).

  3. Cap the tube and vortex for 20 sec.

  4. Centrifuge at 4000 × g at 4 °C for 10 min.

  5. Use a glass pipette to harvest the bottom layer (chloroform) and save it in a new glass tube.

  6. To the previous glass tube, add 2 mL of chloroform and repeat Steps 3 and 4.

  7. Use the glass pipette to harvest the bottom layer and combine it with the previous round. (see Note 10).

  8. Evaporate the solvent under a nitrogen stream to dryness and resuspend the residual with chloroform/methanol (1:1, v/v). The solution is ready for mass spectrometric analysis (see Note 11).

3.4. Mass Spectrometric Analysis

  1. Dilute the as-prepared lipid extraction solution to <50 μM of total lipids with CHCl3/MeOH/IPA (1/2/4/, v/v/v) or CHCl3/MeOH/IPA (1/2/4/, v/v/v) with 4% LiOH, in a Teflon-coated 96-well microplate (see Note 12).

  2. Set the ionization voltage of a nanospray ionization source at 1.3 kV in the positive-ion mode, −1.3 kV in the negative ion mode, and gas pressure at 0.55 psi.

  3. For mass spectrometric analysis, collect 2-min duration of signal averaging in the profile mode for each survey MS scan (see Note 13).

  4. For tandem mass spectrometric analysis, set collision gas pressure at 1.0 mTorr, adjust collision energy with the class of lipids, and collect a 3 to 5 min period of signal averaging in the profile mode for each tandem MS spectrum, including precursor-ion scan (PIS) and neutral-loss scan (NLS), which are sensitive and specific to the lipid class or the category of lipid classes of interest [30].

  5. Processing of MS analysis data including ion peak selection, data transferring, baseline correction, peak intensity comparison, and quantification is conducted by a self-programmed Microsoft Excel macros [30].

Acknowledgement

This work was partially supported by NIH/NIA (RF1 AG061872 and R56 AG061729), the institutional research funds from the University of Texas Health Science Center at San Antonio (UT Health SA), the Mass Spectrometry Core Facility at UT Health SA, and the Methodist Hospital Foundation.

4 Notes

1.

When using the cell scrapers to collect cells, carefully scrape through all the bottom surfaces of the T-75 flasks (or other kinds of cell culture containers). Make sure to harvest as much as possible without damaging the cells.

2.

Perform at a 4 °C environment. Other homogenizing equipment works as well. For example, a typical mechanical tissue grinder Dyna-mix (Fisher Scientific, Ottawa, ON).

3.

Be careful not to disturb the bottom solution. The PNS should be carefully laid on top of the 30% Percoll in Buffer A.

4.

The band should be at about the middle of the centrifuge tube and it should be a visible band. The total protein amount is ~0.6 mg for this band. It can be easily collected by a Pasteur pipette or any kind of standard lab pipette. If an analysis of the Percoll gradients indicates a large degree of contamination of plasma membrane with other intracellular membranes, the position of the plasma membrane can be moved further into the gradient by increasing the pH of the Percoll gradient to 9.6.

5.

Sonication can generate a significant amount of heat in the solvent. The centrifuge tube should be kept in an ice bath all the time. The 1-minute break should cool down the solvent before the next round of sonication (In the original method: total power, 50 J/W per second each time, Vibra Cell sonicator, model VC60S, Sonics & Materials, Danbury, CT [22]).

6.

This should be a distinct opaque band where the designated lipid rafts (and caveolae) are. Typically, there is 0.01 mg of protein in this band. Moreover, this lipid raft fraction can be further separated to flat rafts and caveolae if needed [24].

7.

All of lipid internal standards are purchased from Avanti Polar Lipids, Inc., except if otherwise noted. Other internal standards can be added into the internal standard mixture if the lipid of interest is not listed here.

8.

Prepare the stock solution with chloroform/methanol (1/1, v/v) or with methanol. The concentration of the stock solution could be approximately 1 mg/mL.

9.

In the extraction system, keep the chloroform/methanol/water = 1:1:1 (v/v/v).

10.

The lipids should be extracted and exist in the chloroform layer. The top layer (methanol and water mixture) can be discarded.

11.

The resuspended lipids in chloroform/methanol (1:1, v/v) can be stored in −20 °C until MS analysis.

12.

The total lipid concentration of a lipid extract can be estimated through the dry weight or on the basis of the lipid analysis results from previous experiments [28]. This knowledge is useful for estimation of the concentrations of total lipids from different lipid extracts to prevent lipid aggregation during analysis through mass spectrometry. The 2-6% lithium hydroxide is made from 80-time dilution of a saturated methanol solution.

13.

For the triple-quadrupole mass spectrometer, the first and third quadrupoles are used as an independent mass analyzer with a mass resolution of 0.7 Th, and the second quadrupole serves as a collision cell for tandem mass spectrometry [29].

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