Structural Characterization of NeutralGlycosphingolipids by Thin-Layer ChromatographyCoupled to Matrix-Assisted Laser Desorption/Ionization Quadrupole Ion Trap Time-of-FlightMS/MS

Kyoko Nakamura,† Yusuke Suzuki,† Naoko Goto-Inoue,† Chikako Yoshida-Noro,‡ and Akemi Suzuki*,†

Sphingolipid Expression Laboratory, Supra-Biomolecular System Research Group, Frontier Research System, Institute ofPhysical and Chemical Research (RIKEN), Saitama, Japan, and Advanced Research Institute for the Sciences andHumanities, Nihon University, Tokyo, Japan

Rapid and convenient structural analysis of neutral gly-cosphingolipids (GSLs) was achieved by direct couplingof thin-layer chromatography (TLC) to matrix-assistedlaser desorption/ionization quadrupole ion trap time-of-flight (MALDI-QIT-TOF) MS/MS. Positions of unstainedGSL spots on developed TLC plates were determined bycomparison to orcinol-stained references. A matrix solu-tion of 2,5-dihydroxybenzoic acid (DHB) in acetonitrile/water (1:1 v/v) was then added directly to the unstainedGSL spots, and the GSLs were directly analyzed byMALDI-QIT-TOF MS. The acetonitrile/water DHB solu-tion proved to be suitable for MS/MS structural analysiswith high sensitivity. MS/MS and MS/MS/MS of GSLsyielded simple and informative spectra that revealed theceramide and long-chain base structures, as well as thesugar sequences. Hydroxy fatty acids in ceramide pro-vided characteristic MS/MS fragment ions. GSLs werestained with primuline, a nondestructive dye, after TLCdevelopment, and successfully analyzed by MALDI-QIT-TOF MS/MS with high sensitivity. Immunostaining ofGSLs after TLC development is a powerful method forcharacterizing antibody-specific sugars, but not ceram-ides. By coupling TLC-immunostaining of GSLs to MALDI-QIT-TOF MS/MS, we were able to identify both the sugarand the ceramide structures. The detection limits of asialoGM1 (Galâ1-3GalNAcâ1-4Galâ1-4Glcâ1-1′Cer) were25 and 50 pmol in primuline staining and immunostain-ing, respectively.

Glycosphingolipids (GSLs) are amphipathic molecules consist-ing of a hydrophilic sugar chain and a hydrophobic ceramidemoiety. They are usually located in the outer leaflet of the cellmembrane, in which they are anchored by the ceramide moiety.The sugar chains are directed toward the cell exterior and haveenormous structural diversity. GSLs act as cell-surface recogni-

tion molecules in various biological processes, such as cell-celland cell-pathogen recognition.1,2 Studies on glycosyltransferasegene-targeted mice have clearly demonstrated that GSLs havephysiological functions.3-5 Recently, a new role for cell-surfaceGSLs has been identified, in which they act as a component of a“raft” microdomain.6 Although definitive proof of the existence ofrafts in physiological membranes has not yet been obtained, muchevidence implicates rafts in many cellular processes, includingsignaling, membrane trafficking, cytoskeletal organization, andpathogen entry. The ceramide structure is important in raftformation. The chain length, unsaturation, and hydroxylation ofthe fatty acids and long-chain bases influence raft formation andfunction.7 Hence, the detailed structures of not only the sugarchains but also the ceramides of GSLs are an important focus ofresearch.

Thin-layer chromatography (TLC) is a popular and convenienttechnique for separation and characterization of GSLs, and itrequires only small amounts of GSLs prepared from biologicalsource materials. However, TLC characterization of individualGSLs does not yield unambiguous structural information, becauseGSLs containing the same sugar may migrate at different posi-tions, owing to differences in their ceramide structures. TLC-immunostaining using sugar-specific recognition molecules, suchas antibodies, lectins, and bacterial toxins, is a powerful tool foridentifying parts of the sugar structures.8,9 On the other hand,the introduction of a technique in which TLC is directly coupledto mass spectrometry (TLC-MS) has greatly increased the utility

* Corresponding author. Tel: +81-48-467-9615. Fax: +81-48-462-4692. E-mail:aksuzuki@ riken. jp.

† Institute of Physical and Chemical Research (RIKEN).‡ Nihon University.

(1) Hakomori, S.; Igarashi, Y. Adv. Lipid Res. 1993, 25, 147-162.(2) Karlsson, K. A. Annu. Rev. Biochem. 1989, 58, 309-350.(3) Zhao, J. F. K.; Fukumoto, S.; Okada, M.; Furugen, R.; Miyazaki, H.;

Takamiya, K.; Aizawa, S.; Shiku, H.; Matsuyama, T.; Furukawa, K. J. Biol.Chem. 1999, 274, 13744-13747.

(4) Okada, M.; Itoh, M.; Haraguchi, M.; Okajima, T.; Inoue, M.; Oishi, H.;Matsuda, Y.; Iwamoto, T.; Kawano, T.; Fukumoto, S.; Miyazaki, H.;Furukawa, K.; Aizawa, S.; Furukawa, K. J. Biol. Chem. 2002, 277, 1633-1636.

