Energy-Resolved Structural Details Obtained from Gangliosides

Anal. Chem. 2009, 81, 139–145

Energy-Resolved Structural Details Obtained from Gangliosides Yuki Shioiri,† Ayako Kurimoto,‡ Takuro Ako,‡ Shusaku Daikoku,‡ Atsuko Ohtake,‡ Hideharu Ishida,§ Makoto Kiso,§ Katsuhiko Suzuki,‡ and Osamu Kanie*,†,‡ Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4529 Nagatsuta-cho, Midori-ku, Yokohama 226-0018, Japan, Mitsubishi Kagaku Institute of Life Sciences (MITILS), 11 minami-oya, Machida-shi, Tokyo 194-8511, Japan, and Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan Gangliosides, a family of glycosphingolipids (GSLs) that comprise sialic acid residue(s), are an important class of molecules that exist on the outer surface of the plasma membrane. To assess the functions of a particular series of gangliosides that play important roles in brain functions, their structures and localizations need to be investigated. We studied the structures of these gangliosides by collision-induced dissociation using quadrupole iontrap mass spectrometry. The dissociation processes were investigated in detail based on energy-resolved mass spectrometry using sodiated molecules. The decision of utilization of the positive mode was based on the assumption that it was the generally applicable method for GSLs, including neutral ones. In this investigation, sialic acid residues were esterified to stabilize the linkages and to generate multiple fragment ions for successful structural investigations. A detailed analysis of a series of sodiated species of gangliosides based on energy-resolved mass spectrometry revealed that the GM1-equivelent fragments generated from the precursor ions under low energy CID conditions had the structural characteristics of their individual precursors. It was suggested that this information will be useful in determining the structures of their precursor gangliosides. Glycosphingolipids (GSLs) are present in all cells in the human body, and they are involved in many important biological functions including immunological ones that protect humans from viral and bacterial infections.1 Gangliosides are a family of GSLs that comprise sialic acid residue(s); they have been shown to be involved in proliferation, differentiation, and cell-cell recognition. The plasma membrane of central nervous system cells is particularly rich in gangliosides, and their importance has been recognized.2-4 Mass spectrometric methods have gained considerable importance nowadays because their high sensitivity enables the structural investigation of minute amounts of compounds obtained from biological samples.5-9 The “decoding” of glycan * To whom correspondence should be addressed. E-mail: kokanee@ mitils.jp. Fax: (+81)42-724-6238. † Tokyo Institute of Technology. ‡ Mitsubishi Kagaku Institute of Life Sciences (MITILS). § Gifu University. (1) Miller-Podraza, H. Chem. Rev. 2000, 100, 4663–4681. 10.1021/ac801611z CCC: $40.75  2009 American Chemical Society Published on Web 12/02/2008

