Anal. Chem. 2003, 75, 5719-5725
Discrimination of Neolacto-Series Gangliosides with r2-3- and r2-6-Linked N-Acetylneuraminic Acid by Nanoelectrospray Ionization Low-Energy Collision-Induced Dissociation Tandem Quadrupole TOF MS Iris Meisen, Jasna Peter-Katalinic´,* and Johannes Mu 1 thing
Institute for Medical Physics and Biophysics, University of Mu¨nster, Robert-Koch-Strasse 31, D-48149 Mu¨nster, Germany
A combined strategy of thin-layer chromatography immunostaining and negative ion nanoelectrospray low-energy CID mass spectrometry was established for the differentiation of isomeric r2-3 and r2-6 sialylated neolactoseries monosialogangliosides from human granulocytes. The gangliosides investigated differed in the ceramide moiety by substitution with C16:0 or C24:1 fatty acid and in their oligosaccharide chains due to nLc4 and nLc6 core structures. With respect to the type of sialylation, the homogeneity of the HPLC-purified ganglioside fractions was verified by use of specific anti-Neu5Acr2-3Galβ14GlcNAc-R and anti-Neu5Acr2-6Galβ1-4GlcNAc-R antibodies. A clear-cut series of fragment ions for both types of isomeric gangliosides, carrying r2-3- and r2-6-linked neuraminic acid, respectively, was obtained by low-energy CID. Additionally, a characteristic ring cleavage was detected exclusively in all species with Neu5Acr26Galβ1-4GlcNAc terminus, regardless of ceramide fatty acid and oligosaccharide chain lengths. The diagnostic 0,2X 4/6 ions, generated by ring cleavage of an r2-6-linked neuraminic acid are accompanied by a simultaneous decrease of the corresponding Y4/Y6 ions. These results suggest the unequivocal discrimination of individual r2-3- and r2-6-sialylated neolacto-series monosialogangliosides by distinct fragmentation patterns in lowenergy CID tandem MS. Gangliosides are amphipathic molecules consisting of two structural elements: a lipophilic membrane anchor, the ceramide portion, formed by a long-chain amino alcohol and a fatty acid, and a hydrophilic carbohydrate moiety containing one or more sialic acids. In most vertebrates, sphingosine (d18:1; 4-sphingenine) serves as the lipid backbone, whereas fatty acids of variable chain lengths (mostly C16-C24) are attached via amide bonds to the amino group of sphingosine. Although the nature of the ceramide moiety is relevant for the biological function, e.g., signal transduction and cell regulation,1,2 the oligosaccharide * Corresponding author. Telephone: +49-251-8352308. Fax: +49-251-8355140. E-mail:
[email protected]. (1) Hannun, Y.; Luberto, C. Trends Cell Biol. 2000, 10, 73-80. (2) Spiegel, S.; Merill. A. H., Jr. FASEB J. 1996, 10, 1388-1397. 10.1021/ac0347617 CCC: $25.00 Published on Web 10/03/2003
© 2003 American Chemical Society
moiety of the gangliosides is mainly responsible for their functions such as involvement in cell-cell recognition and regulation of receptor tyrosine kinase activities.3 Most gangliosides can be grouped into one of the four main structural families: the ganglio, globo, lacto, and neolacto series.4 Terminally sialylated monosialogangliosides of the neolacto series act as receptors for lectins,5 viruses,6,7 and bacteria.8 Members of this series have shown differential expression on human lymphocytes,9 and some have been found to be overexpressed on certain tumor cells, e.g., gangliosides with Neu5AcR2-6Galβ1-4GlcNAc residues on hepatocellular carcinomas.10 Determination of the Neu5Ac linkage position, i.e., R2-3- or R2-6-linked to the sugar moiety, usually involves the use of enzymes such as the sialidase from the Newcastle disease virus, cleaving exclusively the R2-3-linked Neu5Ac moiety.11 The most common technique to distinguish glycoconjugates carrying neuraminic acid in either R2-3 or R2-6 linkage is the one by specific lectins in thin-layer chromatography (TLC) immunostains and western blots.12,13 Although mass spectrometry (MS) is routinely used for defining the primary structure of glycoconjugates,14-16 no direct mass spectrometric approach for the discrimination of (3) Hakomori, S.