Anal. Chem. 1998, 70, 4951-4959
Structural Assignment of Permethylated Oligosaccharide Subunits Using Sequential Tandem Mass Spectrometry Nelly Viseux,†,‡ Edmond de Hoffmann,*,† and Bruno Domon§,|
Laboratoire de Spectrome´ trie de Masse, Universite´ Catholique de Louvain, 1348 Louvain-la-Neuve, Belgium, and Universite´ des Sciences et Technologies de Lille, 59655 Villeneuve d’Ascq Cedex, France
The sequential tandem mass spectrometry (MSn) capabilities offered by quadrupole ion trap instruments have been explored in a systematic study of permethylated oligosaccharides. Under collision-induced dissociation, protonated molecular species generated in the electrospray ionization mode yield simple and predictable mass spectra. Information on sequence, branching, and, to some extent, interglycosidic linkages can be deduced from fragments resulting from the cleavage of glycosidic bonds. Simple rules for the structural assignment of carbohydrates have been established for the fragmentation of protonated species and subunits thereof and corroborated by 18O-labeling experiments. Moreover, sequential tandem mass spectrometry was demonstrated to allow the straightforward structural characterization of unknown carbohydrate moieties by comparing their CID spectra with those of a set of references. As the collision-induced dissociation patterns are not dependent on the number of prior tandem mass spectrometric steps, structures can be unambiguously assigned by match of the spectra. These findings establish the basis of MSn performed on a quadrupole ion trap instrument for elucidating structures of large carbohydrates, which can be virtually degraded in the mass spectrometer into smaller entities in one or several steps. This powerful technique has been applied, used in conjunction with specific CD3 labeling, to the characterization of series of subunits generated from fucosylated and sialylated oligosaccharides, which are among the most important structures as far as biological activities are concerned. Carbohydrate moieties, conjugated to lipids or proteins, are widely distributed and play key roles in numerous and various biological processes, such as protein conformation, molecular recognition, and cellular interaction phenomena.1,2 Increasing * Corresponding author: (tel) +32 10.47.29.27; (fax) +32 10.47.29.89; (e-mail)
[email protected]. † Universite ´ Catholique de Louvain. ‡ Current address: Mass Spectrometry Resource, Boston University School of Medicine, 715 Albany St. R806, Boston, MA 02118-2526. § Universite ´ des Sciences et Technologies de Lille. | Current address: Biogen Inc., 14 Cambridge Center, Cambridge, MA 02142. (1) Lis, H.; Sharon, N. Eur. J. Biochem. 1993, 218, 1-27. (2) Varki, A. Glycobiology 1993, 3, 97-130. 10.1021/ac980443+ CCC: $15.00 Published on Web 10/28/1998
© 1998 American Chemical Society
evidence shows the close relationship existing between the functions of glycoconjugates and the glycan structures, involving more specifically the nonreducing end of these entities. It appears that variations of these patterns, such as fucosylation or sialylation, are crucial and have direct implications to carbohydrate biological activities. For full understanding of the specific functions of the glycan portions, a detailed characterization of their structures is mandatory. Carbohydrates present a wide structural diversity because of variability in interglycosidic linkages and branching, even with a very limited set of monosaccharides. Their structural characterization is, therefore, a complex task. At present, there is no universal methodology to determine these structures, in particular for samples available in mixtures or in very small amounts, as is usually the case in glycobiology. Thus, the development of new approaches to characterize the primary structure of carbohydrates remains a challenge for the analytical chemist. The different tandem mass spectrometric (MS/MS) approaches (fast atom bombardment (FAB), electrospray (ES), or matrix-assisted laser desorption/ionization (MALDI); low-energy vs high-energy collision-induced dissociation (CID)) were shown to be powerful means for the structural analysis of biomolecules and more specifically oligosaccharides.3 Most studies published to date deal with FABMS/MS of native4-7 or permethylated7-10 carbohydrates. Several studies on electrospray11,12 and laser desorption in conjunction with reflectron time-of-flight (TOF) (3) Gillece-Castro, B. E.; Burlingame A. L. Methods Enzymol. 