structures using databases is one of the promising approaches that is currently being developed.10-12 We have been attempting to develop a new method for determining anomeric configurations and linkage positions based on energyresolved mass spectrometry (ERMS). This technique provides data about the activation energy of a given analyte under collision-induced dissociation (CID) conditions, which is considered to be advantageous over conventional MS/MS techniques.5,13-15 Thin-layer chromatography-based separation and the subsequent visualization methods have been frequently used to analyze gangliosides.16,17 Immunostaining of organella is a powerful technique with which the distribution of such species can be investigated.18 The observation of GM1 using mass spectrometry imaging is one of the recent important findings.19 These investigations may become extremely important in that they can provide (2) Nagai, Y. Behav. Brain Res. 1995, 66, 99–104. (3) Walkley, S. U.; Siegel, D. A.; Dobrenis, K. Neurochem. Res. 1995, 20, 1287– 1299. (4) Sampathkumar, S.-G.; Li, A.; Yarema, K. J. CNS Neurol. Disord.: Drug Targets 2006, 5, 425–440. (5) Zaia, J. Mass Spectrom. Rev. 2004, 23, 161–227. (6) Fang, T. T.; Bendiak, B. J. Am. Chem. Soc. 2007, 129, 9721–9736. (7) Hofmeister, G. E.; Zhou, Z.; Leary, J. A. J. Am. Chem. Soc. 1991, 113, 5964–5970. (8) Laine, R. A.; Pamidimukkala, K. M.; French, A. D.; Hall, R. W.; Abbas, S. A.; Jain, R. K.; Matta, K. L. J. Am. Chem. Soc. 1988, 110, 6931–6939. (9) Viseux, N.; de Hoffmann, E.; Domon, B. Anal. Chem. 1998, 70, 4951– 4959. (10) Takegawa, Y.; Deguchi, K.; Ito, S.; Yoshioka, S.; Sano, A.; Yoshinari, K.; Kobayashi, K.; Nakagawa, H.; Monde, K.; Nishimura, S.-I. Anal. Chem. 2004, 76, 7294–7303. (11) Kameyama, A.; Kikuchi, N.; Nakaya, S.; Ito, H.; Sato, T.; Shikanai, T.; Takahashi, Y.; Takahashi, K.; Narimatsu, H. Anal. Chem. 2005, 77, 4719– 4725. (12) Ashline, D.; Singh, S.; Hanneman, A.; Reinhold, V. Anal. Chem. 2005, 77, 6250–6262. (13) Zaia, J.; McClellan, J. E.; Costello, C. E. Anal. Chem. 2001, 73, 6030–6039. (14) Kurimoto, A.; Daikoku, S.; Mutsuga, S.; Kanie, O. Anal. Chem. 2006, 78, 3461–3466. (15) Daikoku, S.; Ako, T.; Kurimoto, A.; Kanie, O. J. Mass Spectrom. 2007, 42, 714–723. (16) Kasama, T.; Hisano, Y.; Nakajima, M.; Handa, S.; Taki, T. Glycoconjugate J. 1996, 13, 461–469. (17) Hu ¨ thing, J. TLC in structure and recognition studies of glycosphingolipids. In Methods Mol. Biol. 76, Glycoanalysis Protocols, 2nd ed.; Hounsell, E. F., Ed.; Humana Press Inc.: Totowa, NJ, 1998; pp 183-196. (18) Marconi, S.; De Toni, L.; Lovato, L.; Tedeschi, E.; Gaetti, L.; Acler, M.; Bonetti, B. J. Neuroimmunol. 2005, 170, 115–121. (19) Sugiura, Y.; Shimma, S.; Konishi, Y.; Yamada, M. K.; Setou, M. PLoS One 2008, 3, e3232.

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information about the distribution and amount of GSLs in cells and organs. Mass spectrometric analysis of gangliosides is often performed in the negative ion mode because of the negatively charged character of sialic acid residues.20 Such conditions, however, are not always suitable for observing neutral GSLs, which can be better analyzed under the positive mode. Another problem encountered in the analysis of GSLs is the difficulties involved in MS/MS analysis of species that comprise sialic acid because the linkage is relatively labile and often preferentially cleaved under CID conditions. Therefore, important information about these species cannot be obtained.21 In an attempt to solve this problem, the use of methyl ester and an amide of the carboxylic acid moiety has been reported.21-23 In the present paper, we describe a detailed ERMS analysis of a series of gangliosides. We have found that the dissociations of the glycosidic linkages of HexNAc and sialic acid (Sia) are important in the structural investigation of GSLs. Furthermore, our findings suggested that the structures of precursor species could be estimated by analyzing the generated GM1-equivalent fragment ions.

In our MSn experiments, the end-cap RF amplitude was increased in steps of 0.02 V until the precursor ion could no longer be detected (plateau at less than 0.9% of total ion current). The averages of n - 4 spectra were used for CID experiments (n ) 8-13: where n is the number of spectra obtained during the experiments); the first and last two data sets in an RF amplitude step, which are associated with a transient period to the steady state, were not used to avoid any inaccuracy. Isotopic peaks with [Ii + 1] and [Ii + 2], where Ii indicates a fragment ion, were included in the calculations. (See also Data handling) For the isolation of a product ion, m/z ± 3 (range of isotopes, w ) 6) were isolated and subjected to the CID experiments to include isotopes. Standard MS/MS spectra are the extracts of these ERMS spectra at designated amplitudes. Data Handling. To obtain graphs of the ERMS, the following equations were used. When an ion “IP” produces a series of product ions, I1, I2, I3, . . ., Ii, the relative ion currents for individual ions are defined by the equation i