-I. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 225-232. (4) Mu ¨ thing, J. In Glycoscience: Chemistry and Chemical Biology, Vol. 3: Glycolipids; Fraser-Reid, B., Tatsuta, K., Thiem, J., Eds.; Springer, Heidelberg, Germany, 2001; pp 2220-2249. (5) Mu ¨ thing, J.; Burg, M.; Mo¨ckel, B.; Langer, M.; Metelmann-Strupat, W.; Werner, A.; Neumann, U.; Peter-Katalinic´, J.; Eck, J. Glycobiology 2002, 12, 485-497. (6) Suzuki, Y.; Nagao, Y.; Kato, H.; Matsumoto, M.; Nerome, K.; Nakajima, K.; Nobusawa, E. J. Biol. Chem. 1986, 261, 17057-17061. (7) Mu ¨ thing, J.; Unland, F.; Heitmann, D.; Orlich, M.; Hanisch, F.-G.; PeterKatalinic´, J.; Kna¨uper, V.; Tschesche, H.; Kelm, S.; Schauer, R.; Lehmann, J. Glycoconjugate J. 1993, 10, 120-126. (8) Johansson, L.; Miller-Podraza, H. Anal. Biochem. 1998, 265, 260-268. (9) Kniep, B.; Schakel, K.; Nimtz, M.; Schwartz-Albiez, R.; Schmitz, M.; Northoff, H.; Vilella, R.; Gramatzki, M.; Rieber, E. P. Glycobiology 1999, 4, 399-406. (10) Taki, T.; Yamamoto, K.; Takamatsu, M.; Ishii, K.; Myoga, A.; Sekiguchi, K.; Ikeda, I.; Kurata, K.; Nakayama, J.; Handa, S.; Matsumoto, M. Cancer Res. 1990, 50, 1284-1290. (11) Yohe, H. C.; Wallace, P. K.; Berenson, C. S.; Ye, S.; Reinhold: B. B.; Reinhold, V. N. Glycobiology 2001, 11, 831-841. (12) Johansson, L.; Johansson, P.; Miller-Podraza, H. Anal. Biochem. 1999, 267, 239-241. (13) Haselbeck, H.; Schickaneder, E.; von der Eltz, H.; Ho ¨sel, W. Anal. Biochem. 1990, 191, 25-30. (14) Peter-Katalinic´, J.; Egge, H. Methods Enzymol. 1990, 193, 713-732.
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nonderivatized, isomeric gangliosides with Neu5AcR2-3Gal or Neu5AcR2-6Gal residues has been described. MS-based techniques for the investigation of carbohydrate moieties as their permethylated derivatives are well established.17 The latter give rise to characteristic fragment ions as observed by tandem mass spectrometry experiments on gas-phase ions generated by fast atom bombardment, electrospray, or matrix-assisted laser desorption/ionization (MALDI). Secondary cleavages, e.g., ring cleavages, result in diagnostic A-type ions providing information on, for example, site of sialylation and branching patterns. A direct mass spectrometric approach has been described enabling the distinction of nonderivatized R2-3- and R2-6-sialyllactose by MALDI-PSD in the negative ion mode.18 The relative intensities of the B1 fragment ions has been reported to be the decisive difference due to the preferential cleavage of R2-3-linked neuraminic acid. Recent investigations on R2-3- and R2-6-sialylated synthetic carbohydrates have shown that the position of sialic acid has a dramatic effect on the fragmentation pattern in MALDIPSD and ESI-collision-induced dissociation (CID).19 In particular, 0,4A ions, generated by ring cleavage of a galactose moiety, are 2 considered to be diagnostic for sialic acid R2-6-linked to the galactose residue. In the present study, we report on a combined strategy of TLC immunostaining and nanoESI MS/MS for distinguishing nonderivatized R2-3- and R2-6-sialylated neolactoseries monosialogangliosides