(5) Yamashita, T.; Hashiramoto, A.; Haluzik, M.; Mizukami, H,; Beck, S.; Norton,A,; Kono, M,; Tsuji, S.; Daniotti, J. L.; Werth, N.; Sandhoff, R.; Sandhoff, K.;Proia, R. L. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 3445-3449.

(6) Kasahara, K.; Sanai, Y. Biophys. Chem. 1999, 82, 121-127.(7) Psnasiewicz, M.; Domek, H.; Hoser, G.; Kawalec, M.; Pacuszka, T.

Biochemistry 2003, 42, 6608-6619.

Anal. Chem. 2006, 78, 5736-5743

5736 Analytical Chemistry, Vol. 78, No. 16, August 15, 2006 10.1021/ac0605501 CCC: $33.50 © 2006 American Chemical SocietyPublished on Web 07/19/2006

of TLC for structural characterization. TLC-MS provides both theGSL molecular mass and its structural information withoutrequiring its purification. Advances in MS techniques, such asfast atom bombardment MS,10 secondary ion MS,11 and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)MS,12-14 including technological developments in the transfer ofGSLs to PVDF membranes and MALDI plates,15-17 have alsoincreased the power of TLC-MS.

In TLC-MALDI MS, problems in obtaining good mass accuracyand resolution arise from the difficulties involved in desorbinganalytes from the rough surface of silica gel. Very recently,remarkable advances in MALDI MS ion trap instruments haveenabled TLC-MALDI MS analysis with high mass accuracy andsensitivity.18 Other instruments include an external ion trap, suchas a Fourier transform ion cyclotron resonance ion trap (FTI-CR),19,20 and an orthogonal extracting ion trap.21,22 Using high-sensitivity TLC-FTICR MS, Ivleva et al. successfully identified partsof ganglioside sugar sequences by analyzing its metastable ions.19

In another study, TLC coupled to orthogonal (o)-TOF MS wasused to determine molecular masses and partial sugar sequencesof gangliosides with high resolution; in particular, the attachmentpositions of the sialic acids were determined.21

Successful analytical strategies in which fragment ions ofinterest are selected as the precursor ions in TLC-MALDI MS/MS have not yet been reported. On the other hand, Meisen et al.have successfully applied electrospray ionization (ESI) MS/MSto gangliosides extracted from silica gel after primuline stainingand TLC-immunostaining.23 Using nanoESI-quadrupole TOF MS/MS, they successfully assigned a series of ions arising fromsequential fragmentation. They also reported structural assign-ments for Shiga toxin 1-stained GSLs using this system.24

However, this indirect coupling method also has its disadvantages;the extraction procedure is time-consuming and loss of samplematerial is possible. Glycosphingolipids containing the same sugar

and different ceramides migrate at different position of a spot ona TLC plate. TLC-MALDI-QIT MS/MS is a method directlycoupled to sample position; therefore, it is possible to characterizeGSLs migrating in defined small area within a single spot orclosely migrating spots.

In this report, we describe a method for the rapid andconvenient structural analysis of neutral GSLs in which TLC isdirectly coupled to MALDI-QIT-TOF MS/MS. MALDI MS withan external QIT eliminates the mass accuracy and resolutionproblems that would otherwise arise from the irregular surfaceof the silica gel plate. Furthermore, a MALDI-QIT system easilyperforms sequential MS/MS and MS/MS/MS analyses, providingprecise structural information on GSLs. Although heterogeneityin the ceramide and sugar moieties leads to complex MS spectra,MS/MS overcomes this problem. We were able to obtain fragmentions derived from long-chain bases only by MS/MS. Using 2,5-dihydroxybenzoic acid (DHB) as the matrix and acetonitrile/wateras the solvent, the amount of sample material required for thestructural characterization of GSLs is on the picomole scale.

EXPERIMENTAL SECTIONGSL Nomenclature. The nomenclature was derived from the

recommendations of the IUPAC-IUB Commission on Biochemi-cal Nomenclature25 and the system of Svennerholm.26 The GSLabbreviations are as follows: GlcCer, Glcâ1-1′Cer; LacCer,Galâ1-4Glcâ1-1′Cer; Gb3Cer, GalR1-4Galâ1-4Glcâ1-1′Cer;Gb4Cer, GalNAcâ1-3GalR1-4Galâ1-4Glcâ1-1′Cer; asialo GM1,Galâ1-3GalNAcâ1-4Galâ1-4Glcâ1-1′Cer; and fucosyl asialoGM1, FucR1-2Galâ1-3GalNAcâ1-4Galâ1-4Glcâ1-1′Cer.

Materials. The neutral GSLs for TLC and TLC-MALDI-QIT-TOF MS analyses were GlcCer, LacCer, Gb3Cer, and Gb4Cerpurified from hog erythrocytes. Asialo GM1 prepared from bovinebrain GM1 was purchased from Wako Pure Chemical Industries,Ltd. (Tokyo, Japan), and asialo GM1 containing a unique ceramidewith phytosphingosine and 2-hydroxy fatty acid was purified frommouse intestine in our laboratory.27 Neutral GSLs from mousetestis were prepared in our laboratory. The crude lipid extract oftestis was partially purified using Iatrobeads column chromatog-raphy (Iatron laboratories, Tokyo, Japan), and the partially purifiedfraction containing fucosyl asialo GM1 as a major GSL wassubjected to TLC-MALDI-QIT-TOF MS analysis.