CI

rel

C)

MATERIALS AND METHODS Materials. GM1a, GD1a, and GD1b were purchased from Seikagaku Corp. (Tokyo, Japan). GM3, GT1a, and GT1b were purchased from HyTest Ltd. (Turku, Finland). All commercial gangliosides were purified from bovine brain. Dimethyl sulfoxide (DMSO) and anhydrous 3-methyl-1-p-tolyltriazene (MTT) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO). Synthesized GM1b24 and sialyl lactotetraosyl ceramide25 were used. β-Gal(1f3)-β-GalNAc-(1f4)-β-Gal-(1f4)-Glc-PA and β-Gal-(1f3)-βGlcNAc-(1f3)-β-Gal-(1f4)-Glc-PA were purchased from Takara Bio Inc. (Otsu, Japan). Method of Methyl Esterification. GM1a (4.7 µg, 3 nmol) was dissolved in DMSO (20 µL) containing MTT (0.1M) in an Eppendorf tube. After incubation at 60 °C for 1 h, the mixture was diluted with methanol and analyzed by MS directly. Instrumentation and Data Collection. Samples were analyzed using a quadrupole ion trap mass spectrometer (QIT-MS) coupled with an electrospray interface (Bruker Esquire 3000+, Bruker Daltonics GmbH, Bremen, Germany). The samples were dissolved in a mixture of chloroform, methanol, and H2O (6:4:1), and the solution obtained was diluted with methanol (0.01-0.05 µmol/mL). The solution was introduced into the ion source via infusion (flow rate, 120 µL/h). The parameters for analysis were (1) “dry temperature”, 250 °C; (2) nebulizer gas (N2), 10 psi; (3) dry gas (N2), 4.0 L/min; (4) “Smart frag”, off; (5) scan range, m/z 50-2000; (6) compound stability, 200%; (7) ICC target, 5000; (8) maximum acquisition time, 200 ms; (9) average, 10 spectra; (10) “cut-off”, 28.1%; and (11) collision gas, He (4.4 × 10-6 mbar). Egge, H.; Peter-Katalinic, J. Mass Spectrom. Rev. 1987, 6, 331–393. Handa, S.; Nakamura, K. J. Biochem. 1984, 95, 1323–1329. Sekiya, S.; Wada, Y.; Tanaka, K. Anal. Chem. 2005, 77, 4962–4968. Miura, Y.; Nishimura, S.-I. Chem.sEur. J. 2007, 13, 4797–4804. Prabhanjan, H.; Ishida, H.; Kiso, M.; Hasegawa, A. Carbohydr. Res. 1991, 211, c1-c5. (25) Kameyama, A.; Ishida, H.; Kiso, M.; Hasegawa, A. Carbohydr. Res. 1990, 200, 269–285. (20) (21) (22) (23) (24)

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× 100

n

IP

C +

∑C

(1)

Ii

i)1

where relC denotes the ion current (in percentage) of a given I ion among observed ions; C I , the observed ion current being P considered; and C I , the ion current of a precursor ion. The calculations were performed using a program developed by us in Excel 2000 (Microsoft Co.) that was based on the DSUM function and programmed to select a monoisotope to be taken into consideration. Sigmoidal Curve Fitting. A set of MSn data obtained at various RF amplitudes (end cap) on a mass spectrometer was analyzed using Excel, where sets of peaks having certain m/z values were treated as a series of data. The relative intensities over the total ion current for individual signals were obtained at each amplitude (eq 1). The data were analyzed using Prism 4 software (GraphPad Software, Inc.). Individual data were fitted using the Boltzmann sigmoidal function with nonlinear regression analysis (eq 2, growth, and eq 3, decay). a 1 + exp{(b - x) ⁄ c}

(2)

a 1 + exp{-(b - x) ⁄ c}

(3)

y) y)

where parameters “a”, “b”, and “c”, respectively, indicate the maximum response, half value of the maximum response, and slope factor for each curve. In the series of data used in this paper, the sigmoidal curves and the parameters were obtained by plotting the regression curves by treating all the data obtained with the above mentioned Excel program. RESULTS AND DISCUSSION Structures of Sialylated Tetraosyl Ceramides with Same Molecular Weights. It is important to conduct mass spectrometry studies of stereoisomers that are often found in oligosaccharide

Figure 1. Structure of a series of gangliosides used in this investigation.