by diagnostic fragment ions obtained by low-energy CID. MATERIALS AND METHODS Nomenclature. Abbreviations and corresponding structures of the gangliosides used in this study are as follows: GM3 or Neu5AcR2-3Galβ1-4Glcβ1-1Cer; IV3/6nLc4Cer or Neu5AcR23/6Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1Cer; VI3/6nLc6Cer or Neu5AcR2-3/6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ14Glcβ1-1Cer; sialyl-Lewisx or Neu5AcR2-3Galβ1-4(FucR1-3)GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1Cer or Neu5AcR2-3Galβ1-4(FucR1-3)GlcNAcβ1-3(Galβ1-4GlcNAcβ13)2Galβ1-4Glcβ1-1Cer; VIM-2 or Neu5AcR2-3Galβ14GlcNAcβ1-3Galβ1-4(FucR1-3)GlcNAcβ1-3Galβ1-4Glcβ11Cer or Neu5AcR2-3Galβ1-4GlcNAcβ1-3Galβ1-4(FucR1-3)GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1Cer; GM1 or Galβ1-3GalNAcβ1-4(Neu5AcR2-3)Galβ1-4Glcβ1-1Cer; GD1a or Neu5AcR2-3Galβ1-3GalNAcβ1-4(Neu5AcR2-3)Galβ14Glcβ1-1Cer; GD1b or Galβ1-3GalNAcβ1-4(Neu5AcR28Neu5AcR2-3)Galβ1-4Glcβ1-1Cer; GT1b or Neu5AcR23Galβ1-3GalNAcβ1-4(Neu5AcR2-8Neu5AcR2-3)Galβ14Glcβ1-1Cer. Gangliosides. A ganglioside mixture containing GM3 and neolactoseries gangliosides IV3nLc4Cer(d18:1, C16:0/C24:1), IV6nLc4Cer(d18:1, C16:0/C24:1), VI3nLc6Cer(d18:1, C16:0/ C24:1), and VI6nLc6Cer(d18:1, C16:0/C24:1) as major components was isolated from human granulocytes () total human granulocyte gangliosides, HGG) and further separated by TMAE-fractogel anion-exchange HPLC as already reported.20 The fractions HGG1 (15) Reinhold, V. N.; Reinhold, B. B.; Costello, C. E. Anal. Chem. 1995, 67, 1772-1784. (16) Dell, A.; Morris, H. R. Science 2001, 291, 2351-2356. (17) Peter-Katalinic´, J. J. Mass Spectrom. Rev. 1994, 13, 77-98. (18) Yamagaki, T.; Nakanishi, H. Glycoconjugate J. 1999, 16, 385-389. (19) Wheeler, S. F.; Harvey, D. J. Anal. Chem. 2000, 72, 5027-5039. (20) Mu ¨ thing, J. Methods Enzymol. 2000, 312, 45-64.
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Figure 1. Orcinol stain (A), anti-Neu5AcR2-3Galβ1-4GlcNAc-R (AB2-3) antibody (B), and anti-Neu5AcR2-6Galβ1-4GlcNAc-R (AB2-6) antibody TLC overlay assay (C) with HPLC-purified neolactoseries monosialogangliosides. (A): lane a, 15 µg of total HGG; lane b, 6 µg of HGG1 (IV3nLc4Cer/VI3nLc6Cer); lane c, 4 µg of HGG2 (VI3nLc6Cer); lane d, 4 µg of HGG3 (IV6nLc4Cer); lane e, 6 µg of HGG4 (IV6nLc4Cer/VI6nLc6Cer); lane f, 15 µg of human brain gangliosides. For TLC overlay assays (B, C), half quantities of gangliosides were applied, respectively. Only the most prominent compounds of each HPLC fraction are mentioned. Minor fucosylated neolacto-series gangliosides are referred to in Results and Discussion.
and HGG2 contained as major compounds R2-3-sialylated IV3nLc4Cer and VI3nLc6Cer, respectively. Fraction HGG3 represents IV6nLc4Cer whereas fraction HGG4 contained IV6nLc4Cer, VI6nLc6Cer, and to a minor extent VIII6nLc8Cer, all carrying terminal Neu5Ac in R2-6 linkage. A preparation of human brain gangliosides (HBG), composed of GM1, GD1a, GD1b, and GT1b, used as negative control in the overlay assays, was purchased from Supelco Inc. (Bellefonte, PA). High-Performance Thin-Layer Chromatography (HPTLC). Gangliosides were separated on glass-backed Silica Gel 60 precoated HPTLC plates (size 10 cm × 10 cm, thickness 0.2 mm, Merck, Darmstadt, Germany) for 25 min using a mixture of chloroform/methanol/water (120/85/20, each by volume) supplemented with 2 mM CaCl2 and visualized with orcinol.21 TLC Immunostaining. After TLC of gangliosides, the silica gel was fixed with poly(isobutyl methacrylate) (Plexigum P28, (21) Svennerholm, L. J. Neurochem. 1956, 1, 42-53.