1990, 193, 689712. (4) Zhou, Z.; Ogden, S.; Leary, J. A. J. Org. Chem. 1990, 55, 5444-5446. Hofmeister, G. E.; Zhou, Z.; Leary, J. A. J. Am. Chem. Soc. 1991, 113, 59645970. Fura, A.; Leary, J. A. Anal. Chem. 1993, 65, 2805-2811. (5) Garozzo, D.; Giuffrida, M.; Impallomeni, G. Anal. Chem. 1990, 62, 279286. Garrozzo, D.; Impallomeni, G.; Montaudo, G.; Spina, E. Rapid Commun. Mass Spectrom. 1992, 6, 550-552. (6) Orlando, R.; Bush, C. A.; Fenselau, C. Biomed. Mass Spectrom. 1990, 19, 747-754. (7) Egge, H.; Peter-Katalinic, J. Mass Spectrom. Rev. 1987, 6, 331-339. (8) Laine, R. A.; Pamidimukkala, K. M.; French, A. D.; Hall, R. W.; Abbas, S. A.; Jain, R. K.; Matta, J. L. J. Am. Chem. Soc. 1988, 110, 6931-6939. (9) Domon, B.; Mueller, D. R.; Richter, W. J. Org. Mass Spectrom. 1994, 29, 713-719. (10) Baldwin M. A.; Stahl, N.; Reinders, L. G.; Gibson, B. W.; Prusiner, S. B.; Burlingame, A. L. Anal. Biochem. 1990, 191, 174-182. (11) Garozzo, D.; Green, B. N. Carbohydr. Res. 1991, 221, 253-257. (12) Reinhold: B. B.; Chan, S. Y.; Reuber, L.; Walker, G. C.; Reinhold: V. N. J. Bacteriol. 1994, 176, 1997-2002. (13) Spengler, B.; Kirsch, D.; Kaufmann, R.; Lemoine, J. Org. Mass Spectrom. 1994, 29, 782-787.
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analyzer13,14 or sector15 instruments have also been reported. New emerging techniques such as quadrupole-time-of-flight (Q-TOF), Fourier transform (FT), and ion trap MS have also been applied to glycans.16-19 Many of the MS/MS reports have demonstrated that derivatization of oligosaccharides by permethylation remains a prerequisite in order to generate valuable structural information. Despite the drawback of involving an additional wet chemistry step, methylation increases the MS response by several orders of magnitude in all ionization modes (FAB, ES, MALDI). In addition, it allows the distinction between primary fragment ions generated by cleavage of one single glycosidic bond and inner fragments resulting from the rupture of two glycosidic linkages. In contrast to the analysis of underivatized compounds, for which both types of fragments are isobaric, labeling of hydroxyl residues by methylation allows alleviation of such ambiguities, which may compromise structural elucidation. Permethylated oligosaccharides when analyzed in the electrospray mode, as in FAB or in MALDI, usually yield intense sodium cationized molecular species. The main fragmentation process observed in the CID spectra of these ions involves the cleavage of glycosidic bonds and gives rise to pertinent data on sequence and branching.20 In addition, signals originating from sugar ring cleavages, although of much lower intensities, carry information on interglycosidic linkages. Recently, the authors reported that protonated molecular species can be generated in high yield by desalting the samples by on-line reversed-phase liquid chromatography prior to ESMS analysis.21 Under low-energy CID conditions, these species, significantly more labile than the corresponding sodium adducts, yield very simple fragmentation patterns allowing immediate and unambiguous assignment of sequence and branching. In addition, a very specific fragmentation pathway indicative of the substitution of HexNAc units has been observed. In the current study, the CID behavior of protonated molecular ions of permethylated oligosaccharides has been investigated by electrospray quadrupole ion trap mass spectrometry. The sequential MS/MS capabilities of this new type of instrument were probed for the structural characterization of carbohydrates. Special attention was devoted toward a systematic study of isomeric subunits generated from fucosylated and sialylated oligosaccharides, by comparing and eventually matching the CID patterns of fragment ions. EXPERIMENTAL SECTION Sample Preparation. Disaccharide and LNnH and LNDFH2 oligosaccharides (Table 1) were purchased from Sigma (St. Louis, (14) Lemoine, J.; Chirat, F.; Domon, B. J. Mass Spectrom. 1996, 31, 908-912. (15) Harvey, D. J.; Bateman, R. H.; Green, M. R. J. Mass Spectrom. 1997, 32, 167-187. (16) Solouki, T.; Reinhold: B. B.; Costello, C. E.; O’Malley, M.; Guan, S.; Marshall, A. G. Anal. Chem. 1998, 70, 857-864. (17) Penn, S. G.; Cancilla, M. T.; Lebrilla, C. T. Anal. Chem. 1996, 68, 23312339. (18) Asam, M. R.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1997, 8, 987-995. (19) Weiskopf, A. S.; Vouros, P.; Harvey, D. J. Rapid Commun. Mass Spectrom. 1997, 11, 1493-1504. (20) Reinhold: V. N.; Reinhold: B. B.; Costello, C. E. Anal. Chem. 1995, 67, 1772-1784. (21) Viseux, N.; de Hoffmann, E.; Domon, B. Anal. Chem. 1997, 69, 31933198.