Figure 2. Dissociation curves (ERMS) obtained for three sodiated monosialylated gangliosides, namely, GM1a (open square), GM1b (open triangle), and IV3NeuAcLc4Cer (open circle).

structures. We selected a series of gangliosides that comprise various numbers of Sia residues, namely, GM1a, GM1b, IV3NeuAcLc4Cer (sialyl lactotetraosyl ceramide), GD1a, GD1b, GT1a, and GT1b, and investigated their structural characteristics using ERMS. GM1a and GM1b can be discriminated by the linkage position of a Sia residue, as well as by branch versus linear glycans. With regard to the comparison of GM1b and IV3NeuAcLc4Cer, one of the components (HexNAc) is different, although there is a similarity in that both have linear structures. With regard to higher gangliosides, additional linkage patterns exist because of the presence of a dimer of Sia. (Figure 1) Methyl Esterification of a Series of Gangliosides and the Effects on ERMS. For the analysis of gangliosides using MS, the application of the negative ion mode to observe negatively charged ion species is advantageous because the Sia-containing glycans tend to carry negative charges.20 However, the neutral glycans and GSLs tend to generate positive ions. The nature of GSLs generally makes it difficult to decide the experimental mode (positive vs negative) to be used. Further, the MS/MS of the three abovementioned monosialylated gangliosides under positive mode resulted in the confirmation of preferential cleavages of sialyl glycosidic linkages, which is not suitable for structural investigations. We considered the possibility of applying ERMS even in such a case. We expected to observe some differences because of the activation energies of these gangliosides under CID conditions. The results of ERMS experiments of the precursor ions (m/z 1569) indicated that there were notable differences in the activation energy profiles. (Figure 2) It was found that such differences might be described by two parameters of the sigmoidal-shaped

decay curves. One is the half value of the required activation energy, and the other is the relative “width” of energy required during the dissociation. According to these parameters, the sodiated IV3NeuAcLc4Cer required the least energy for dissociation, indicating that it is the most labile species among the three gangliosides and is easily discriminated. GM1a and GM1b had very similar energy requirements (half values); however, they appeared to have different “energy widths.” Although the interpretation of the width remains undetermined, it was apparent that for the first time, it was possible to distinguish three isomeric gangliosides under positive ERMS conditions. One of the alternatives is to stabilize the labile sialyl glycoside by esterification and amidation, which enables positive ion generation and also provides various fragment ion species.21-23 Thus, we examined the ERMS analysis of esterified gangliosides. First, the effect of esterification was investigated using GM1a under the positive mode. The ERMS analyses of sodiated molecules [m/z 1569, GM1a (Figure 3 a) and m/z 1611, GM1a methyl ester (Figure 3b; closed circle)] were carried out. The Y2β ion (m/z ) 1306) associated with the cleavage of the Sia residue was observed exclusively for GM1a, whereas Y2R ion (m/z 1246), the structure resembling GM3, was the major signal, and the Y2β (m/z 1306) and Y2R/2β (m/z 941; lactosyl ceramide equivalent) ions followed. These results indicate the obvious advantage of the methyl esterification of the carboxyl function, which resulted in the stabilization of the sialyl glycosidic linkage becoming comparable to the bond energies of other glycosidic linkages. It was also obvious that the sodiated GM1a dissociated at a lower CID energy as compared to its methyl ester. Thus, it was shown that methyl esterification is useful in the analysis of gangliosides under the positive ion mode and provides more fragment ions that can be further analyzed at MSn stages. It remains unclear whether the observed stabilizing effect of esterification was actually caused by the chemical modification or by an increase in the m/z value since it has been reported that the dissociation energy is proportional to the m/z value.26 To clarify this issue, we also analyzed a methyl ester of GM1a that comprises a ceramide with two missing carbons (m/z 1583). (Figure 3b; open circle) The ERMS of the two esters was almost identical except for a slight shift associated with the difference in m/z values. Note that the fragmentation profiles of these com(26) Harvey, D. J. J. Mass Spectrom. 2000, 35, 1178–1190.


