Structural Elucidation of Zwitterionic Sugar Cores from Glycosphingolipids by Nanoelectrospray Ionization–Ion-Trap Mass Spectrometry

Analytical Biochemistry 284, 279 –287 (2000) doi:10.1006/abio.2000.4681, available online at http://www.idealibrary.com on

Structural Elucidation of Zwitterionic Sugar Cores from Glycosphingolipids by Nanoelectrospray Ionization–Ion-Trap Mass Spectrometry Claudia H. Friedl,* Gu¨nter Lochnit,* Rudolf Geyer,* Michael Karas,† and Ute Bahr† ,1 *Institute of Biochemistry, University of Giessen, Friedrichstrasse 24, D-35392 Giessen, Germany; and †Division for Instrumental Analytical Chemistry, J.-W. Goethe University Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany

Received February 3, 2000

The use of electrospray ionization (ESI)–ion-trap mass spectrometry (ITMS) for analysis of zwitterionic, glycolipid-derived sugar cores of glycosphingolipids is described. The capability of the method to perform multiple steps of fragmentation (MS n) allows structural characterization of these compounds. No derivatization of the released oligosaccharides is necessary when using nano-ESI with sample solution flow rates of about 30 nL/min. Investigations of positive as well as negative ions in fragmentation experiments up to MS 4 permit determination of the sequence of sugar units, their linkage positions, and the exact location of the substituents phosphocholine and phosphoethanolamine. In the case of phosphocholine, chemical cleavage of this substituent was necessary to obtain all the linkage information. Approximately 150 –250 ng of sample was needed for each analysis. © 2000 Academic Press Key Words: structural analysis; carbohydrates; glycosphingolipids; ESI-MS; ion-trap MS; phosphocholine; phosphoethanolamine; nematodes.

The introduction of soft ionization techniques such as matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF-MS) 2 and electrospray ionization mass spectrometry (ESI-MS) in the 1980s has created the possibility of analyzing intact biomolecules with high sensitivity, speed, and accuracy. The analysis of fragment ions by postsource decay 1 To whom correspondence should be addressed. Fax: ⫹49 69-6313 984. E-mail: [email protected]. 2 Abbreviations used: MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; ESI-MS, electrospray ionization mass spectrometry; PC, phosphocholine; PE, phosphoethanolamine; PGC, porous graphitic carbon; HexNAc, Nacetylhexosamine.

0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

(PSD) MALDI-TOF-MS (1) and electrospray ionization collision-induced dissociation mass spectrometry (ESICID-MS) (2) has allowed further detailed structural analyses of, for example, peptide sequences, posttranslational modifications, and, in part, linkage positions of monosaccharide constituents of glycans. The development of quadrupole MS with orthogonal TOF analyzers (QTOF) (3, 4) and the coupling of MALDI and ESI sources with Fourier-transform ion cyclotron resonance analyzers (FT-ICR) (5, 6) have allowed extremely sensitive and high-resolution mass spectrometric analyses. It was the introduction of quadrupole ion-trap (IT) technology (7–10) which now permits sequential MS/MS operations up to MS 11 by selection of a precursor ion even from complex sample mixtures. The MS n spectra contain much more information than single MS/MS spectra and can provide insight into the origin and identity of fragments. Hence, detailed structural analysis of compounds containing labile substituents, the loss of which often dominates the MS/MS spectrum, can be performed only by multiple steps of fragmentation. The mass spectrometric structural analysis of glycans with their potential heterogeneity in branching, substitution positions, and anomeric configurations as well as the simultaneous presence of labile and more stable glycosidic bonds is a promising application for ESI-ITMS; however, the hydrophilic character of glycans reduces the ionization efficiency and requires derivatization by methylation or acetylation prior to conventional LC–MS coupling with high flow rates (11– 13). The introduction of nanospray technology (14, 15) with its improved desolvation process and higher ionization efficiency has created the possibility of analyzing underivatized glycans. Localization of noncarbohydrate substituents of glycoconjugates has gained increasing interest in the past 279

