Structural Characterization of Neutral Glycosphingolipids by Thin

Levery, S. B.; Toledo, M. S.; Doong, R. L.; Straus, A. H.; Takahashi, H. K. Rapid Commun. Mass Spectrom. 2000, 14, 551−563. [Crossref], [PubMed], [C...
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Anal. Chem. 2006, 78, 5736-5743

Structural Characterization of Neutral Glycosphingolipids by Thin-Layer Chromatography Coupled to Matrix-Assisted Laser Desorption/ Ionization Quadrupole Ion Trap Time-of-Flight MS/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 of Physical and Chemical Research (RIKEN), Saitama, Japan, and Advanced Research Institute for the Sciences and Humanities, Nihon University, Tokyo, Japan

Rapid and convenient structural analysis of neutral glycosphingolipids (GSLs) was achieved by direct coupling of thin-layer chromatography (TLC) to matrix-assisted laser desorption/ionization quadrupole ion trap time-offlight (MALDI-QIT-TOF) MS/MS. Positions of unstained GSL spots on developed TLC plates were determined by comparison to orcinol-stained references. A matrix solution of 2,5-dihydroxybenzoic acid (DHB) in acetonitrile/ water (1:1 v/v) was then added directly to the unstained GSL spots, and the GSLs were directly analyzed by MALDI-QIT-TOF MS. The acetonitrile/water DHB solution proved to be suitable for MS/MS structural analysis with high sensitivity. MS/MS and MS/MS/MS of GSLs yielded simple and informative spectra that revealed the ceramide and long-chain base structures, as well as the sugar sequences. Hydroxy fatty acids in ceramide provided characteristic MS/MS fragment ions. GSLs were stained with primuline, a nondestructive dye, after TLC development, and successfully analyzed by MALDI-QITTOF MS/MS with high sensitivity. Immunostaining of GSLs after TLC development is a powerful method for characterizing antibody-specific sugars, but not ceramides. By coupling TLC-immunostaining of GSLs to MALDIQIT-TOF MS/MS, we were able to identify both the sugar and the ceramide structures. The detection limits of asialo GM1 (Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1′Cer) were 25 and 50 pmol in primuline staining and immunostaining, respectively. Glycosphingolipids (GSLs) are amphipathic molecules consisting of a hydrophilic sugar chain and a hydrophobic ceramide moiety. They are usually located in the outer leaflet of the cell membrane, in which they are anchored by the ceramide moiety. The sugar chains are directed toward the cell exterior and have enormous structural diversity. GSLs act as cell-surface recogni* 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.

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tion molecules in various biological processes, such as cell-cell and cell-pathogen recognition.1,2 Studies on glycosyltransferase gene-targeted mice have clearly demonstrated that GSLs have physiological functions.3-5 Recently, a new role for cell-surface GSLs has been identified, in which they act as a component of a “raft” microdomain.6 Although definitive proof of the existence of rafts in physiological membranes has not yet been obtained, much evidence implicates rafts in many cellular processes, including signaling, membrane trafficking, cytoskeletal organization, and pathogen entry. The ceramide structure is important in raft formation. The chain length, unsaturation, and hydroxylation of the fatty acids and long-chain bases influence raft formation and function.7 Hence, the detailed structures of not only the sugar chains but also the ceramides of GSLs are an important focus of research. Thin-layer chromatography (TLC) is a popular and convenient technique for separation and characterization of GSLs, and it requires only small amounts of GSLs prepared from biological source materials. However, TLC characterization of individual GSLs does not yield unambiguous structural information, because GSLs containing the same sugar may migrate at different positions, owing to differences in their ceramide structures. TLCimmunostaining using sugar-specific recognition molecules, such as antibodies, lectins, and bacterial toxins, is a powerful tool for identifying parts of the sugar structures.8,9 On the other hand, the introduction of a technique in which TLC is directly coupled to mass spectrometry (TLC-MS) has greatly increased the utility (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, 16331636. (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. 10.1021/ac0605501 CCC: $33.50