Thin-Layer Chromatography. Neutral GSLs dissolved inchloroform/methanol (1:1, v/v) were applied as 3-5 mm spotsto silica gel-coated plates with aluminum backing (Merck, Darm-stadt, Germany). Plates were developed with a solvent system ofchloroform/methanol/water (65:35:8, v/v/v, solvent A). Whensamples on a TLC plate were to be directly analyzed by MALDI-QIT-TOF MS without staining, duplicate, side-by-side spots weremade for each sample, and the plate was cut into two pieces afterdevelopment. One piece was used as a reference for the positionsof the GSLs, which were detected with orcinol reagent, and theother piece was used for MS analysis. The positions of the GSLson the latter piece were determined by comparison with theorcinol-stained GSLs on the former piece and then marked with

(8) Hansson, G. C.; Karlsson, K. A.; Larson, G.; McKibbin, J. M.; Blaszczyk,M.; Herlyn, M.; Steplewski, Z.; Koprowski, H. J. Biol. Chem. 1983, 258,4091-4097.

(9) Muthing, J. In Glycoanalysis Protocols; Hounsell, E., Ed.; Humana PressInc.: Totowa, NJ, 1998; pp 183-195.

(10) Karlsson, K. A.; Lanne, B.; Pimlott, W.; Teneberg, S. Carbohydr. Res. 1991,221, 49-61.

(11) Kushi, Y.; Rokukawa, C.; Handa, S. Anal. Biochem. 1988, 175, 167-176.(12) Gusev, A. I.; Proctor, A.; Rabinovich, Y. I.; Hercules, D. M. Anal. Biochem.

1995, 67, 4565-4570(13) Gusev, A. I. Fresenius J. Anal. Chem. 2000, 366, 691-700.(14) Mowthorpe, S.; Clench, M. R.; Cricelius, A.; Richards, D. S.; Parr, V.; Tetler,

L. W. Rapid Commun. Mass Spectrom. 1999, 13, 264.(15) Guittard, J.; Hronowski, X. L.; Costello, C. E. Rapid Commun. Mass Spectrom.

1999, 13, 1838-1849.(16) Taki, T.; Ishikawa, D.; Handa, S.; Kasama, T. Anal. Biochem. 1995, 225,

24-27.(17) Mehl, J. T.; Hercules, D. M. Anal. Biochem. 2000, 72, 68-73.(18) Wilson, I. D. J. Chromatogr., A 1999, 856, 429-442.(19) Ivleva, V. B.; Elkin, Y. N.; Budnic, B. A.; Moyer, S. C.; O’Connor, P. B.;

Costello, C. E. Anal. Chem. 2004, 76, 6484-6491.(20) O’Connor, P. B.; Budnik, B. A.; Ivleva, V. B.; Kaur, P.; Moyer, S. C.; Pittman,

J. L.; Costello, C. E. J. Am. Soc. Mass Spectrom. 2004, 15, 128-132.(21) Dreisewerd, K.; Muthing, J.; Rohlfing, A.; Meisen, I.; Vukelic, Z.; Peter-

Katalinic, J.; Hillenkamp, F.; Berkenkamp, S. Anal. Chem. 2005, 77, 4098-4107.

(22) Ivleva, V. B.; Sapp, L. M.; O’Connor, P. B.; Costello, C. E. J. Am. Soc. MassSpectrom. 2005, 16, 1552-1560.

(23) Meisen, I.; Peter-Katalinic, J.; Muthing, J. Anal. Chem. 2004, 76, 2248-2255.

(24) Meisen, I.; Friedrich, A. W.; Karch, H.; Witting, U.; Peter-Katalinic, J.;Muthing, J. Rapid Commun. Mass Spectrom. 2005, 19, 3659-3665.

(25) IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCNB). Eur.J. Biochem. 1998, 257, 293-298.

(26) Svennerholm, L. J. Neurochem. 1963, 10, 613-623.(27) Umesaki, Y.; Suzuki, A.; Kasama, T.; Tohyama, K.; Mutai, M.; Yamakawa,

T. J. Biochem. 1981, 90, 1731-1738.

Analytical Chemistry, Vol. 78, No. 16, August 15, 2006 5737

a pencil. For primuline staining, developed plates were dried andthen sprayed with 0.01% primuline in acetone/water (4:1, v/v).The fluorescent spots were visualized using a Fujifilm LAS-1000imaging system (Tokyo, Japan) and marked with a pencil.

TLC-Immunostaining. TLC-immunostaining was performedas previously described with a minor modification.28 In brief,neutral GSLs were separated on a TLC plate with aluminumbacking by development with solvent A. The plate was cut intotwo pieces, one of which was sprayed with orcinol reagent todetermine the positions of the GSLs. The other piece, which wasused for immunostaining, was soaked in 0.1% poly(isobutylmethacrylate) in cyclohexane for 1 min, dried, and preincubatedwith 1% bovine serum albumin-phosphate-buffered saline (BSA-PBS) for 30 min. The plate was then incubated with anti-asialoGM1 antibody solution (Wako Pure Chemical Industries, Ltd.;diluted 1:2000 with 1% BSA-PBS) for 1.5 h. The plate was washedwith PBS and then incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody [F(ab′)2] solution (Amersham,Arlington Heights, IL; diluted 1:5000 with 1% BSA-PBS) for 1 h.The plate was washed again with PBS, and the GSL bands werevisualized by enhanced chemiluminescence with Super Signal(Pierce, Rockford, IL) using a Fujifilm LAS-1000 imaging system.For further analysis by MALDI-QIT-TOF MS, the plate waswashed with PBS and water and dried with cold air. The dry platewas dipped twice in chloroform for 1 min to remove the coatingpolymer and bound antibody.23 When the plate was dry, spotpositions were determined by reference to the detected LAS-1000chemiluminescence image and marked with a pencil.