Table 1. Potential Antibody-Binding Epitopes of Neolacto-Series Monosialogangliosides from Human Granulocytes
a AB2-3: anti-Neu5AcR2-3Galβ1-4GlcNAc-R antibody; AB2-6: anti-Neu5AcR2-6Galβ1-4GlcNAc-R antibody. b R ) (Galβ1-4GlcNAcβ13)x Galβ1-4Glcβ1-1Cer(d18:1, C16:0/24:1) with x ) 0 for nLc4 core and x ) 1 for nLc6 core. c R ) (Galβ1-4GlcNAcβ1-3)y Galβ1-4Glcβ11Cer(d18:1, C16:0/24:1) with y ) 1 for nLc6 core and y ) 2 for nLc8 core. d R ) (Galβ1-4GlcNAcβ1-3)z Galβ1-4Glcβ1-1Cer(d18:1, C16:0/24:1) with z ) 0 for nLc6 core and z ) 1 for nLc8 core.
Ro¨hm, Darmstadt, Germany) followed by incubation of the plates in phosphate-buffered saline (PBS) at 37 °C overnight. The blocking procedure was performed in 1% (w/v) bovine serum albumin (BSA) in PBS for 15 min. Two polyclonal chicken antiNeu5AcR2-3Galβ1-4GlcNAc-R (AB2-3) and anti-Neu5AcR26Galβ1-4GlcNAc-R (AB2-6) antibodies were used in 1:1000 dilution in 1% BSA in PBS. AB2-3 and AB2-6 were prepared as follows: At the age of 12 weeks, two chickens were immunized according to the method of Kasai et al.22 HPLC-purified IV3nLc4Cer and IV6nLc6Cer were prepared from human granulocytes as described.20 One milligram of each ganglioside was adsorbed to 1 mg of permethylated BSA (Serva, Heidelberg, Germany) in PBS. The solutions were emulsified with an equal part of Freund’s adjuvant (Difco, Detroit, MI) in a final volume of 1 mL and administered at multiple intramuscular sites. Preimmune sera were taken just before immunization. After 4 weeks, the chickens were boostered and exsanguinated 14 days later. After 1 h of incubation, the plates were overlayed with a secondary rabbit anti-chicken IgY antibody labeled with alkaline phosphatase (Dianova, Hamburg, Germany), 1:2000 diluted in 1% BSA in PBS, 2-h incubation. Bound antibodies were visualized by color development using 0.05% (w/v) 5-bromo-4-chloro-3-indolyl phosphate (Biomol, Hamburg, Germany) in glycine buffer (0.1 M glycine, 1 mM ZnCl2, 1 mM MgCl2, pH 10.4). Between each incubation step the TLC plates were washed three times with 0.05% (v/v) Tween 21 in PBS. Additionally, two washing steps with glycine buffer were performed before the substrate solution was added. Mass Spectrometry. All MS experiments were performed using a QTOF mass spectrometer equipped with a nanospray (22) Kasai, M.; Iwamori, M.; Nagai, Y.; Okumura, K.; Tada, T. Eur. J. Immunol. 1980, 10, 175-180.
manipulator (Micromass, Manchester, U.K.). Capillaries were made in-house from borosilicate glass (Hilgenberg, Malsfeld, Germany) using a vertical pipet puller (model 720, David Kopf Instruments, Tujunga, CA). High voltage was applied via a steel wire to the sample solution. Negative ion mode was used exclusively for the mass spectrometric investigation of the neolacto-series monosialogangliosides. The capillary voltage was set to 1.1 kV, and the voltage on the counter electrode was set to 140 V. For MS/MS experiments, the singly charged precursor ions were selected with the first quadrupole by increasing the dc/ac ratio. CID was performed using argon as collision gas whereby the pressure in the collision cell was ∼3.5 × 10-5 mbar. The collision energy applied was set to values reducing the intensity of the precursor ion signal to one-third of its original intensity, at best. The HPLC-purified fractions HGG1-HGG4 were dissolved in pure distilled methanol (Merck) to a concentration of ∼0.05 µg/µL. MS spectra were obtained by combining 36-51 scans/spectrum, resulting in an acquisition time of ∼1.5 min. For MS/MS spectra, acquisition time was 5-15 min and 213694 single scans were combined. RESULTS AND DISCUSSION Gangliosides from Human Granulocytes. Besides GM3, the predominant ganglioside species of granulocytes belong to the neolacto series based on the neutral core (Galβ1-4GlcNAcβ13)nGalβ1-4Glcβ1-1Cer (n ) 1 for nLc4Cer and n ) 2 for nLc6Cer).4,7 The attachment of one sialic acid to the terminal galactose residue gives rise to the neolacto-series monosialogangliosides IV3/6Neu5Ac-nLc4Cer and VI3/6Neu5Ac-nLc6Cer. The orcinol stain of the total ganglioside fraction is shown in Figure 1A, lane a. Ganglioside species containing the same sugar moiety separate in double bands, due to the substitution of the sphinAnalytical Chemistry, Vol. 75, No. 21, November 1, 2003
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Figure 2. NanoESI mass spectra of HPLC-purified R2-3- and R2-6-sialylated neolacto-series monosialogangliosides obtained in the negative ion mode. MS1 spectra of HGG1 (A), HGG2 (B), HGG3 (C), and HGG4 (D) correspond to the TLC lanes b-e of Figure 1, respectively. Species, further investigated by MS/MS, are marked with asterisks (see Figures 3 and 5).