© 2006 American Chemical Society Published on Web 07/19/2006

of TLC for structural characterization. TLC-MS provides both the GSL molecular mass and its structural information without requiring its purification. Advances in MS techniques, such as fast atom bombardment MS,10 secondary ion MS,11 and matrixassisted laser desorption/ionization time-of-flight (MALDI-TOF) MS,12-14 including technological developments in the transfer of GSLs to PVDF membranes and MALDI plates,15-17 have also increased the power of TLC-MS. In TLC-MALDI MS, problems in obtaining good mass accuracy and resolution arise from the difficulties involved in desorbing analytes from the rough surface of silica gel. Very recently, remarkable advances in MALDI MS ion trap instruments have enabled TLC-MALDI MS analysis with high mass accuracy and sensitivity.18 Other instruments include an external ion trap, such as a Fourier transform ion cyclotron resonance ion trap (FTICR),19,20 and an orthogonal extracting ion trap.21,22 Using highsensitivity TLC-FTICR MS, Ivleva et al. successfully identified parts of ganglioside sugar sequences by analyzing its metastable ions.19 In another study, TLC coupled to orthogonal (o)-TOF MS was used to determine molecular masses and partial sugar sequences of gangliosides with high resolution; in particular, the attachment positions of the sialic acids were determined.21 Successful analytical strategies in which fragment ions of interest 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/MS to gangliosides extracted from silica gel after primuline staining and TLC-immunostaining.23 Using nanoESI-quadrupole TOF MS/ MS, they successfully assigned a series of ions arising from sequential fragmentation. They also reported structural assignments 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 sample material is possible. Glycosphingolipids containing the same sugar (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) Mu ¨ thing, J. In Glycoanalysis Protocols; Hounsell, E., Ed.; Humana Press Inc.: 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.; Mu ¨ thing, J.; Rohlfing, A.; Meisen, I.; Vukelic´, Zˇ .; PeterKatalinic´, J.; Hillenkamp, F.; Berkenkamp, S. Anal. Chem. 2005, 77, 40984107. (22) Ivleva, V. B.; Sapp, L. M.; O’Connor, P. B.; Costello, C. E. J. Am. Soc. Mass Spectrom. 2005, 16, 1552-1560. (23) Meisen, I.; Peter-Katalinic´, J.; Mu ¨ thing, J. Anal. Chem. 2004, 76, 22482255. (24) Meisen, I.; Friedrich, A. W.; Karch, H.; Witting, U.; Peter-Katalinic´, J.; Mu ¨ thing, J. Rapid Commun. Mass Spectrom. 2005, 19, 3659-3665.

and different ceramides migrate at different position of a spot on a TLC plate. TLC-MALDI-QIT MS/MS is a method directly coupled to sample position; therefore, it is possible to characterize GSLs migrating in defined small area within a single spot or closely migrating spots. In this report, we describe a method for the rapid and convenient structural analysis of neutral GSLs in which TLC is directly coupled to MALDI-QIT-TOF MS/MS. MALDI MS with an external QIT eliminates the mass accuracy and resolution problems that would otherwise arise from the irregular surface of the silica gel plate. Furthermore, a MALDI-QIT system easily performs sequential MS/MS and MS/MS/MS analyses, providing precise structural information on GSLs. Although heterogeneity in the ceramide and sugar moieties leads to complex MS spectra, MS/MS overcomes this problem. We were able to obtain fragment ions derived from long-chain bases only by MS/MS. Using 2,5dihydroxybenzoic acid (DHB) as the matrix and acetonitrile/water as the solvent, the amount of sample material required for the structural characterization of GSLs is on the picomole scale. EXPERIMENTAL SECTION GSL Nomenclature. The nomenclature was derived from the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature25 and the system of Svennerholm.26 The GSL abbreviations are as follows: GlcCer, Glcβ1-1′Cer; LacCer, Galβ1-4Glcβ1-1′Cer; Gb3Cer, GalR1-4Galβ1-4Glcβ1-1′Cer; Gb4Cer, GalNAcβ1-3GalR1-4Galβ1-4Glcβ1-1′Cer; asialo GM1, Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1′Cer; and fucosyl asialo GM1, FucR1-2Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1′Cer. Materials. The neutral GSLs for TLC and TLC-MALDI-QITTOF MS analyses were GlcCer, LacCer, Gb3Cer, and Gb4Cer purified from hog erythrocytes. Asialo GM1 prepared from bovine brain GM1 was purchased from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan), and asialo GM1 containing a unique ceramide with phytosphingosine and 2-hydroxy fatty acid was purified from mouse intestine in our laboratory.27 Neutral GSLs from mouse testis were prepared in our laboratory. The crude lipid extract of testis was partially purified using Iatrobeads column chromatography (Iatron laboratories, Tokyo, Japan), and the partially purified fraction containing fucosyl asialo GM1 as a major GSL was subjected to TLC-MALDI-QIT-TOF MS analysis. Thin-Layer Chromatography. Neutral GSLs dissolved in chloroform/methanol (1:1, v/v) were applied as 3-5 mm spots to silica gel-coated plates with aluminum backing (Merck, Darmstadt, Germany). Plates were developed with a solvent system of chloroform/methanol/water (65:35:8, v/v/v, solvent A). When samples on a TLC plate were to be directly analyzed by MALDIQIT-TOF MS without staining, duplicate, side-by-side spots were made for each sample, and the plate was cut into two pieces after development. One piece was used as a reference for the positions of the GSLs, which were detected with orcinol reagent, and the other piece was used for MS analysis. The positions of the GSLs on the latter piece were determined by comparison with the orcinol-stained GSLs on the former piece and then marked with (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.