Mass Spectrometry. An Axima-QIT mass spectrometerequipped with a nitrogen laser (337 nm, 3 ns pulse width)(Shimadzu, Kyoto, Japan) was used. The ion trap chamber wassupplied with two separate and independent gases; He gas withcontinuous flow was used for collisional cooling, and pulsed Argas was used for imposing collision-induced fragmentation.29 Thepressure in the ionization chamber was maintained at less than 6× 10-6 Torr. Precursor and fragment ions obtained by collision-induced dissociation (CID) were ejected from the ion trap andanalyzed by a reflectron TOF detector operated in positive ionmode. The mass spectra were assembled from 200 to 1000accumulations of the profile obtained by two laser shots using alaser power of 30-60 arbitrary units. Stronger laser powerincreased sensitivity of MS and provided fragment ions used asthe precursor ion for sequential MS/MS analysis. Externalcalibration of MS spectra was performed using angiotensin II ([M+ H]+, m/z 1046.5) and ACTH ([M + H]+, m/z 2465.2) on aMALDI sample plate rather than a TLC plate.

TLC plates for MS analysis were cut into small piecescontaining the analytes, and the pieces were attached to a MALDIsample plate with double-sided adhesive tape.19 Several smalldroplets of matrix solution (totaling 1-2 µL) were then depositedon the spot area using a microsyringe, with immediate drying witha cold air stream after each droplet. The matrix solution contained0.1 mg of DHB/µL in acetonitrile/water (1:1, v/v).

RESULTS AND DISCUSSIONSelection of a Matrix Solution for TLC-MALDI-QIT-TOF

MS. Many kinds of matrixes and solvents have been used in TLC-MALDI MS analysis. The selected matrix is a critical factor inobtaining useful spectra with high sensitivity, and the matrixsolvent is also important. In general, matrixes are dissolved involatile solvents at saturated concentrations to make matrix-analyte crystals and to minimize sample diffusion.19 DHB is oneof the best matrixes discovered thus far for analysis of neutralGSLs by TLC-MALDI MS, and a hydrophobic solvent (chloroform/methanol) has been successfully used as a matrix solvent.15 Whenwe attempted to analyze small amounts of GSL (e.g., 8 pmol ofasialo GM1), however, the formation of matrix clusters in thechloroform/methanol solution was a serious problem (Figure 1A,B).

Surprisingly, we found that hydrophilic solvents containingwater provided excellent MS spectra with the DHB matrix. Asshown in Figure 1C-F, sodiated molecular ions at m/z 1278 or1306 were clearly present in the MS spectra derived from asialoGM1 with d18:1 sphingosine and C18:0 fatty acid (d18:1-C18:0)or d18:1-C20:0, respectively. Fragment ions indicating LacCer(Y2) were observed at m/z 913 and 941 as well. A comparison ofthe spectra indicates that acetonitrile/water (1:1, v/v) yielded thebest spectra of asialo GM1 with good sensitivity. The detectionlimit was similar to that of orcinol staining, in which 5 pmol ofasialo GM1 was the approximate limit of detection (Figure 2A).Because acetonitrile/water is not volatile, the matrix solutionshould be deposited as several very small droplets on GSL spots.

(28) Nakamura, K.; Suzuki, H.; Hirabayashi, Y.; Suzuki, A. J. Biol. Chem. 1995,270, 3876-3881.

(29) Martin, R. L.; Brancia, F. L. Rapid Commun. Mass Spectrom. 2003, 17,1358-1365.

Figure 1. TLC-MALDI-QIT-TOF MS spectra of asialo GM1 obtainedwith DHB dissolved in different solvents. DHB was dissolved in (A)chloroform/methanol (1:1, v/v), (B) chloroform/methanol/water (30:60:8, v/v/v), (C) methanol/water (1:1, v/v), (D) acetone/water (4:1, v/v),(E) acetonitrile/water (1:1, v/v), or (F) acetonitrile/water (1:2, v/v).MALDI-QIT-TOF MS was performed using 8 pmol of bovine brainasialo GM1. The annotation of fragment ions is shown in Figure 4.