gosine moiety (d18:1) with different fatty acids, C24:1 and C16:0, resulting in upper and lower bands, respectively. The total ganglioside mixture was further separated by TMAE-fractogel HPLC20 resulting in four fractions (HGG1-HGG4) containing either R2-3- (HGG1, HGG2) or R2-6-linked sialic acid species (HGG3, HGG4) exclusively. The orcinol stain of the four HPLC fractions obtained is shown in Figure 1A, lanes b-e. TLC Immunostaining of Isomeric Gangliosides. To assess sample homogeneity with respect to the type of sialylation of fractions HGG1-HGG4, TLC overlay assays with an antiNeu5AcR2-3Galβ1-4GlcNAc-R (AB2-3) antibody (Figure 1B) and an anti-Neu5AcR2-6Galβ1-4GlcNAc-R (AB2-6) antibody (Figure 1C) were performed. It could be clearly shown that HGG1 (Figure 1B and C, lane b) and HGG2 fraction (Figure 1B and C, lane c) contain neolacto-series monosialoganglioside species with R2-3-linked Neu5Ac exclusively, i.e., IV3nLc4Cer and VI3nLc6Cer, whereas fractions HGG3 (Figure 1B and C, lane d) and HGG4 (Figure 1B and C, lane e) contain the isomeric species IV6nLc4Cer or VI6nLc6Cer with neuraminic acid in the R2-6 position. The additional double bands detected by reaction with AB2-3 in fraction HGG1 (Figure 1B, lane b), are known to represent fucosylated VI3nLc6Cer(d18:1, C16:0/C24:1) and VIII3nLc8Cer(d18:1, C16:0/C24:1) species with a VIM-2 structure,23 respectively, carrying the fucose at the inner GlcNAc residue. This carbohy(23) Metelmann, W.; Peter-Katalinic´, J.; Mu ¨ thing, J. J. Am. Soc. Mass Spectrom. 2001, 12, 964-973.
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drate moiety exhibits a binding epitope recognizable by AB2-3, whereas fucosylation of the subterminal GlcNAc residue, characteristic for the sialyl-Lewisx structure, prevents antibody binding (Table 1). MS1 Spectra of Fractions HGG1-HGG4. The fractions HGG1-HGG4, each containing the Neu5Ac moiety of a defined linkage exclusively, were further investigated by nanoESI MS in the negative ion mode. Figure 2 shows the MS1 spectra obtained from the HPLC-purified ganglioside fractions. The species further investigated by low-energy CID mass spectrometry are marked by asterisks. Under the conditions used, only singly charged molecular ions [M - H]- were detected. As already shown by the TLC orcinol stain and the TLC overlay assays, HGG1 contains IV3nLc4Cer(d18:1, C16:0/C24:1) as major compounds (Figure 2A) whereas the VI3nLc6Cer(d18:1, C16:0/C24:1) species are enriched in fraction HGG2 (Figure 2B). The chromatographic behavior of the neolacto-series monosialogangliosides observed in TLC, i.e., the separation into double bands, is reflected in the mass spectra by the existence of two peaks for each species differing by 110 Da in their molecular masses. This mass shift is due to the substitution of the sphingosine moiety with either a C16:0 or C24:1 fatty acid. The MS1 spectra of the fractions HGG3 and HGG4 containing neolacto-series monosialogangliosides with Neu5Ac in R2-6 linkage exclusively are shown in Figure 2C and D, respectively. As marked by asterisks, the species IV6nLc4Cer(d18:1, C16:0/
Figure 3. Negative ion mode MS/MS spectra of the gangliosides IV3nLc4Cer(d18:1, C16:0) (A) and IV6nLc4Cer(d18:1, C16:0) (B) with m/z 1516.85 and 1516.99, respectively. The corresponding m/z values of the CID-derived fragment ions are assigned in the spectra. Y4 and diagnostic ions are printed in boldface type.