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a pencil. For primuline staining, developed plates were dried and then sprayed with 0.01% primuline in acetone/water (4:1, v/v). The fluorescent spots were visualized using a Fujifilm LAS-1000 imaging system (Tokyo, Japan) and marked with a pencil. TLC-Immunostaining. TLC-immunostaining was performed as previously described with a minor modification.28 In brief, neutral GSLs were separated on a TLC plate with aluminum backing by development with solvent A. The plate was cut into two pieces, one of which was sprayed with orcinol reagent to determine the positions of the GSLs. The other piece, which was used for immunostaining, was soaked in 0.1% poly(isobutyl methacrylate) in cyclohexane for 1 min, dried, and preincubated with 1% bovine serum albumin-phosphate-buffered saline (BSAPBS) for 30 min. The plate was then incubated with anti-asialo GM1 antibody solution (Wako Pure Chemical Industries, Ltd.; diluted 1:2000 with 1% BSA-PBS) for 1.5 h. The plate was washed with 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 were visualized 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 was washed with PBS and water and dried with cold air. The dry plate was dipped twice in chloroform for 1 min to remove the coating polymer and bound antibody.23 When the plate was dry, spot positions were determined by reference to the detected LAS-1000 chemiluminescence image and marked with a pencil. Mass Spectrometry. An Axima-QIT mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse width) (Shimadzu, Kyoto, Japan) was used. The ion trap chamber was supplied with two separate and independent gases; He gas with continuous flow was used for collisional cooling, and pulsed Ar gas was used for imposing collision-induced fragmentation.29 The pressure in the ionization chamber was maintained at less than 6 × 10-6 Torr. Precursor and fragment ions obtained by collisioninduced dissociation (CID) were ejected from the ion trap and analyzed by a reflectron TOF detector operated in positive ion mode. The mass spectra were assembled from 200 to 1000 accumulations of the profile obtained by two laser shots using a laser power of 30-60 arbitrary units. Stronger laser power increased sensitivity of MS and provided fragment ions used as the precursor ion for sequential MS/MS analysis. External calibration of MS spectra was performed using angiotensin II ([M + H]+, m/z 1046.5) and ACTH ([M + H]+, m/z 2465.2) on a MALDI sample plate rather than a TLC plate. TLC plates for MS analysis were cut into small pieces containing the analytes, and the pieces were attached to a MALDI sample plate with double-sided adhesive tape.19 Several small droplets of matrix solution (totaling 1-2 µL) were then deposited on the spot area using a microsyringe, with immediate drying with a cold air stream after each droplet. The matrix solution contained 0.1 mg of DHB/µL in acetonitrile/water (1:1, v/v). (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.

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Figure 1. TLC-MALDI-QIT-TOF MS spectra of asialo GM1 obtained with 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 brain asialo GM1. The annotation of fragment ions is shown in Figure 4.