5738 Analytical Chemistry, Vol. 78, No. 16, August 15, 2006

TLC-MALDI-QIT-TOF MS/MS and MS/MS/MS Analysesof Unstained Asialo GM1 and Fucosyl Asialo GM1. TLC-MALDI-QIT-TOF MS and MS/MS spectra of asialo GM1 frombovine brain are shown in Figure 3. Two molecular ions are clearlydetected at m/z 1278.0 and 1306.1 in Figure 3A. A series of Y-typeions are also detected as three pairs of ions at m/z 1116.0 and1144.0 (Y3), 912.8 and 940.9 (Y2), and 750.8 and 778.8 (Y1),indicating the sequential loss of Gal, GalNAc, and Gal from thenonreducing end of asialo GM1. The fragment ions at m/z 912.8and 940.9, which indicate LacCer, were abundant. The fragmenta-tion annotations of asialo GM1 are shown in Figure 4 accordingto the nomenclature of Domon and Costello.30,31 These MS spectra

demonstrate that mass accuracy and resolution are not affectedby the irregular surface of the silica gel.

A comparison of mass spectra obtained by the TLC-MALDI-QIT-TOF MS method described herein and by MALDI-QIT-TOFMS using a MALDI sample plate revealed differences in the resultsfrom these two systems. All the ions from TLC-MALDI MS weredetected as monosodiated forms, whereas MALDI MS yieldedboth sodium and potassium adduct ions (data not shown). In TLC-infrared MALDI-o-TOF MS, GM3 ganglioside was detected asmono- and disodiated ions in positive ion mode.21 Therefore, theTLC-MALDI-QIT-TOF MS system has the advantage of yieldingsimpler spectra with monosodiated ions.

The MS/MS spectrum of asialo GM1, which is shown in Figure3B, was obtained by selecting the molecular ion at m/z 1278.0 asthe precursor ion. An abundant ion at m/z 912.9 (Y2) and an ionat m/z 750.8 (Y1) were detected. To obtain structural informationin a low-molecular-mass region, MS/MS analysis was sequentiallyperformed using LacCer ions at m/z 940.9 and 912.8 as precursorions and abundant GlcCer (Y1) ions were detected at m/z 778.8and 750.8, respectively (Figure 3C and D). The ceramide ion wasdetected at m/z 588.3 (Y0) in (D), and the ceramide minus H2Oions (Z0) are at m/z 598.4 in (C) and 570.5 in (D). Fragment ionsat m/z 304.9 (d1b or d1b′ in Figure 4) in both (C) and (D) indicatethe presence of 4-sphingenine (d18:1) as the long-chain base, aspreviously reported by Hsu et al.32 Although we did not observeany fragment ions from the fatty acid, the fatty acid molecular

(30) Domon, B.; Costello, C. E. Biochemistry 1988, 27, 1534-1543.(31) Domon, B.; Costello, C. E. Glycoconjugate J. 1988, 5, 397-409.

(32) Hsu, F.-F.; Turk, J.; Stewart, M. E.; Downing, D. T. J. Am. Soc. Mass Spectrom.2002, 13, 680-695.

Figure 2. Thin-layer chromatography of asialo GM1 and fucosylasialo GM1. (A) Lane 1 contains GlcCer, LacCer, Gb3Cer, and Gb4-Cer. Lanes 2-8 contain asialo GM1 from bovine brain: (lane 2) 500,(lane 3) 250, (lane 4) 100, (lane 5) 50, (lane 6) 25, (lane 7) 10, and(lane 8) 5 pmol. (B) Lane 1 contains GlcCer, LacCer, Gb3Cer, andGb4Cer. Lanes 2-5 contain neutral GSLs: (lane 2) bovine brain asialoGM1; (lane 3) mouse intestine asialo GM1; (lane 4) crude lipid extractfrom mouse testis; (lane 5) partially purified mouse testis lipid fractioncontaining fucosyl asialo GM1 (indicated by arrow). TLC plates weredeveloped with solvent A and detected with orcinol reagent, asdescribed in the Experimental Section. Asterisks indicate orcinol-negative bands.

Figure 3. TLC-MALDI-QIT-TOF MS/MS spectra of bovine brainasialo GM1. (A) MS spectrum; (B-D) MS/MS spectra obtained withthe precursor ions at (B) m/z 1278.0, (C) 940.9, and (D) 912.8. Theannotation of fragment ions is shown in Figure 4.

Figure 4. Molecular structure and fragmentation scheme of asialoGM1 and fucosyl asialo GM1. The annotation of fragment ions isaccording to the nomenclature of Domon and Costello30,31 and thatof sphingosine base the nomenclature of Hsu et al.32

Analytical Chemistry, Vol. 78, No. 16, August 15, 2006 5739

mass could be calculated from the masses of the ceramide andthe long-chain base. The abundant fragment ions at m/z 347 and365 detected in both (C) and (D) are characteristic ions assignedas Gal-Glc (B2) and Gal-Glc + H2O (C2), respectively, as shownin the bottom of Figure 4.