C24:1) of HGG3 (Figure 2C) and VI6nLc6Cer(d18:1, C16:0/C24: 1) of HGG4 (Figure 2D) were chosen for MS/MS experiments. The low-abundant fucosylated VI3nLc6Cer(d18:1, C16:0/C24: 1) and VIII3nLc8Cer(d18:1, C16:0/C24:1) species with VIM-2 structures, observed in fraction HGG1 by reaction with AB2-3 (Figure 1B, lane b), could be detected neither by TLC orcinol stain (see Figure 1A, lane b) nor by mass spectrometry under the conditions used. On the other hand, fraction HGG2 showed beside the expected molecular ions of IV3nLc4Cer(d18:1, C16:0/ 24:1) and VI3nLc6Cer(d18:1, C16:0/24:1) two additional molecular ions at m/z 2028.17 and 2138.30 (Figure 2B), also visible by TLC orcinol stain (see Figure 1A, lane c) but not detectable with AB2-3 (Figure 1B, lane c). The mass difference of 146 Da in comparison to the VI3nLc6Cer(d18:1, C16:0/C24:1) gangliosides gives evidence for fucosylated VI3nLc6Cer(d18:1, C16:0/C24:1) species. Additionally, the structural relationship within these substances is deducible from the difference of 110 Da in their molecular masses as described above. These gangliosides, each containing the sialyl-Lewisx structure with a fucose residue attached to the penultimate GlcNAc moiety,23,24 are not recognized by AB2-3 in the overlay assay (Figure 1B, lane c). Thus, AB2-3 binds well to VIM-2 determinants (Figure 1B, lane b), whereas the sialyl-Lewisx
structure is not a binding epitope for this antibody (Figure 1B, lane c). A survey of the antibody specificities is given in Table 1. MS/MS Spectra of Neolacto-Series Monosialogangliosides. The low-energy CID spectra of nLc4Cer(d18:1, C16:0) carrying neuraminic acid in R2-3 and R2-6 linkage, respectively, are depicted in Figure 3A and B. Their structures with the corresponding fragmentation schemes are shown in Figure 4A and B. The fragment ions are assigned according to the nomenclature for carbohydrate fragmentation by Domon and Costello.25 In both spectra, a series of high-abundant Y-type ions is present, starting with loss of sialic acid (Y4 at m/z 1225.84 and 1225.92, respectively) and followed by consecutive cleavage of the glycosidic bonds ending up with the Y0 fragment ion representing the ceramide moiety (d18:1, C16:0). A few B-type ions and internal double bond cleavages give further evidence for the sequence of the oligosaccharide chain. Besides these well-characterized ions, three additional fragment ions with m/z 306.21, 468.29, and 1295.93 are detected in the CID spectrum of R2-6-sialylated IV6nLc4Cer(d18:1, C16:0) (Figure 3B), hardly distinguishable from the noise in the CID spectrum of R2-3-sialylated IV3nLc4Cer(d18:1, C16:0) (Figure 3A). The fragment ion at m/z 1295.93 shows a mass difference of 221 Da to the precursor ion. We propose that this ion is generated by a neutral loss of 221 Da occurring via X-type cleavage of the sialic acid being highly specific for Neu5Ac in R2-6 linkage. According to the nomenclature for carbohydrate fragmentation by Domon and Costello,25 this first diagnostic fragment ion at m/z 1295.93 can be assigned to a 0,2X4 ion. Additionally, in the case of the R2-6-sialylated species, the relative abundance of the Y4 ion is, as a consequence of the ring cleavage, markedly decreased in comparison to the spectrum of the R2-3-sialylated species, where the Y4 ion is beside B1 ion the most prominent fragment ion. For the species with R2-6-linked Neu5Ac, the formation of the 0,2X4 and Y4 fragment ions occurs at similar relative abundances, probably representing competitive processes. These results are in good agreement with the wellknown fact that R2-3-linked sialic acid is cleaved much easier from the sugar moiety than R2-6-linked sialic acid.18 The preferred fragmentation of the R2-3 glycosidic bond gives rise to high-abundant B1 and Y4 ions for the IV3nLc4Cer species. The higher stability of R2-6 glycosidically linked sialic acid results in occurrence of two competitive processes, i.e., cleavage of the glycosidic bond and ring cleavage of the sialic acid moiety. The fragment ion at m/z 306.21 is referred to as the second diagnostic ion for a Neu5AcR2-6 linkage, since it is, in addition to the 0,2X4 ion, detected exclusively within the R2-6-sialylated neolacto-series ganglioside species. This fragment ion, also generated via a specific ring cleavage, can be assigned to a 0,4A2 ion, accompanied by loss of CO2 of the R2-6-linked sialic acid as already described for synthetic carbohydrates.19 A third specific ring cleavage, represented by the ion at m/z 468.29, is again detected only for the R2-6-sialylated species in significant abundance. This diagnostic 2,4A3 ion, generated by ring cleavage within the GlcNAc residue, is also accompanied by loss of CO2 from the R2-6-linked sialic acid. (24) Mu ¨ thing, J.; Spanbroek, R.; Peter-Katalinic´, J.; Hanisch, F.-G.; Hanski, C.; Hasrgawa, A.; Unland, F.; Lehmann, J.; Tschesche, H.; Egge, H. Glycobiology 1996, 6, 147-156. (25) Domon, B.; Costello, C. E. Glycoconjugate J. 1988, 5, 397-409.