RESULTS AND DISCUSSION Selection of a Matrix Solution for TLC-MALDI-QIT-TOF MS. Many kinds of matrixes and solvents have been used in TLCMALDI MS analysis. The selected matrix is a critical factor in obtaining useful spectra with high sensitivity, and the matrix solvent is also important. In general, matrixes are dissolved in volatile solvents at saturated concentrations to make matrixanalyte crystals and to minimize sample diffusion.19 DHB is one of the best matrixes discovered thus far for analysis of neutral GSLs by TLC-MALDI MS, and a hydrophobic solvent (chloroform/ methanol) has been successfully used as a matrix solvent.15 When we attempted to analyze small amounts of GSL (e.g., 8 pmol of asialo GM1), however, the formation of matrix clusters in the chloroform/methanol solution was a serious problem (Figure 1A, B). Surprisingly, we found that hydrophilic solvents containing water provided excellent MS spectra with the DHB matrix. As shown in Figure 1C-F, sodiated molecular ions at m/z 1278 or 1306 were clearly present in the MS spectra derived from asialo GM1 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 of the spectra indicates that acetonitrile/water (1:1, v/v) yielded the best spectra of asialo GM1 with good sensitivity. The detection limit was similar to that of orcinol staining, in which 5 pmol of asialo GM1 was the approximate limit of detection (Figure 2A). Because acetonitrile/water is not volatile, the matrix solution should be deposited as several very small droplets on GSL spots.

Figure 2. Thin-layer chromatography of asialo GM1 and fucosyl asialo GM1. (A) Lane 1 contains GlcCer, LacCer, Gb3Cer, and Gb4Cer. 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, and Gb4Cer. Lanes 2-5 contain neutral GSLs: (lane 2) bovine brain asialo GM1; (lane 3) mouse intestine asialo GM1; (lane 4) crude lipid extract from mouse testis; (lane 5) partially purified mouse testis lipid fraction containing fucosyl asialo GM1 (indicated by arrow). TLC plates were developed with solvent A and detected with orcinol reagent, as described in the Experimental Section. Asterisks indicate orcinolnegative bands.

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

TLC-MALDI-QIT-TOF MS/MS and MS/MS/MS Analyses of Unstained Asialo GM1 and Fucosyl Asialo GM1. TLCMALDI-QIT-TOF MS and MS/MS spectra of asialo GM1 from bovine brain are shown in Figure 3. Two molecular ions are clearly detected at m/z 1278.0 and 1306.1 in Figure 3A. A series of Y-type ions are also detected as three pairs of ions at m/z 1116.0 and 1144.0 (Y3), 912.8 and 940.9 (Y2), and 750.8 and 778.8 (Y1), indicating the sequential loss of Gal, GalNAc, and Gal from the nonreducing end of asialo GM1. The fragment ions at m/z 912.8 and 940.9, which indicate LacCer, were abundant. The fragmentation annotations of asialo GM1 are shown in Figure 4 according to the nomenclature of Domon and Costello.30,31 These MS spectra

demonstrate that mass accuracy and resolution are not affected by the irregular surface of the silica gel. A comparison of mass spectra obtained by the TLC-MALDIQIT-TOF MS method described herein and by MALDI-QIT-TOF MS using a MALDI sample plate revealed differences in the results from these two systems. All the ions from TLC-MALDI MS were detected as monosodiated forms, whereas MALDI MS yielded both sodium and potassium adduct ions (data not shown). In TLCinfrared MALDI-o-TOF MS, GM3 ganglioside was detected as mono- and disodiated ions in positive ion mode.21 Therefore, the TLC-MALDI-QIT-TOF MS system has the advantage of yielding simpler spectra with monosodiated ions. The MS/MS spectrum of asialo GM1, which is shown in Figure 3B, was obtained by selecting the molecular ion at m/z 1278.0 as the precursor ion. An abundant ion at m/z 912.9 (Y2) and an ion at m/z 750.8 (Y1) were detected. To obtain structural information in a low-molecular-mass region, MS/MS analysis was sequentially performed using LacCer ions at m/z 940.9 and 912.8 as precursor ions and abundant GlcCer (Y1) ions were detected at m/z 778.8 and 750.8, respectively (Figure 3C and D). The ceramide ion was detected at m/z 588.3 (Y0) in (D), and the ceramide minus H2O ions (Z0) are at m/z 598.4 in (C) and 570.5 in (D). Fragment ions at m/z 304.9 (d1b or d1b′ in Figure 4) in both (C) and (D) indicate the presence of 4-sphingenine (d18:1) as the long-chain base, as previously reported by Hsu et al.32 Although we did not observe any 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 3. TLC-MALDI-QIT-TOF MS/MS spectra of bovine brain asialo GM1. (A) MS spectrum; (B-D) MS/MS spectra obtained with the precursor ions at (B) m/z 1278.0, (C) 940.9, and (D) 912.8. The annotation of fragment ions is shown in Figure 4.