Asialo GM1 from mouse intestine contains a unique ceramideconsisting of phytosphingosine and 2-hydroxy fatty acids, aspreviously reported.27 Because its ceramide has two additionalhydroxy groups, mouse intestine asialo GM1 migrates more slowlyon a TLC plate than does asialo GM1 prepared from bovine brainGM1 (Figure 2B, lanes 2 and 3). In the MS spectrum of mouseintestine asialo GM1, which is shown in Figure 5A, a series ofions carrying different fatty acid residues were detected at m/z1268.0, 1368.1, 1382.1, 1394.1, and 1396.1. These ions correspondto asialo GM1 (phytosphingosine; t18:0) substituted with C16:0,hC22:0, hC23:0, hC24:1, and hC24:0 fatty acids, respectively. Aset of Y-series fragment ions arising from the sequential elimina-tion of Gal-GalNAc and Gal were also detected. The MS/MSspectrum obtained from selection of MS ion at m/z 1002.9 as aprecursor ion is shown in Figure 5A′. The abundant fragment ion

at m/z 840.8 is assigned as GlcCer (Y1). The fragment ionsdetected at m/z 346.9 and 364.9 are the same ions detected inthe spectrum of bovine brain asialo GM1 (Figure 3C, D) andcorrespond to B2 and C2 ions, respectively. Two characteristicfragment ions were clearly detected at m/z 664.3 and 502.1. The162.2 atomic mass unit (amu) difference between these ionssuggests the presence of a hexose. Further analysis, in which theMS/MS ion at m/z 664.3 was selected as the precursor ion forMS/MS/MS, confirmed that the fragment ion at m/z 502.1 arosefrom degradation of the ion at m/z 664.3 (data not shown). Thesetwo characteristic ions are assigned as O ions containing Gal-Glc (m/z 664.3) and Glc (m/z 502.1), produced by cleavage ofthe amide NH-CO bond (Figure 4). This characteristic cleavagewas previously reported by Ann and Adams as a result of CIDfragmentation of ceramide lithium adduct ions.33,34 Only the GSLscontaining 2-hydroxy fatty acids yielded these O ions in abun-dance, and GSLs containing nonhydroxy fatty acids yielded nodetectable O ions (Figure 3C, D). The abundant cleavage at theO position prevented detection of the ceramide fragment ion inFigure 5A′. When MS/MS/MS analysis was performed withselection of the MS/MS ion at m/z 840.8 as the precursor ion, Y0

and Z0 ions could be detected at m/z 679.3 and 661.3, respectively(Figure 5A′′). Only one O ion was detected at m/z 502.4. In Figure5A′, a fragment ion was detected at m/z 305.0 (d1b or d1b′); thision could be assigned as 4-sphingenine (d18:1) or as t18:0 minusH2O. Since the presence of phytosphingosine was confirmed byits two characteristic fragment ions at m/z 664.3 and 502.1, asdescribed above, the selected precursor ion for MS/MS/MS mustcontain only t18:0.

Fucosyl asialo GM1 with unique ceramide structures wasrecently reported by Sandhoff et al.,35 who purified GSLs frommouse testis and identified novel fucosylated GSLs containingpolyunsaturated and very long-chain fatty acids. These GSLs maybe essential for spermatogenesis and male fertility.35 We detectedfour neutral GSLs in the crude lipid extract of mouse testis, andtheir mobilities on a TLC plate were similar to those reported bySandhoff et al. (Figure 2B, lane 4). We obtained a partially purifiedfraction that contained fucosyl asialo GM1 as its dominant GSL(Figure 2B, lane 5). We subjected this fraction to TLC-MALDI-QIT-TOF MS and MS/MS, as shown in Figure 5B and B′. Notably,the quality of the MS spectra was not negatively affected by theabundant remaining impurities in the analyzed fraction. Twomolecular ions were observed at m/z 1570.3 and 1598.4 (Figure5B). On the basis of previously reported results,35 these ions areassigned as sodiated molecular ions of fucosyl asialo GM1 (d18:1) with hC28:5 and hC30:5 fatty acids. A series of Y-type ions werealso detected as three pairs of ions at m/z 1424.3 and 1452.3 (Y4),1059.0 and 1087.0 (Y2), and 896.8 and 924.9 (Y1), indicating thesequential loss of Fuc, Gal-GalNAc, and Gal from the nonreduc-ing end of the molecule. The structure and the fragmentationscheme are shown in Figure 4. MS/MS analysis with selectionof the ion at m/z 1087.0 as the precursor ion resulted in anabundant fragment ion (Y1) at m/z 924.9 (Figure 5B′). The

(33) Ann, Q.; Adams, J. Anal. Chem. 1993, 65, 7-13.(34) Levery, S. B.; Toledo, M. S.; Doong, R. L.; Straus, A. H.; Takahashi, H. K.

Rapid Commun. Mass Spectrom. 2000, 14, 551-563.(35) Sandhoff, R.; Geyer, R.; Jennemann, R.; Paret, C.; Kiss, E.; Yamashita, T.;

Gorgas, K.; Sijmonsma, T. P.; Iwamori, M.; Finaz, C.; Proia, R. L.; Wiegandt,H.; Grone, H.-J. J. Bioi. Chem. 2005, 29, 27310-27318.

Figure 5. TLC-MALDI-QIT-TOF MS/MS and MS/MS/MS spectraof mouse intestine asialo GM1 and MS/MS spectrum of mouse testisfucosyl asialo GM1. Spectra for asialo GM1 purified from mouseintestine are shown in (A) (A′), and (A′′). (A) MS spectrum; (A′) MS/MS spectrum obtained with the precursor ion at m/z 1002.9; (A′′) MS/MS/MS spectrum obtained with the second precursor ion at m/z 840.8.Spectra for fucosyl asialo GM1 purified from mouse testis are shownin (B) and (B′). (B) MS spectrum; (B′) MS/MS spectrum obtained withthe precursor ion at m/z 1087.0. Asterisks indicate the characteristicfragment ions produced by degradation of GSLs at the O position,as shown in Figure 4. The annotation of fragment ions is shown inFigure 4.