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Figure 3. ERMS spectra of native (a) and methyl ester (b) of GM1a. The half values of the dissociation curves are indicated by arrows. The open and closed symbols in (b) respectively indicate GM1a carrying C36 and C38 ceramides.

pounds were indistinguishable. We concluded that the observed difference in CID energies reflects the effect of esterification. ERMS Analysis and Consideration of Activation Energies of Sodiated Methyl Esters of GM1a, GM1b, and IV3NeuAcLc4Cer. The results of ERMS analyses of GM1a, GM1b, and IV3NeuAcLc4Cer are shown in Figures 3b and 4. These results indicate a preferential cleavage of Sia and HexNAc (either GalNAc or GlcNAc). This suggests that these linkages were less stable as compared to the other linkages. Further, this suggests that the energy requirement for the dissociation of esterified sialic glycoside is similar to those of HexNAcs. The linkage positions of HexNAcs in the above series of gangliosides are different, and thus, we might have misinterpreted the phenomena. Therefore, it is important to use compounds from a combinatorial library to confirm if the glycoside of HexNAc is labile as compared to that of Hex. We selected β-GalNAc-(1f4)R-GlcNAc-OR and β-Gal-(1f4)-R-GlcNAc-OR (R: octyloxyphenyl), where the difference in structure is the functional group at C-2 (NHAc vs OH) of the nonreducing end monosaccharide.27 For the dissociations of sodiated molecules, the relationship between the sum of signal intensities of B+Y ion species and CID energy were plotted. (Figure 5) The maximum response value of the curve for the dissociation of the compound containing GalNAc was approximately two times that for the compound with Gal, suggesting the lability of the glycosidic linkage of GalNAc. We now discuss the contribution of the activation energies of the glycosidic linkages of Sia and HexNAc in the dissociation of the precursors. It is necessary to consider both B- and Y-ion species associated with the cleavage of a certain glycosyl bond. These counterions are generated depending on the coordination (27) Compounds were synthesized, synthetic details will be reported elsewhere.

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Figure 4. ERMS spectra of sodiated methyl esters of GM1b (a) and IV3NeuAcLc4Cer (b). The half values of the dissociation curves are indicated by arrows.

Figure 5. Structures and ERMS spectra with an emphasis on the dissociations corresponding to the B/Y rupture.

position of Na+. Thus, we will use the sum of intensities (plateau values) of B- and Y-ions. The relative ion yields for the cleavage of HexNAc and Sia are 65 and 20 (HexNAc/Sia ) 65/20) for the fragmentation of the methyl ester of GM1a. Those for GM1b and IV3NeuAcLc4Cer were HexNAc/Sia ) 70/25 and 10/60, respectively. These results can be explained when the activation energy of the precursor ions is described by the following equation. Ea )

∑nE

i i

(4)

The activation energy (Ea) of the precursor ions can be expressed as the sum of the product of the activation energies of individual chemical bonds (Ei) and a specific factor associated with the bond cleavage (ni). The specific factor (ni) can be a product of the restriction factor through-space interaction and the probability factor. Here, Ei can be affected by confor-

Figure 6. Schematic representation of the reaction coordinates of the dissociation of sodiated native GM1a (a), methyl esters of GM1a and GM1b (b), and methyl ester of IV3NeuAcLc4Cer (c).