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Table 1. Structures of Oligosaccharides
MO) and Dextra Laboratories Ltd. (Reading, U.K.), respectively. 2AGN1 oligosaccharide was a generous gift from Dr. G. Strecker (University of Lille). All other oligosaccharides were obtained from Oxford Glycosciences (Abingdon, U.K.). H218O, CD3I, and 2-aminopyridine were purchased from Aldrich Chemical Co. (Milwaukee, WI), NaOH and CH3I were from Fluka Chemie AG (Buchs, CH), and anhydrous DMSO was from Pierce Chemical Co. (Rockford, IL). Oligosacharides were permethylated using the protocol of Ciucanu and Kerek.22 Briefly, 10-50 µg of native oligosaccharide was dissolved in 10 µL of anhydrous DMSO, and 100 µg of dried powdered NaOH was added. The mixture was sonicated for 30 min and frozen prior to the addition of 10 µL of CH3I and another sonication step for 1 h. Permethylated oligosaccharide was recovered from the reaction mixture by extractions with chloroform and was extensively washed prior to mass spectrometric analysis. In the case of sialylated oligosaccharides, the aqueous solution was cooled in dry ice and acidified with acetic acid prior to extraction. The 18O labeling of oligosaccharides was performed, as described earlier,21 by dissolving native compounds (200 µg), previously dried overnight on P2O5, in 100 µL of H218O, 10 µL of the catalyst solution, and 2 µL of AcOH. The catalyst solution was prepared by dissolving 2.7 mg of 2-aminopyridine in 1.0 mL of anhydrous MeOH. The reaction mixture was stirred for 5 h prior to evaporation to dryness and permethylation. (22) Ciucanu, I.; Kerek, F. Carbohydr. Res. 1984, 131, 209-217.
Figure 1. ESMSn of permethylated tetrasaccharides: MS2 spectra of [M + H]+ ions at m/z 904 of LNT (a) and LNnT (b) (collision energy 18%); MS3 spectra of B2 fragments at m/z 464 of LNT (c) and LNnT (d) (collision energy 15%). Substructures derived from oligosaccharides are depicted with black symbols. Symbols: half circle, fucose; 2, sialic acid; b, galactose; 9, N-acetylglucosamine; [, glucose; O, mannose. Fragments were labeled according to the nomenclature of Domon and Costello.23
Prior to desialylation, permethylated oligosaccharide (30 µg) was subjected to alkaline hydrolysis to cleave the ester bond of sialic acid residue(s) by dissolving the derivative in 200 µL of ethanol/H2O (90:10) containing 0.1 M NaOH. The mixture was allowed to stay at room temperature for 4 h and was neutralized by 0.5 N ethanolic HCl. The desialylation step was carried out by adding 400 µL of ethanol/H2O (90:10) containing 1.0 M HCOOH and stirring the vial overnight at 80 °C. After evaporation under N2, the partially methylated asialooligosaccharide was specifically alkylated with CD3I. Mass Spectrometry. Sequential tandem mass spectrometry (MSn) spectra were measured on a quadrupole ion trap instrument (LCQ, Finnigan MAT, San Jose, CA) using electrospray ionization. Samples were dissolved at a concentration of 10 pmol/µL in MeOH/H2O (90:10) containing 1 mM ammonium acetate and injected through a capillary into the instrument source. Experiments were carried out using a source capillary temperature of 250 °C; the electrospray voltage was set at 3.8 kV, the capillary voltage at 2 V, and the voltage offset of the first octopole at -7.2 V. The CID spectra were obtained using helium as a damping gas introduced in the trap at a flow rate of 1 mL/min. The collision energy, expressed in an arbitrary unit, ranging between 0.1 and 20%, was optimized to generate a maximum of pertinent information. The maximum ion collection time was set at 100 ms. The mass selection window was commonly set to 10 u,
