Convenient and rapid removal of detergent from glycolipids in detergent-resistant membrane microdomains.

Although detergents are often essential in protocols, they are usually incompatible with further biochemical analysis. There are several methods for detergent removal, but the procedures are complicated or suffer from sample loss. Here, we describe a convenient and rapid method for detergent removal from sialic acid-containing glycosphingolipids (gangliosides) and neutral glycolipids in detergent-resistant membrane (DRM) microdomain. It is based on selective detergent extraction, in which the sample is dried on a glass tube, followed by washing with organic solvent. We investigated 18 organic solvents and used high performance thin-layer chromatography (HPTLC) and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS) to confirm that dichloroethane (DCE) was the most suitable solvent and completely removed the nonionic detergent Triton X-100. Furthermore, DCE extraction effectively removed interference caused by other nonionic, zwitterionic, or ionic detergents in MALDI-QIT-TOF MS analysis.

The samples were centrifuged at 39,000 rpm for 18 h in an SW41 rotor (Beckman Instruments, Palo Alto, CA); 1 ml fractions were collected from the top, desalted by a Sep-Pak C18 cartridge, and analyzed by high performance thin-layer chromatography (HPTLC) and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-fl ight mass spectrometry (MALDI-QIT-TOF MS). All steps were carried out at 4°C.

Classical preparative column chromatography
DEAE A-25 sephadex, Iatrobeads, and Florisil column beads were packed into a standard glass Pasteur pipette (60 mm × 6 mm i.d.). To confi rm the detergent removal ratio, GM3 (4 g) and Triton X-100 (4 mg) mixtures were applied and washed with each solvent system as described previously ( 19 ).

Detergent extraction with organic solvent
To confi rm the detergent extraction ability from the ganglioside of the organic solvent, GM3 (4 g) and Triton X-100 (4 mg for MS, 30 g for HPTLC) were mixed and dried in Pyrex glass tubes. The GM3-Triton X-100 mixture was washed three times with 2 ml of various organic solvents. The washing fractions were combined and dried by N 2 fl ow, and the washing and residue fractions were applied to HPTLC or MALDI-QIT-TOF MS, respectively. The fractions of 3T3-L1 preadipocyte cells after the sucrose gradient and desalting by the Sep-Pak C18 cartridge were washed three times with 2 ml of DCE. The residues were analyzed by MALDI-QIT-TOF MS.

Cell line and culture conditions
Murine 3T3-L1 preadipocytes were cultured and maintained as described previously ( 18 ). Briefl y, cells were seeded and grown in Dulbecco's Modifi ed Eagle's Medium (DMEM) supplemented with 10% calf serum and passaged when the culture reached 70% confl uence.

Sucrose gradient centrifugation
DRM microdomains were fractionated from 3T3-L1 preadipocytes as described previously ( 6 ). Briefl y, 3T3-L1 preadipocytes were washed with phosphate buffered saline (PBS) and lysed in 2 ml of TNE buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM ethylenediamine tetra-acetic acid) containing protease inhibitors, 2 mM Na 3 VO 4 , and 0.08% Triton X-100. Lysates were centrifuged for 5 min at 1,300 g to remove nuclei and large cellular debris, and the supernatants were diluted with equal volumes of 85% (w/v) sucrose in TNE buffer. The diluted lysates in an ultracentrifuge tube were overlaid with 4 ml of 30% sucrose (w/v) in TNE buffer, followed by 4 ml of 5% sucrose (w/v) in TNE buffer. the cholesterol and glycosphingolipids on the plate were visualized by spraying with orcinol/H 2 SO 4 reagent followed by heating.

MALDI-QIT-TOF MS/MS analysis of glycolipids
MALDI-QIT-TOF MS was performed on an AXIMA MALDI-QIT-TOF mass spectrometer (SHIMADZU, Kyoto, Japan) equipped with a 337 nm nitrogen laser. MS and MS n spectra were calibrated externally using a peptide calibration standard mixture containing bradykinin ([M+H] + 757.40) and human ACTH (fragments 18-39) ([M+H] + 2465.20) as 1 pmol/µl solutions. The matrix was 2,5-dihydroxybenzoic acid (DHB) at a concentration of 10 mg/ml in water. The gangliosides were dissolved in 2 l of C/M (1:1, v/v), and matrix solutions were mixed and placed on a target plate for crystallization. Crystallization was accelerated by a gentle stream of cold air.