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few years, due to their implication in biological function and/or activity (16). For diagnosis, screening assays, and drug development, there is a great need for rapid structural elucidation methods applicable to low amounts of material. Phosphocholine (PC) and phosphoethanolamine (PE) substituents on protein and lipid structures fulfil these characteristics for method development, since these modifications can be regarded as markers for nematode and bacterial infections, although detailed structural information is often lacking, and may be considered as potential targets for anthelmintic and antibacterial therapeutics (17). Mass spectrometric characterization of zwitterionic glycosphingolipids from the porcine nematode parasite Ascaris suum by MALDI-TOF-MS revealed a characteristic fragmentation of the phosphocholine and phosphoethanolamine substituents (18). The exact localization of these residues was not possible by this method, due to low fragmentation yield of the carbohydrate backbone and the absence of ring-cleavage products. Methylation analyses before and after HF treatment of the major components revealed PC and PE substituents at C-6 of N-acetylglucosamine and C-6 of mannose, respectively (18). This experimental approach, however, proved to be time-consuming and required comparatively large amounts of material. The aim of this study is to show that ESI-MS n with an ion-trap instrument is a valuable tool for identification and structural elucidation of glycans carrying zwitterionic substituents such as PE and/or PC. Nano-ESI with sample spray rates of about 30 nL/min has been chosen not only because of its low sample consumption but also because of a higher ionization efficiency of carbohydrates compared to ESI with flow rates in the ␮l/min range (17), thus eliminating the need for derivatization (11–13). Two zwitterionic compounds, components A and C, the structures of which have been previously elucidated (18), were selected to find out which structural information could be obtained by ESI-MS n in an iontrap mass spectrometer. The final goal was to develop a MS protocol allowing structural analyses of similar compounds in the future with reduced time and lower amounts of material consumed. MATERIALS AND METHODS

Isolation of Zwitterionic Glycosphingolipids Glycolipids were extracted from A. suum worms (1250 g wet weight) and purified as described previously (18, 20). Cleavage with Endoglycoceramidase Glycosphingolipids (100 mg) were dissolved by sonication in 25 mL of 50 mM acetate buffer (pH 5.0)

containing 0.1% sodium taurodeoxycholate and incubated at 37°C for 24 h, employing a final concentration of 10 mU/mL of recombinant endoglycoceramidase from Rhodococcus sp. (BioWhittaker, Verviers, Belgium). Enzyme addition was repeated after 24 h. Released oligosaccharides and remaining glycolipids were separated by phase partition with butanol–water and reverse-phase chromatography using C18ec cartridges as described elsewhere (21). Noncleaved glycolipids were subjected to repeat endoglycoceramidase treatment. Free oligosaccharides (20 mg) were desalted on porous graphitic carbon (PGC) cartridges. Desalting of Oligosaccharides PGC solid-phase extraction cartridges (200 mg of HyperSep; MZ-Analysentechnik, Mainz, Germany) were conditioned with 9 mL of water, 9 mL of H 2O– acetonitrile (5:95, v/v), and 9 mL of water consecutively. Oligosaccharides were applied to the cartridge, desalted with 9 mL of water and eluted with 6 mL of H 2O–acetonitrile (5:95, v/v) (22). Isolation of Individual Oligosaccharides by High-pH Anion-Exchange Chromatography (HPAEC) Oligosaccharides (20 ␮g) were fractionated on a CarboPak PA-100 column (Dionex, Sunnyvale, CA) with a linear gradient from 10 to 20 mM sodium acetate in 80 mM sodium hydroxide for 35 min at a flow rate of 1 mL/min using the instrumentation described elsewhere (23) and desalted directly on PGC cartridges. HF Treatment The PC substituent from component A was released by HF treatment as described elsewhere (21). Mass Spectrometry ESI-MS and ESI-MS n experiments were performed with an LCQ quadrupole ion-trap mass spectrometer (Finnigan MAT, San Jose, CA) equipped with a nanoESI source (Protana, Odense, Denmark). A 3- to 5-␮L aliquot of oligosaccharide solution (50 ng/␮L in methanol–water (1:1, v/v)) was loaded into a laboratorymade, gold-coated glass capillary and electrosprayed at a voltage of 700 –1000 V. The heated metal-transfer capillary was held at 200°C. The capillary and tube lens were held at the same potential (48 V). Positiveand negative-ion spectra were averaged over 10 scans, each scan consisting of 3 microscans. For consecutive MS–MS (MS n ) experiments, the relative collision energy in the trap varied between 30 and 70% (corresponding to the LCQ software (LCQ 1.0 software) settings defining the amplitude of the resonance