mational and rotational freedom, chirality, and so forth. The total activation energy (Ea) required for the dissociation of a precursor ion is given by the sum of energies of individual chemical bonds (Ei) regardless of which single bond ruptures. The energy provided to a precursor ion diffuses throughout the ion; therefore, the sum of the bond energies of the constituent chemical bonds is required for the dissociation of a given ion. If sufficient energy is provided to an ion, any chemical bond can cleave. The probability of the occurrence of cleavage, and not the order, is considered to be governed by the order of individual bond energies. We observe the average of the molar fractions of individual ion species because of the differences in the coordination locations of cationic species such as metals. We assume that only the glycosidic linkages associated with HexNAc and Sia are responsible for the dissociation of the above gangliosides. This assumption was based on the fact that most of the observed fragment ions were related to the cleavages of these linkages. Further, the elimination of other minor factors helps us to understand the overall phenomena involved in the dissociation of gangliosides. The activation energy can be given as Ea ) EHexNAc + ESia. The unmodified GM1a dissociated at the glycosidic bond of Sia suggests EHexNAc . ESia (Figure 6a). Methyl esterification would result in an increase in ESia, and this would cause EHexNAc ≈ ESia. When we assume that EHexNAc in the dissociation of both precursors is similar, Ea increases by a factor of the difference between ESia for methylated and unmodified Sia. (Figure 6b) Therefore, fragment ions associated with the cleavage of the glycosidic linkages of HexNAc and Sia would be produced, and the dissociation energy would increase. Similarly, the methylesters of GM1a and IV3NeuAcLc4Cer can be compared. ESia for both precursors is considered to be similar. However, the preferential dissociation of the glycosidic linkage of Sia suggests that EGlcNAc > EGalNAc. (Figure 6c) The differences in Ea among these compounds can be interpreted as being those of CID energies although the applied energy

Figure 7. ERMS spectra of sodiated molecules of pyridylaminated asialogangliotetraoses, namely, Gg4-PA (a) and Lc4-PA (b).

and the observed energy dependence (apparent energy) is not the exact activation energy. The ERMS analysis provides considerable information about structurally isomeric compounds that is particularly useful in the case of oligosaccharides;14,15,28,29 in addition, it also suggests that detailed mechanistic interpretations are involved in the observed dissociation reactions under CID conditions. Analysis of Dissociations of Asialoganglio- And AsialolactoTetraose. It was observed in the above experiments that the dissociation of a linkage between GalNAc and Gal is one of the dominant cleavages. Since this was observed in both branched and linear oligosaccharides, it might be a special characteristic of the particular linkage. We were interested in examining whether the above phenomena can be observed in glycans without a Sia residue. (Figure 7) Let us focus on the CID of a linkage between HexNAc and Gal (B2/Y2) for pyridylaminated (PA-) asialogangliotetraose (Gg4-PA; β-Gal-(1f3)-β-GalNAc-(1f4)-β-Gal-(1f4)-Glc-PA) and PAasialolactotetraose (Lc4-PA; β-Gal-(1f3)-β-GlcNAc-(1f3)-β-Gal(1f4)-Glc-PA). The sum of the intensities of the B2- and Y2 ions (B2 + Y2) for Gg4-PA was 94.8%, whereas that for Lc4-PA (28) Suzuki, K.; Daikoku, S.; Ako, T.; Shioiri, Y.; Kurimoto, A.; Ohtake, A.; Sarkar, S. K.; Kanie, O. Anal. Chem. 2007, 79, 9022–9029. (29) Daikoku, S.; Ako, T.; Kato, R.; Ohtsuka, I.; Kanie, O. J. Am. Soc. Mass Spectrom. 2007, 18, 1873–1879.