Confi rmation of detergent interference for MALDI-QIT-TOF MS analysis of gangliosides
The presence of detergents is known to interfere with many analytical techniques, including mass spectrometry (14)(15)(16)(17)20 ). To determine the detection limit of Triton X-100 interference, various concentrations of Triton X-100 backing (Merck, Darmstadt, Germany). The HPTLC plates were developed with a solvent system of C/M/0.2% aqueous CaCl 2 (60:40:9, v/v/v). The plates were dried, and 0.001% primuline in acetone/H 2 O (8:2, v/v) was sprayed evenly onto the plate. The plate was dried and visualized by densitometry (Atto Densitograph, Tokyo, Japan). Identities of the stained lipids and Triton X-100 bands were ascertained by referring to standards. Finally,  GM3 were detected at m/z 1225.5 and 1253.5, and ions derived from NeuAc-Hex-dissociated ions were detected at m/z 750.6 and 778.7 ( Fig. 2A ). Triton X-100 was detected in positive ion mode by a characteristic ion pattern at 44 Da intervals ( Fig. 2B ). After purifi cation by Sep-Pak C18 cartridge, DEAE sephadex A-25, Iatrobeads, and Florisil column chromatography methods, the peaks derived from residual Triton X-100 were detected in all spectra ( Fig.  2C-F ). The spectrum obtained after purifi cation by the Sep-Pak C18 cartridge showed a similar pattern to the spectrum of the Triton X-100 standard ( Fig. 2B, C ). The spectra developed after purifi cation by other chromatography methods showed that the +32-shifted peaks derived from residual Triton X-100 and methanol adduct ions increased after purifi cation by other chromatography methods ( Fig. 2D-F ). These results indicated that classical column chromatography, except for Sep-Pak C18 cartridge, can remove almost all Triton X-100 from the GM3-Triton X-100 mixture but that the residual Triton X-100 is still too great for MALDI-QIT-TOF MS analysis.
(1 mg, 100 g, 10 g, 1 g, and 100 ng) were analyzed by MALDI-QIT-TOF MS in positive ion mode ( Fig. 1A -E ). In the MS spectra, the lower detection limit of Triton X-100 was 10 g ( Fig. 1C ). Furthermore, the GM3 (100 pmol)derived ions were detected in the presence of less than 10 g Triton X-100 (data not shown). These results indicated that Triton X-100 needs to be removed at a concentration range of 1-10 g for ganglioside analysis by MALDI-QIT-TOF MS.
between the oligosaccharide moiety of gangliosides and the hydroxy group on a glass surface are thought to form stronger bonds compared to the interactions between detergents and the hydroxy groups on a glass surface. Therefore, we attempted to establish a new method based on selective detergent extraction by washing absorbed ganglioside on a glass tube with organic solvents. In this study, we used 18 organic solvents ( Table 1 ). We confi rmed the detergent removal effi ciency and sample loss by HPTLC using the described method ( Fig. 3 ). Although

HPTLC analysis of detergent removal effi ciency after washing with various organic solvents
Various organic solvents are used in column chromatography to remove detergents, salt, and other contaminants, including chloroform, methanol, hexane, and DCE, that are absorbed onto the columns. Furthermore, ethyl acetate extraction or DCE extraction is used for detergent removal from peptides (14)(15)(16)(17). Gangliosides have several hydroxy groups in the oligosaccharide moiety, and therefore, the hydrogen bond interactions diisopropylether. Almost all the GM3 was retained on the glass tubes by washing with hexane, heptane, benzene, dichloromethane, or DCE. The nonpolar organic solvents, including hexane, cyclohexane, and heptane, which have low permittivity and low solubility to water, showed incomplete removal of Triton X-100 ( Fig. 3 , nos. 9-11). Therefore, DCE was the most suitable solvent, and it completely removed the nonionic detergent Triton X-100.