except for MS2 experiments on ions below 500 u for which it was set at 1.5 u. RESULTS AND DISCUSSION Permethylated carbohydrate derivatives when analyzed in the electrospray ionization mode form preferentially sodiated molecular adducts. Protonated species can be nevertheless easily generated in high abundance by using an on-line reversed-phase HPLC desalting step prior to MS analysis21 or, alternatively, by dissolving samples in an ammonium acetate buffer as described in the Experimental Section. The protonated molecular ions of permethylated oligosaccharides, or fragments thereof, produced in this way were subjected to CID in the ion trap cell, to study their fragmentation by sequential tandem mass spectrometry (MSn). The MS/MS spectra of the molecular species at m/z 904 of the two isomeric tetrasaccharides LNT and LNnT (Table 1) present striking differences, as illustrated in Figure 1a and b, respectively. The cleavage of the glycosidic bonds, favored at the HexNAc residues, yields B ions at m/z 464 (B2), 668 (B3), and 872 (B4) (for nomenclature, see Scheme 1a and ref 23). The B nature of these fragments, i.e., bearing the nonreducing end of the oligosaccharide, was corroborated by MS experiments on 18O(23) Domon, B.; Costello, C. E. Glycoconjugate J. 1988, 5, 397-409.
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Scheme 1. Fragmentation of Protonated Permethylated Oligosaccharides (a) and Permethylated N-Acetylhexosamine-Containing B Ions (b)
labeled derivatives. A heavy oxygen atom was incorporated into the hemiacetal group of the reducing end of the native oligosaccharides by exchange in H218O prior to methylation. In a MS/ MS experiment of the labeled oligosaccharides (parent ions at m/z 906), none of the fragments mentioned above did exhibit a mass shift (data not shown). Both spectra of Figure 1 are dominated by the B2 ion at m/z 464, which corresponds to the Hex-HexNAc subunit, but only the LNnT isomer is characterized by an intense signal at m/z 432. This ion results from a subsequent loss of a molecule of methanol from the B2 ion to yield an E-type conjugated diene fragment, as tentatively formulated in Scheme 1b. This fragment reflects the structural difference between these two disaccharidic subunits, i.e., (1-3) vs (14) interglycosidic linkage on the HexNAc unit for LNT and LNnT, respectively. However, with the ion trap instrument used, only fragments with a mass exceeding 30% of that of the parent ion can be detected. This means that, in case of a substitution in position 3, the elimination of a glycosyl residue from a B2 ion leads to a fragment going beyond the detection window. However, this problem can be easily overcome by including an additional MS step, i.e., a MS3 experiment. The parent ion at m/z 904 is then subjected to collision to yield the ion at m/z 464, which is subsequently mass-selected and fragmented in the next step to yield the mass spectra shown in Figure 1c and d. The differences observed between these spectra reflect the structural variation between the two B2 fragments previously mentioned. The specific elimination of the substituents linked in position 3 of the HexNAc residues leads to the E ions at m/z 228 and 432 for LNT and LNnT, respectively. This fragmentation was reported earlier in FABMS/ MS7,9,24 and ESMS/MS.21 In both cases, a subsequent elimination of the substituent in position 4 gives rise to the E2′ ion at m/z 196. To establish the CID conditions that ensure an optimal differentiation of both isomers, the spectra were measured under different collision energies as shown in Figure 2. The pseudo(24) Dell, A. Adv. Carbohydr. Chem. Biochem. 1987, 45, 19-72.
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Figure 2. Pseudobreakdown curves of B ions at m/z 464 from permethylated LNT (a) and LNnT (b) derivatives. Lines: ‚‚‚, m/z 464; s, m/z 228 or 432; - - -, m/z 196. Markers: b, MS2; 2, MS3; [, MS4.
breakdown curve diagram of the LNT B2 ion is characterized by strong intensity values of the ion at m/z 228 for energies exceeding 12% (Figure 2a). At very low collision energies (