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Figure 5. TLC-MALDI-QIT-TOF MS/MS and MS/MS/MS spectra of mouse intestine asialo GM1 and MS/MS spectrum of mouse testis fucosyl asialo GM1. Spectra for asialo GM1 purified from mouse intestine 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 shown in (B) and (B′). (B) MS spectrum; (B′) MS/MS spectrum obtained with the precursor ion at m/z 1087.0. Asterisks indicate the characteristic fragment ions produced by degradation of GSLs at the O position, as shown in Figure 4. The annotation of fragment ions is shown in Figure 4.

mass could be calculated from the masses of the ceramide and the long-chain base. The abundant fragment ions at m/z 347 and 365 detected in both (C) and (D) are characteristic ions assigned as Gal-Glc (B2) and Gal-Glc + H2O (C2), respectively, as shown in the bottom of Figure 4. Asialo GM1 from mouse intestine contains a unique ceramide consisting of phytosphingosine and 2-hydroxy fatty acids, as previously reported.27 Because its ceramide has two additional hydroxy groups, mouse intestine asialo GM1 migrates more slowly on a TLC plate than does asialo GM1 prepared from bovine brain GM1 (Figure 2B, lanes 2 and 3). In the MS spectrum of mouse intestine asialo GM1, which is shown in Figure 5A, a series of ions carrying different fatty acid residues were detected at m/z 1268.0, 1368.1, 1382.1, 1394.1, and 1396.1. These ions correspond to asialo GM1 (phytosphingosine; t18:0) substituted with C16:0, hC22:0, hC23:0, hC24:1, and hC24:0 fatty acids, respectively. A set of Y-series fragment ions arising from the sequential elimination of Gal-GalNAc and Gal were also detected. The MS/MS spectrum obtained from selection of MS ion at m/z 1002.9 as a precursor ion is shown in Figure 5A′. The abundant fragment ion 5740 Analytical Chemistry, Vol. 78, No. 16, August 15, 2006

at m/z 840.8 is assigned as GlcCer (Y1). The fragment ions detected at m/z 346.9 and 364.9 are the same ions detected in the spectrum of bovine brain asialo GM1 (Figure 3C, D) and correspond to B2 and C2 ions, respectively. Two characteristic fragment ions were clearly detected at m/z 664.3 and 502.1. The 162.2 atomic mass unit (amu) difference between these ions suggests the presence of a hexose. Further analysis, in which the MS/MS ion at m/z 664.3 was selected as the precursor ion for MS/MS/MS, confirmed that the fragment ion at m/z 502.1 arose from degradation of the ion at m/z 664.3 (data not shown). These two characteristic ions are assigned as O ions containing GalGlc (m/z 664.3) and Glc (m/z 502.1), produced by cleavage of the amide NH-CO bond (Figure 4). This characteristic cleavage was previously reported by Ann and Adams as a result of CID fragmentation of ceramide lithium adduct ions.33,34 Only the GSLs containing 2-hydroxy fatty acids yielded these O ions in abundance, and GSLs containing nonhydroxy fatty acids yielded no detectable O ions (Figure 3C, D). The abundant cleavage at the O position prevented detection of the ceramide fragment ion in Figure 5A′. When MS/MS/MS analysis was performed with selection 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 Figure 5A′, a fragment ion was detected at m/z 305.0 (d1b or d1b′); this ion could be assigned as 4-sphingenine (d18:1) or as t18:0 minus H2O. Since the presence of phytosphingosine was confirmed by its two characteristic fragment ions at m/z 664.3 and 502.1, as described above, the selected precursor ion for MS/MS/MS must contain only t18:0. Fucosyl asialo GM1 with unique ceramide structures was recently reported by Sandhoff et al.,35 who purified GSLs from mouse testis and identified novel fucosylated GSLs containing polyunsaturated and very long-chain fatty acids. These GSLs may be essential for spermatogenesis and male fertility.35 We detected four neutral GSLs in the crude lipid extract of mouse testis, and their mobilities on a TLC plate were similar to those reported by Sandhoff et al. (Figure 2B, lane 4). We obtained a partially purified fraction that contained fucosyl asialo GM1 as its dominant GSL (Figure 2B, lane 5). We subjected this fraction to TLC-MALDIQIT-TOF MS and MS/MS, as shown in Figure 5B and B′. Notably, the quality of the MS spectra was not negatively affected by the abundant remaining impurities in the analyzed fraction. Two molecular ions were observed at m/z 1570.3 and 1598.4 (Figure 5B). On the basis of previously reported results,35 these ions are assigned as sodiated molecular ions of fucosyl asialo GM1 (d18: 1) with hC28:5 and hC30:5 fatty acids. A series of Y-type ions were also 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 the sequential loss of Fuc, Gal-GalNAc, and Gal from the nonreducing end of the molecule. The structure and the fragmentation scheme are shown in Figure 4. MS/MS analysis with selection of the ion at m/z 1087.0 as the precursor ion resulted in an abundant 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.; Gro ¨ne, H.-J. J. Bioi. Chem. 2005, 29, 27310-27318.