MALDI-QIT-TOF MS analysis of detergent removal effi ciency after washing with various organic solvents
Although there are neutral lipids as well as gangliosides in the DRM of biomaterials, NeuAc-dissociated ions were mainly detected in the MALDI-QIT TOF MS Triton X-100 was completely isolated from mixtures of GM3-Triton X-100 by washing with polar and bipolar organic solvents, almost all the GM3 tended to be lost from the glass tubes ( Fig. 3 , nos. 1-8). GM3 and Triton X-100 were removed together from the glass surface by washing with methanol, 1-butanol, pyridine, or tetrahydrofuran. GM3 remained on the glass tubes to some extent after washing with acetonitrile, acetone, or methyl acetate; GM3 loss was greater than 50% ( Fig. 3 , nos. 1-8). However, Triton X-100 was removed by washing with nonpolar organic solvents, and the ganglioside tended to be retained on the glass tubes ( Fig. 3 , nos. 9-18). Almost all the GM3 was removed from the glass surface by washing with diethyl ether, xylene, or chloroform, and equal quantities of GM3 were removed by cyclohexane, toluene, or rived from GM3 were detected in residues of acetone and DCE. In the lower phase of Svennerholm's partition, residues of methanol or hexane, only the peaks derived from Triton X-100 were detected. These results indicated that the removal effi ciencies of Triton X-100 by washing with acetone or DCE were high enough to use MALDI-QIT-TOF MS ( Fig. 4D, F ). However, in Svennerholm's partition, the effi ciency of washing with methanol or hexane was not high enough for MALDI-QIT-TOF MS analysis ( Fig. 4B ). Almost all the GM3 was removed from the glass surface by washing with acetone; therefore, DCE was confi rmed as the most suitable solvent for Triton X-100 removal by MALDI-QIT-TOF MS as well as HPTLC analysis. spectrum ( Fig. 2A ). To distinguish between the peaks derived from NeuAc-dissociated or neutral lipids, the residues were divided into gangliosides and neutral lipids by Svennerholm's partition and analyzed by HPTLC and MALDI-QIT-TOF MS ( Fig. 4A , no. 1, and B). Furthermore, we selected methanol, acetone, hexane, and DCE to analyze the removal effi ciency of Triton X-100 by HPTLC and MALDI-TOF MS ( Fig. 4A , nos. 2-5, and C-F). Using HPTLC, the Triton X-100 was distributed into the upper phase, and almost all the GM3 was distributed into the lower phase of the Svennerholm's partition ( Fig. 4A , no.1). Extraction with methanol, acetone, hexane, or DCE showed reproducible results for HPTLC ( Fig. 3 ). MALDI-QIT-TOF MS results showed that the peaks de- Half of the quantity was applied to HPTLC; the remainder was separated into gangliosides or other lipids via Svennerholm's partition, and the resulting lower phase was applied to MALDI-QIT-TOF MS for analysis ( Fig. 6 ). Using HPTLC, Triton X-100, cholesterol, and other simple lipids were completely removed by DCE washing ( Fig. 6A ). In the MS spectra, the peaks derived from GM3 were detected in fraction nos. 4-6 and 6-9 ( Fig.  6B-E ). Furthermore, the detailed structural analyses of the detected GM3 were confi rmed by MALDI-QIT-TOF MS n and LC-IT-TOF MS n analyses. The ceramide components of GM3 derived from 3T3-L1 preadipocytes were composed of d18:1-C16:0, C18:0, C20:0, C22:0, C22:1, C23:0, C24:0, and C24:1 (data not shown). These results indicated that DCE extraction was also useful for Triton X-100 removal of gangliosides derived from biological sources.