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fragment ions B2 and C2 were detected at m/z 346.9 and 364.9,respectively. Two characteristic fragment ions indicating thepresence of 2-hydroxy fatty acid were clearly detected at m/z 646.2and 484.1. These O ions are 18 amu smaller than those of asialoGM1 (m/z 664.3 and 502.1) in Figure 5A′. This 18-amu differencecorresponds to a hydroxy group derived from phytosphingosine(t18:0). Therefore, if GSL ceramides contain 2-hydroxy fatty acids,the structures of the long-chain bases can be determined by thedetection of these characteristic fragment ions. The fragment ionat m/z 305.0 (d1b or d1b′) indicates the presence of 4-sphingenine(d18:1) as a long-chain base.35

TLC-MALDI-QIT-TOF MS/MS Analyses of Primuline-Stained Asialo GM1. Primuline is a nondestructive fluorochromethat detects lipids and has been used to locate GSLs on a TLCplate prior to MS analysis.21,23 In the present study, the detectionlimit of primuline-stained asialo GM1 was determined by serialdilution. Seven different TLC spots containing asialo GM1 from 5to 500 pmol, were directly subjected to MS/MS analyses afterdeposition of the DHB matrix solution onto the spots (Figure 6,top). The resulting spectra of asialo GM1 (250, 100, 50, and 25pmol) are shown in Figure 6A-D (MS) and A′-D′ (MS/MS).All four MS spectra exhibited the same, clear fragmentationpattern consisting of molecular ions and a series of Y-type ions.Further MS/MS analyses with the m/z 913 ion as a precursorion provided the spectra shown in Figure 6A′-D′. The MS/MSfragmentation pattern was the same for amounts of asialo GM1ranging from 25 to 250 pmol and allowed characterization of theasialo GM1 structure.

TLC-MALDI-QIT-TOF MS/MS Analyses of ImmunostainedAsialo GM1. TLC-immunostaining has been widely used as aconvenient technique for partial characterization of GSL sugarstructures, but it cannot be used for characterizing ceramidestructures. To overcome this disadvantage, MALDI MS analysiswas performed directly on anti-asialo GM1 antibody-stained TLCspots. Meisen et al. previously reported that chloroform treatmentremoves the coating polymer and antibodies on the plate andresults in adequate TLC-MALDI MS spectra.23 As shown in Figure7, we were able to detect as little as 10 pmol of asialo GM1 byTLC-immunostaining with anti-asialo GM1 rabbit antibody andHRP-anti-rabbit IgG antibody. After chloroform treatment, the platewas directly subjected to MALDI-QIT-TOF MS analysis. Theresulting spectra are shown in Figure 7A and B (100 and 50 pmolof asialo GM1, respectively). These spectra reflect the samefragmentation patterns as those obtained with primuline staining,as described above, consisting of molecular ions and a series ofY-type ions. Further MS/MS analyses with the ion at m/z 913 asa precursor ion also provided spectra consistent with thoseobtained with primuline staining (Figure 7A′, B′). The MS/MSdetection limit of asialo GM1 was 50 pmol (∼60 ng), in contrastto a microgram scale of a single GSL in the extraction andnanoESI-QTOF-MS/MS method reported by Meisen et al.24 Theloss of samples in the procedures of scraping and elution mightbe one of the causes of the difference.

TLC-MALDI-QIT-TOF MS/MS Analyses of Neutral, Prim-uline-Stained GSLs. Finally, neutral GSLs with various sugarchains and fatty acids were subjected to TLC-MALDI-QIT-TOFMS/MS analyses (Figure 8). The MS spectrum of GlcCerindicates molecular ions at m/z 806.7 and 834.8, corresponding

to GlcCer containing d18:1-C22:0 and d18:1-C24:0, respectively(Figure 8A). MS/MS analysis of GlcCer revealed structuralinformation regarding the ceramide but not the long-chain base(Figure 8A′). Ionization of GSLs with short sugar chains, such asGlcCer, was difficult under these conditions. The MS spectrumof LacCer, shown in Figure 8B, clearly indicates two molecularions at m/z 969.0 and 997.0, corresponding to LacCer containingd18:1-C22:0 and d18:1-C24:0, respectively. The loss of Gal isindicated as a set of Y1 ions at m/z 806.9 and 834.9. The sequentialelimination of Glc could be observed as Y0 ion at m/z 664.5 byMS/MS analysis with the ion at m/z 969.0 as a precursor ion(Figure 8B′). Simple and reasonable MS and MS/MS spectra were

Figure 6. TLC-MALDI-QIT-TOF MS/MS spectra of primuline-stainedasialo GM1. Upper panel: thin-layer chromatogram stained withprimuline. Lane 1 contains GlcCer, LacCer, Gb3Cer, and Gb4Cer, asreferences. Lanes 2-8 contain bovine brain asialo GM1: (lane 2)500, (lane 3) 250, (lane 4) 100, (lane 5) 50, (lane 6) 25, (lane 7) 10,and (lane 8) 5 pmol. The plate was developed with solvent A, andGSL spots were detected with primuline reagent. Lower panel: TLC-MALDI-QIT-TOF MS and MS/MS spectra of asialo GM1 detected withprimuline. MS spectra are shown in (A-D), and MS/MS spectraobtained with the precursor ion at m/z 913 are shown in (A′-D′). (A)and (A′), (B) and (B′), (C) and (C′), and (D) and (D′) are spectraderived from 250, 100, 50, and 25 pmol of asialo GM1, respectively,corresponding to lanes 3-6 in the upper panel. The annotation offragment ions is shown in Figure 4.