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FIG. 1. MS 2 spectra of the 18O-labeled model compound cellopentaose. The typical glycosidic bond cleavages as well as the cross-ring fragments are indicated. (a) Positive-ion spectrum; (b) negative-ion spectrum.

excitation ac voltage), depending on the chemical nature of the precursor ion. RESULTS AND DISCUSSION

For structural elucidation of carbohydrates, ESI-MS of positive and negative ions gave complementary information. This was exemplified by cellopentaose, a linear neutral oligosaccharide composed of ␤(1– 4)linked glucose residues. This test molecule was chosen because the components under investigation contained mostly ␤-linked monosaccharides. For the differentiation of isomeric fragment ions originating from the reducing and/or nonreducing end of the oligosaccharide, the anomeric OH group of cellopentaose was labeled with 18O (24). Fragmentation of positive ions (here, [M ⫹ Na] ⫹ at m/z 853) leads mainly to Y and B fragments (for nomenclature, see (25)) from glycosidic bond cleavages (Fig. 1a). Thus, the sequence of the oligosaccharide can be read from the nonreducing end by consecutive Y fragments and from the reducing end by sequential B fragments. The linkage types between sugar residues are reflected in specific cross-ring frag-

ments (24). In the positive-ion mode, the only information concerning linkages is given for the sugar unit at the reducing end (see Fig. 1a). Here, an 0,2A 5 fragment resulting from loss of 62 Da and an 2,4A 5 fragment from loss of 122 Da appear and indicate a (1– 4) linkage. It should be mentioned here that (1–3)-linked sugar unit ring fragmentation is hindered, thus preventing the formation of ring fragments. A reducing end seems to be a prerequisite for ring fragmentation of underivatized sugars, to occur presumably via ring opening. Fragmentation of negative ions provides, in addition to sequence data, information on all linkage positions in the sugar chain. Figure 1b shows the fragment-ion spectrum of the chloride adduct [M ⫹ Cl] ⫺ (m/z 864.7) of cellopentaose labeled with 18O. In contrast to positive ions, C ions are produced by collision-induced dissociation in the ion trap together with B ions of lower abundance. C ions have a new reducing end and show typical cross-ring cleavages, which in the case of (1– 4)linked hexoses are 0,2A x (C x ⫺ 60 Da), 0,2A x ⫺ H 2O (C x ⫺ 78 Da), and 2,4A x (C x ⫺ 120 Da). The ring fragments in Fig. 1b indicate that all sugar units are (1– 4)-linked.

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FIG. 2. Positive-ion MS n spectra of component A. (a) The full MS spectrum shows the predominant pseudomolecular ion [M ⫹ Na] ⫹; (b) MS 2 of [M ⫹ Na] ⫹ at m/z 1098.7 shows only one fragment ion resulting from the loss of trimethylamine (⫺59 Da); (c) MS 3 of the fragment ion at m/z 1039.3; (d) MS 4 of the Y 3 fragment present in the MS 3 spectrum leading to a series of B and Y fragments which can be used for sequence analysis.

The remaining linkage position between both sugars at the nonreducing end is determined as (1– 4) by MS 3 of the C 2 ion (spectrum not shown). With this approach, all kinds of linkages can be differentiated by their specific fragmentation patterns (26, 27).

The study of the fragmentation behavior described above was a prerequisite for the interpretation of the spectra obtained in the case of the following native molecules. Figure 2 shows the positive-ion MS and MS n spectra

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FIG. 3. Negative-ion MS n spectra of component A. (a) The full MS spectrum shows the [M ⫹ Cl] ⫺-attached molecule; (b) MS 2 of [M ⫹ Cl] ⫺ at m/z 1111.0 shows only one fragment on the loss of methyl chloride; (c) MS 3 of the fragment ion at m/z 1060.5 shows cross-ring cleavages (A fragments) within C fragments which contain information of linkage positions.