Recovery confi rmation of gangliosides and neutral glycolipids after extraction with DCE
To determine whether the DCE washing can be applied to detergent removal from other gangliosides and neutral glycolipids, we analyzed HPTLC of gangliosides or neutral glycolipids-Triton X-100 mixtures after washing with DCE. Although faint bands derived from ganglioside GD1a and GT1b were detected in the wash fraction, almost all gangliosides and neutral glycolipids were recovered in residue

HPTLC and MALDI-QIT-TOF MS analysis of crude extract in 3T3-L1 preadipocytes after fractionation by sucrose gradient
To confi rm the detergent removal effi ciency from biological material by DCE extraction, we extracted gangliosides in DRM from 3T3-L1 preadipocytes. The 3T3-L1 preadipocytes were lysed with lysis buffer containing 0.08% Triton X-100 and fractionated into 12 tubes using a sucrose gradient. These were desalted by the Sep-Pak C18 cartridge, and the presence of Triton X-100 and glycolipids was confi rmed by HPTLC and MALDI-QIT-TOF MS (see Experimental Procedures ). As shown in Fig. 5 , Triton X-100 was detected in the top HPTLC phase, and the concentrations gradually increased with increasing sucrose concentrations. Cholesterol and glycosphingolipids containing gangliosides were detected in fraction nos. 4 and 5 by HPTLC ( Fig. 5A ). MALDI-QIT-TOF MS spectra showed that the peaks derived from GM3 were not detected due to background peaks derived from Triton X-100 ( Fig. 5B-E ).

Confi rmation of detergent interference for ganglioside analysis by MALDI-QIT-TOF MS after fractionation using a sucrose gradient
Fractions of 3T3-L1 preadipocytes after fractionation using a sucrose gradient and desalting with the Sep-Pak C18 cartridge were washed three times with 2 ml of DCE. The residues were dissolved in 100 l of C:M (1:1, v/v). were developed as hardware ( 21,22 ). Fluorescence resonance energy transfer (FRET), fl uorescence recovery after photo bleaching (FRAP), single-particle tracking (SPT), fl uorescence correlation spectroscopy (FCS), point scan-FCS, and raster image correlation spectroscopy (RICS) were developed for fl uorescence microscopy applied to the dynamics of membrane molecules ( 21,22 ). Although these new approaches are powerful tools for the aforementioned research fi elds, unresolved problems still remain. For example, fl uorescence labeling of the ganglioside is challenging because of steric hindrance, and there are thousands of species in membrane lipids. Furthermore, it is well known that membrane lipids are mobile even after chemical fi xation ( 23 ). Therefore, other classical approaches, including biochemical isolation and analysis of molecules in the DRM, are still required for functional elucidation of gangliosides. Here, we described the successful application of detergent removal using DCE extraction in the analysis of gangliosides and neutral glycolipids in DRM. The established method does not require expensive instruments or machines and can effectively remove nonionic, ionic, or zwitterionic detergents using a simple procedure.

CONCLUSIONS
We presented results that suggest that DCE extraction is a convenient and rapid method for detergent removal fractions ( Fig. 7A ). However, the recovery of GM3 was gradually decreased with increasing Triton X-100 amount ( Fig.  7B ). These results indicated that DCE extraction was useful for Triton X-100 removal from other gangliosides and neutral glycolipids and that some ganglioside loss was observed in the presence of much higher amounts of Triton X-100.

Removal of other nonionic, zwitterionic, or ionic detergents by DCE extraction
We applied the established DCE extraction method to the removal other nonionic, zwitterionic, or ionic detergents. We mixed Brij 58/97, Nonidet P-40 (NP-40), CHAPS, taurocholic acid, and deoxycholic acid with GM3 and extracted their detergents with DCE. As shown in Fig. 8 , the DCE extraction effectively removed interference of other nonionic, zwitterionic, or ionic detergents in MALDI-QIT-TOF MS analysis. These results indicated that the DCE extraction can be applied not only to detergent removal from DRM fractions using a variety of nonionic detergents but also to other experiments, such as detergent removal from glycolipids after synthesis by glycosyltransferases.
Recently, to evaluate how membrane heterogeneity regulates receptor distributions or signal transductions, hardware and software of fl uorescence microscope imaging have been developed. Spectroscope-stimulated emission depletion microscopy (STED), photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and structured illumination microscopy (SIM) from glycolipids. Although some sample loss was observed in the presence of a much higher amount of Triton X-100, the peaks derived from glycolipids can be detected in the MS spectrum after DCE extraction. This method can also be used in broader detergent removal applications, such as glycolipid biosynthesis. In the future, this method should be refi ned and shown to be an automatic method.