fragment ions B2 and C2 were detected at m/z 346.9 and 364.9, respectively. Two characteristic fragment ions indicating the presence of 2-hydroxy fatty acid were clearly detected at m/z 646.2 and 484.1. These O ions are 18 amu smaller than those of asialo GM1 (m/z 664.3 and 502.1) in Figure 5A′. This 18-amu difference corresponds 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 the detection of these characteristic fragment ions. The fragment ion at 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 PrimulineStained Asialo GM1. Primuline is a nondestructive fluorochrome that detects lipids and has been used to locate GSLs on a TLC plate prior to MS analysis.21,23 In the present study, the detection limit of primuline-stained asialo GM1 was determined by serial dilution. Seven different TLC spots containing asialo GM1 from 5 to 500 pmol, were directly subjected to MS/MS analyses after deposition of the DHB matrix solution onto the spots (Figure 6, top). The resulting spectra of asialo GM1 (250, 100, 50, and 25 pmol) are shown in Figure 6A-D (MS) and A′-D′ (MS/MS). All four MS spectra exhibited the same, clear fragmentation pattern consisting of molecular ions and a series of Y-type ions. Further MS/MS analyses with the m/z 913 ion as a precursor ion provided the spectra shown in Figure 6A′-D′. The MS/MS fragmentation pattern was the same for amounts of asialo GM1 ranging from 25 to 250 pmol and allowed characterization of the asialo GM1 structure. TLC-MALDI-QIT-TOF MS/MS Analyses of Immunostained Asialo GM1. TLC-immunostaining has been widely used as a convenient technique for partial characterization of GSL sugar structures, but it cannot be used for characterizing ceramide structures. To overcome this disadvantage, MALDI MS analysis was performed directly on anti-asialo GM1 antibody-stained TLC spots. Meisen et al. previously reported that chloroform treatment removes the coating polymer and antibodies on the plate and results in adequate TLC-MALDI MS spectra.23 As shown in Figure 7, we were able to detect as little as 10 pmol of asialo GM1 by TLC-immunostaining with anti-asialo GM1 rabbit antibody and HRP-anti-rabbit IgG antibody. After chloroform treatment, the plate was directly subjected to MALDI-QIT-TOF MS analysis. The resulting spectra are shown in Figure 7A and B (100 and 50 pmol of asialo GM1, respectively). These spectra reflect the same fragmentation patterns as those obtained with primuline staining, as described above, consisting of molecular ions and a series of Y-type ions. Further MS/MS analyses with the ion at m/z 913 as a precursor ion also provided spectra consistent with those obtained with primuline staining (Figure 7A′, B′). The MS/MS detection limit of asialo GM1 was 50 pmol (∼60 ng), in contrast to a microgram scale of a single GSL in the extraction and nanoESI-QTOF-MS/MS method reported by Meisen et al.24 The loss of samples in the procedures of scraping and elution might be one of the causes of the difference. TLC-MALDI-QIT-TOF MS/MS Analyses of Neutral, Primuline-Stained GSLs. Finally, neutral GSLs with various sugar chains and fatty acids were subjected to TLC-MALDI-QIT-TOF MS/MS analyses (Figure 8). The MS spectrum of GlcCer indicates molecular ions at m/z 806.7 and 834.8, corresponding