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Figure 4. Molecular structures and fragmentation schemes of IV3nLc4Cer(d18:1, C16:0) (A) and IV6nLc4Cer(d18:1, C16:0) (B). Y4 and diagnostic ions are printed in boldface type. Table 2. Type of Fragment Ions and Corresponding m/z Values of Individual Neolacto-Series Monosialogangliosides with nLc6 Core Obtained by Low-Energy CIDa m/z values fragment ion 0,4A
2
- CO2b CO2b
30,2X b 6 2,4A
Y0; Z0 Y1; Z1 Y2; Z2 Y3; Z3 Y4; Z4 Y5; Z5 Y6; Z6 B1 B2; B2 - CO2 B3; B3 - CO2 B4; C4 B5 B6 HexHexHexNAcY6/B4 Z5/B5 Y5/B5 Y6/2,4A5 Y5/2,4A6 Y6/B5 Y6/B6 a
VI3nLc6Cer(d18:1,
C16:0)
ndc “468.27”d “1661.20” 536.64; “518.61” 698.70; 680.69 860.80; “842.77” 1063.91; 1045.87 1225.98; nd 1429.11; 1411.06 1591.18; nd 290.18 “452.25”; 408.27 “655.36”; “611.37” nd; nd nd nd 179.13 364.22 526.30 549.33 567.34 586.33 “628.36” 729.41 “891.51”
VI6nLc6Cer(d18:1,
C16:0)
306.23 468.32 1661.25 536.66; 518.64 698.73; 680.72 860.83; “842.84”d 1063.94; 1045.95 1226.03; nd 1429.15; 1411.15 1591.22; nd 290.18 452.27; 408.29 655.39; “611.40” 817.49; 799.46 1020.61 1182.71 179.13 364.24 526.33 549.36 567.37 586.36 628.39 729.44 891.54
VI3nLc6Cer(d18:1, C24:1)
VI6nLc6Cer(d18:1, C24:1)
nd “468.30” “1771.34” 646.78; nd 808.86; 790.83 970.94; nd 1174.07; 1156.04 1336.16; nd 1539.28; 1521.28 1701.34; nd 290.18 “452.27”; 408.27 655.39; nd nd; nd “1020.61” nd 179.13 364.24 526.33 549.36 567.37 586.36 628.39 729.44 891.51
306.23 468.32 1771.44 646.81; nd 808.90; 790.90 970.98; nd 1174.11; 1156.08 1336.20; “1318.16” 1539.32; 1521.33 1701.43; nd 290.20 452.30; 408.29 655.42; nd 817.52; 799.49 1020.61 1182.71 179.14 364.26 526.36 549.36 567.40 586.38 628.39 729.48 891.58
Spectra not shown. b Diagnostic ion. c nd, not detected. d Low-abundant ions in quotation marks.