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was 49.6%. It was confirmed that the glycosidic linkage of GalNAc is more labile than that of GlcNAc. Analysis of Higher Gangliosides. It is known that oligosialylated SGLs exist on vertebrate cells. The structures of most of these species can be determined by means of traditional MS/ MS; however, some highly labile structures might be overlooked. It was reported that GM1, GD1a, GD1b, GT1b, and GQ1b were major sialo SGLs in the brain.30 Therefore, it is important to observe their distributions and the changes associated with development and disease. We believe that MS imaging may provide crucial information in this regard, and it has been used to observe the presence of GM1, GD1, and GT1. However, a description of isomeric forms such as GM1a and GM1b has not been provided.19 In the future, a method for discriminating these isomeric gangliosides will certainly be required. A method for observing and discriminating isomers of higher gangliosides is also required. On the basis of these considerations, we analyzed the fragmentation patterns of gangliosides. First, the maximum response values of the fragment signals in ERMS were obtained (Figure 8a). Although some differences existed in the m/z values because of the chain length of the ceramide, we treated them as the same ion species on the assumption that these subtle differences do not affect the fragmentations at the glycan moiety. The basis for this assumption has been described above. Therefore, it was clearly shown that all sialylated GSLs used in the experiment provided different fragmentation patterns, and they can be easily distinguished. Next, we focused on fragment (m/z 1611) ions obtained from GD1a, GD1b, GT1a, and GT1b. (Figure 8b) Although the fragment ions share the same m/z value, it is possible that these ions are either isomers (GM1a or GM1b) or a mixture of these ions. The GM1 equivalent generated from GD1a under CID conditions was further analyzed by ERMS where two possibilities regarding the cleavage of Sia exist. The observed fragmentation pattern, however, was different from those of GM1a and GM1b, indicating that the observed GM1 equivalent was a mixture of both species. Next, we examined the sodiated methyl ester of GD1b. The possible structure for the GM1 equivalent fragment ion in this case is only GM1a. In fact, the patterns matched each other well, which suggested that the structure of the fragment ion was GM1a. Experiments with GT1a and GT1b were then performed. It is possible to yield GM1a- and GM1b-equivalents in these cases; thus we might expect to observe spectra of a mixture of these species. However, it was found that GT1a and GT1b produced GM1a- and GM1b-equivalents, respectively. This indicates that the dissociation of a single linkage is preferred to multiple dissociations under the given conditions, although it is evident that multiple bond cleavages can occur, as indicated by the generation of an ion whose structure resembles a lactosyl ceramide. It was found that the GM1 equivalent ion species generated independently from higher gangliosides under CID conditions differ in terms of their structures. To summarize, it was found for the first time that GD1b and GT1a produced only GM1a, (30) Kotani, M.; Kawashima, D.; Ozawa, H.; Terashima, T.; Tai, T. Glycobiology 1993, 3, 137–146.

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Figure 8. Fragmentation profiles obtained from ERMS spectra of sodiated individual compounds. (a) Fragmentation profiles of a series of gangliosides at MS2 stage. (b) Fragmentation profiles (MS3) of GM1 equivalent obtained at MS2 resulted in a few patterns including those observed for GM1a and GM1b.

GT1b produced only GM1b, and GD1a produced a mixture of GM1a and GM1b. These findings suggest the possibility of determining the precursor ion species of a GM1 equivalent ion. Similar considerations might be applied to the conditions for in-source or post-source decays. CONCLUSION We investigated ERMS spectra of a series of gangliosides systematically and observed the following important phenomena. (1) Energy-related factors are useful in the analysis of carboxylate-free gangliosides in the positive mode although the MS/MS spectra are identical, showing only an ion that has lost sialic acid. (2) Methyl esterification is very useful in that it provides more fragment ions, and thus, more information is

obtained. (3) The dissociation reaction can be analyzed using the m/z of the produced fragment ions as well as the energyrelated factors. (4) The higher gangliosides provided distinguishable fragment patterns.5 An ion species whose m/z value is equal to that of GM1 was produced from all higher gangliosides and the structures depended on the precursor ions. At this stage of investigation, an analysis based on ERMS might not be appropriate for de novo analysis; however, a method that minimizes the sample consumption and that can potentially be automated is currently being developed.

ACKNOWLEDGMENT The authors acknowledge the financial support obtained from Mitsubishi Chemical Co. (MCC) and the Key Technology Research Promotion Program of the New Energy and Industrial Development Organization (NEDO) of the Ministry of Economy, Trade and Industry of Japan. Received for review July 31, 2008. Accepted November 11, 2008. AC801611Z


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Energy-Resolved Structural Details Obtained from Gangliosides

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