Figure 6. TLC-MALDI-QIT-TOF MS/MS spectra of primuline-stained asialo GM1. Upper panel: thin-layer chromatogram stained with primuline. Lane 1 contains GlcCer, LacCer, Gb3Cer, and Gb4Cer, as references. 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, and GSL spots were detected with primuline reagent. Lower panel: TLCMALDI-QIT-TOF MS and MS/MS spectra of asialo GM1 detected with primuline. MS spectra are shown in (A-D), and MS/MS spectra obtained 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 spectra derived from 250, 100, 50, and 25 pmol of asialo GM1, respectively, corresponding to lanes 3-6 in the upper panel. The annotation of fragment ions is shown in Figure 4.

to GlcCer containing d18:1-C22:0 and d18:1-C24:0, respectively (Figure 8A). MS/MS analysis of GlcCer revealed structural information regarding the ceramide but not the long-chain base (Figure 8A′). Ionization of GSLs with short sugar chains, such as GlcCer, was difficult under these conditions. The MS spectrum of LacCer, shown in Figure 8B, clearly indicates two molecular ions at m/z 969.0 and 997.0, corresponding to LacCer containing d18:1-C22:0 and d18:1-C24:0, respectively. The loss of Gal is indicated as a set of Y1 ions at m/z 806.9 and 834.9. The sequential elimination of Glc could be observed as Y0 ion at m/z 664.5 by MS/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 Analytical Chemistry, Vol. 78, No. 16, August 15, 2006

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Figure 7. TLC-MALDI-QIT-TOF MS/MS spectra of immunostained asialo 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 Gb4Cer as 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 orcinol detection or immunostaining, as described in the Experimental Section. Lower panel: MALDI-QIT-TOF MS (A) and (B) and MS/MS spectra (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. The annotation of fragment ions is shown in Figure 4.

obtained for Gb3Cer and Gb4Cer (Figure 8C, C′, D, D′). For Gb3Cer, three molecular ions (m/z 1131.0, 1159.1, and 1173.1) were clearly indicated, corresponding to Gb3Cer containing d18:1-C22: 0, d18:1-C24:0, and d18:1-C25:0, respectively, as well as a series of Y-type ions (Figure 8C). For Gb4Cer, two molecular ions (m/z 1333.9 and 1361.9) were detected, corresponding to Gb4Cer containing d18:1-C22:0 and d18:1-C24:0, respectively, as well as a series of Y-type ions (Figure 8D). MS/MS analyses of Gb3Cer and Gb4Cer performed with selection of the LacCer ion at m/z 997 as the precursor ion resulted in the spectra shown in Figure 8C′ (Gb3Cer) and D′ (Gb4Cer), respectively. In both spectra, abundant Y1 ion was detected at m/z 835. Z0 ion was detected at m/z 655 in (C′) and (D′). Fragment ions present at m/z 305 (d1b or d1b′) in (B′), (C′), and (D′) represent the long5742 Analytical Chemistry, Vol. 78, No. 16, August 15, 2006

Figure 8. TLC-MALDI-QIT-TOF MS/MS spectra of primuline-stained neutral GSLs. Neutral GSLs on a TLC plate after primuline staining were 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 ions at (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 is according to the nomenclature of Domon and Costello30,31 and is shown in Figure 4 also.

chain base 4-sphingenine (d18:1). Fragment ions at m/z 347 and 365 were detected in large amounts in (B′), (C′), and (D′) and assigned as B2 and C2 ions, respectively. CONCLUSIONS We 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 matrix solution is DHB dissolved in acetonitrile/water (1:1 v/v), which yields MS/MS spectra of sufficient quality for structural analysis with high sensitivity. The positions of GSL spots on TLC plates were determined by comparison to orcinol-stained references. The unstained GSL spots were directly subjected to MALDI-QIT-TOF MS after DHB solution was added to the spots. MS/MS and MS/ MS/MS spectra of asialo GM1 (Galβ1-3GalNAcβ1-4Galβ14Glcβ1-1′Cer) revealed simple and informative fragmentation patterns, allowing structural characterization of the ceramide and long-chain base, as well as the sugar sequence. The presence of

2-hydroxy fatty acids in ceramide was determined by the presence of characteristic fragment ions in MS/MS spectra of mouse intestine asialo GM1 and mouse testis fucosyl asialo GM1. Furthermore, both primuline-stained and anti-asialo GM1 antibodystained asialo GM1 were successfully analyzed by TLC-MALDIQIT-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-TOF MS/MS after primuline staining.

Received for review March 27, 2006. Accepted June 19, 2006. AC0605501

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