To investigate whether these fragment ions are truly diagnostic for R2-6-sialylated neolacto-series gangliosides, further MS/MS experiments with gangliosides varying either in the lipid or in 5724 Analytical Chemistry, Vol. 75, No. 21, November 1, 2003
the sugar moiety were performed. The low-energy CID spectra of IV3nLc4Cer and IV6nLc4Cer carrying a C24:1 instead of C16:0 fatty acid in the ceramide moiety are shown in Figure 5A and B,
VI3/6nLc6Cer(d18:1, C16:0) and VI3/6nLc6Cer(d18:1, C24:1). The R2-3- and R2-6-sialylated isomeric gangliosides were derived from fractions HGG2 and HGG4, respectively. The spectra are not shown, but the fragment ions obtained and their corresponding m/z-values are summarized in Table 2. Diagnostic ions for R26-sialylated gangliosides are printed in boldface type and m/z values of low-abundant ions are in quotation marks. The data clearly reveal that neither the ceramide fatty acid nor the oligosaccharide chain length exhibits any influence on the specific fragmentation pattern of Neu5AcR2-6-substituted gangliosides. All diagnostic ions, i.e., the fragment ions at m/z 306.23 and 468.32 and in case of the VI6nLc6Cer(d18:1, C16:0/C24:1) the 0,2X6 ion at m/z 1661.25 and 1771.44, respectively, were detected only within the R2-6-sialylated species. As a consequence of the first diagnostic fragment ion obtained by ring cleavage, the relative abundance of the Y6 ion is again decreased conspicuously in the spectra of the species with R2-6-linked Neu5Ac. The higher stability of R2-6 glycosidically linked sialic acid, giving rise to the competitive ring cleavage reaction, is reflected in all spectra by the presence of a complete series of B-type ions detected only partially or in lower abundance in gangliosides with R2-3-linked sialic acid. The distinct fragmentation spectra obtained, enabled a clear distinction between R2-3- and R2-6-sialylated gangliosides. Diagnostic decarboxylated 0,4A2-type ions at m/z 306.21/306.23, 2,4A -type ions at m/z 468.29/468.32, and the presence of 3 0,2X /0,2X fragment ions obtained by sialic acid ring cleavage 4 6 accompanied by a clearly decreased relative intensity of Y4/Y6 ions can be correctly assigned to gangliosides with the Neu5AcR2-6Gal terminus. Figure 5. Negative ion mode MS/MS spectra of the gangliosides IV3nLc4Cer(d18:1, C24:1) (A) and IV6nLc4Cer(d18:1, C24:1) (B) with m/z 1627.14 and 1627.19, respectively. The corresponding m/z values of the CID-derived fragment ions are assigned in the spectra. Y4 and diagnostic ions are printed in boldface type.
respectively. The substitution of the ceramide moiety with a C24:1 fatty acid results in a mass shift of 110 Da for all Y- and Z-type ions compared to the spectra of both gangliosides with a C16:0 fatty acid (see Figure 3). In the spectrum of the R2-3-sialylated species (Figure 5A), the most prominent Y4 ion is generated by loss of neuraminic acid whereas the relative intensity of this ion is much lower in the spectrum of the R2-6-sialylated species (Figure 5B). As stated above, the fragmentation leading to the Y4 ion seems to be in competition with the linkage-specific 0,2X4 ring cleavage of the neuraminic acid resulting in similar relative abundances of both ions. Additionally the second and third diagnostic ion, i.e., 0,4A2 - CO2 ion at m/z 306.21 and 2,4A3 - CO2 at m/z 468.29, were again detected exclusively in the spectrum of the ganglioside with R2-6-linked neuraminic acid. These results clearly indicate that there is no impact of the fatty acid chain length on formation of the diagnostic fragment ions, specific for neolactoseries gangliosides with terminally R2-6-linked neuraminic acid. Final investigations concerning the different fragmentation behavior of R2-3- and R2-6-sialylated gangliosides with respect to an oligosaccharide chain prolongation were performed with
CONCLUSIONS The HPLC-purified neolacto-series monosialoganglioside fractions analyzed in this study provide an excellent model for investigating the fragmentation patterns of gangliosides containingneuraminic acid of different, well-defined linkages. The combination of immunochemical methods and tandem mass spectrometry enabled the unambigious discrimination of isomeric compounds such as IV3/IV6nLc4Cer and VI3/VI6nLc6Cer. The purity of the HPLC fractions with respect to the sialylation type, a prerequisite for our study, was first demonstrated in TLC overlay assays using highly specific antibodies. The subsequent mass spectrometric investigation by nanoESI-QTOF low-energy CID in the negative ion mode gave clear-cut results for distinction of neolacto-series monosialogangliosides with Neu5AcR2-6Gal and Neu5AcR2-3Gal termini according to their specific diagnostic fragmentation behavior. This is the first report on differentiation of isomeric gangliosides with R2-3- and R2-6-linked Neu5Ac by mass spectrometry without prior chemical modification of the carbohydrate moiety.
Received for review July 8, 2003. Accepted August 11, 2003. AC0347617
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