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obtained for Gb3Cer and Gb4Cer (Figure 8C, C′, D, D′). For Gb3-Cer, three molecular ions (m/z 1131.0, 1159.1, and 1173.1) wereclearly indicated, corresponding to Gb3Cer containing d18:1-C22:0, d18:1-C24:0, and d18:1-C25:0, respectively, as well as a seriesof Y-type ions (Figure 8C). For Gb4Cer, two molecular ions (m/z1333.9 and 1361.9) were detected, corresponding to Gb4Cercontaining d18:1-C22:0 and d18:1-C24:0, respectively, as wellas a series of Y-type ions (Figure 8D). MS/MS analyses of Gb3-Cer and Gb4Cer performed with selection of the LacCer ion atm/z 997 as the precursor ion resulted in the spectra shown inFigure 8C′ (Gb3Cer) and D′ (Gb4Cer), respectively. In bothspectra, abundant Y1 ion was detected at m/z 835. Z0 ion wasdetected at m/z 655 in (C′) and (D′). Fragment ions present atm/z 305 (d1b or d1b′) in (B′), (C′), and (D′) represent the long-

chain base 4-sphingenine (d18:1). Fragment ions at m/z 347 and365 were detected in large amounts in (B′), (C′), and (D′) andassigned as B2 and C2 ions, respectively.

CONCLUSIONSWe have described here a procedure for rapid and convenient

structural analysis of neutral GSLs by TLC-MALDI-QIT-TOF MS/MS. One of the key features of this procedure is that the matrixsolution is DHB dissolved in acetonitrile/water (1:1 v/v), whichyields MS/MS spectra of sufficient quality for structural analysiswith high sensitivity. The positions of GSL spots on TLC plateswere determined by comparison to orcinol-stained references. Theunstained GSL spots were directly subjected to MALDI-QIT-TOFMS after DHB solution was added to the spots. MS/MS and MS/MS/MS spectra of asialo GM1 (Galâ1-3GalNAcâ1-4Galâ1-4Glcâ1-1′Cer) revealed simple and informative fragmentationpatterns, allowing structural characterization of the ceramide andlong-chain base, as well as the sugar sequence. The presence of

Figure 7. TLC-MALDI-QIT-TOF MS/MS spectra of immunostainedasialo GM1. Upper panel: TLC of asialo GM1 with orcinol staining(lanes 1 and 2) or immunostaining with anti-asialo GM1 antibody(lanes 3-7). Lane 1 contains GlcCer, LacCer, Gb3Cer, and Gb4Ceras references. Lanes 2-7 contain bovine brain asialo GM1: (lane 2)400, (lane 3) 100, (lane 4) 50, (lane 5) 25, (lane 6) 10, and (lane 7)5 pmol. The plate was developed with solvent A, followed by orcinoldetection or immunostaining, as described in the ExperimentalSection. Lower panel: MALDI-QIT-TOF MS (A) and (B) and MS/MSspectra (A′) and (B′) of asialo GM1 after TLC-immunostaining. MS/MS spectra were obtained with the precursor ion at m/z 913. (A) and(A′) 100 pmol of asialo GM1; (B) and (B′) 50 pmol of asialo GM1,corresponding to lanes 3 and 4 of immunostaining, respectively. Theannotation of fragment ions is shown in Figure 4.

Figure 8. TLC-MALDI-QIT-TOF MS/MS spectra of primuline-stainedneutral GSLs. Neutral GSLs on a TLC plate after primuline stainingwere analyzed by MALDI-QIT-TOF MS/MS: (A) and (A′) GlcCer; (B)and (B′) LacCer; (C) and (C′) Gb3Cer; (D) and (D′) Gb4Cer. (A-D)MS spectra. (A′-D′) MS/MS spectra obtained with the precursor ionsat (A′) m/z 806.7, (B′) 969.0, (C′) 997.0, and (D′) 996.8. Asterisks in(A′) indicate unidentified peaks. The annotation of fragment ions isaccording to the nomenclature of Domon and Costello30,31 and isshown in Figure 4 also.

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2-hydroxy fatty acids in ceramide was determined by the presenceof characteristic fragment ions in MS/MS spectra of mouseintestine asialo GM1 and mouse testis fucosyl asialo GM1.Furthermore, both primuline-stained and anti-asialo GM1 antibody-stained asialo GM1 were successfully analyzed by TLC-MALDI-QIT-TOF MS/MS, with detection limits of 25 and 50 pmol,respectively. Other neutral GSLs (GlcCer, LacCer, Gb3Cer, Gb4-

Cer) were also successfully analyzed by TLC-MALDI-QIT-TOFMS/MS after primuline staining.

Received for review March 27, 2006. Accepted June 19,2006.

AC0605501

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