of component A (for structure, see Fig. 2b). The molecular ion appears predominantly as [M ⫹ Na] ⫹ ion, as known from unmodified oligosaccharides (m/z 1098.7 in Fig. 2a). By MS 2 of this ion, only one fragment ion resulting from the loss of 59 u is detected, which can be explained by the loss of trimethylamine from the PC group (Fig. 2b). This type of fragmentation is characteristic for all phosphocholine-substituted glycoconjugates tested so far in positive-ion nano-ESI MS/MS. MS 3 of this fragment ion (m/z 1039.3) (Fig. 2c) shows second-generation fragmentation of the oligosaccharide chain leading to Y 3, Y 4, B 3, and B 4 ions; smaller Y and B fragments are not observed. These fragments are sufficient to determine the sequence as hexose fol-

lowed by N-acetylhexosamine (HexNAc) from the nonreducing end and two hexoses from the reducing end. The remaining sugar residue has a mass of 309 Da. A mass increment of 124 Da, not known from unmodified oligosaccharides, appears twice, as loss from the precursor ion as well as from the Y 3 fragment ion; it can be explained as phosphotriester, which in this case is linked to a HexNAc. The sequence can be confirmed by a MS 4 step (Fig. 2d) of the third-generation fragment m/z 674.2, interpreted before as a Y 3 fragment. This ion loses the modified HexNAc unit (Y 2) from one end and two hexoses (B 2, B 1) from the other end. Again, a fragment ion from the loss of 124 Da is observed. The strong fragment-ion signals at m/z 979.3 in Fig. 2c and

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FIG. 4. Negative-ion MS n spectra of component A after chemical treatment with HF. (a) MS 2 of [M ⫹ Cl] ⫺ at m/z 946.2; (b) MS 3 of the C 2 fragment in (a).

m/z 614.2 in Fig. 2d are due to ring fragmentation of the reducing end sugar. To determine the linkage positions between the sugar units, the negative ions were analyzed (Fig. 3). The base peak in Fig. 3a is the chloride adduct [M ⫹ Cl] ⫺ at m/z 1111.0/1113.2 (two peaks due to the isotopic distribution of chlorine, 3:1 35Cl/ 37Cl). Upon fragmentation, this ion predominantly produces an ion at m/z 1060.5 resulting from the loss of methyl chloride (⫺50 Da, Fig. 3b). The following MS 3 spectrum (Fig. 3c) shows that the negative charge remains attached to the phosphate group and that, in this case, fragmentation is different from the one described above for unmodified oligosaccharides. Since cross-ring fragmentation, implying information about the sugar linkages, requires a reducing end (i.e., C fragmentation), from the spectrum in Fig. 3c, only three linkages can be deduced, as only the fragments C 3 and C 4 appear. The ring fragments observed are 0,2A 5, 0,2A 5 ⫺ H 2O, and 2,4 A 5 from losses of 60, 78, and 120 Da, typical for

(1– 4)-linked hexoses. In the case of the C 4 fragment, there are no ring fragments pointing to a (1–3) linkage, which hinders ring fragmentation. The C 3 fragment loses 101 and 119 Da to give the 0,2A 3 and the 0,2A 3 ⫺ H 2O fragment ions characteristic for (1– 4)-linked HexNAc residues. Thus, from the reducing end a (1– 4) linkage, followed by a (1–3) and again a (1– 4) linkage can be determined. From the appearance of the 0,2A 3 fragment, it can be concluded that this sugar unit carries a (1– 4)- and a (1– 6)-linked substituent, i.e., the PC group and the remaining disaccharide. To define their individual positions and to determine the linkage between the remaining hexose and HexNAc at the nonreducing end, treatment of the sample with HF was necessary to cleave the PC from the sugar and obtain the unmodified oligosaccharide. Fragmentation of the respective [M ⫹ Cl] ⫺ pseudomolecular ion now displays all linkages (see Fig. 4). The MS 2 spectrum (Fig. 4a) confirms a (1– 4) linkage followed by a (1–3) linkage and shows that the HexNAc is (1– 4)-linked to the

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FIG. 5. Positive-ion MS n spectra of component C. (a) MS 2 shows a base peak at m/z 1098.2 from loss of 123 Da; (b) MS 3 of m/z 1162.3. The location of PE on the second hexose unit can be elucidated from the mass increment between B 4 and B 3.

following HexNAc, thus implying that the PC residue in the original molecule was linked to C-6 of the sugar unit. MS 3 of m/z 381.9 (C 2) now reveals a (1–3) linkage between both sugar units at the nonreducing end of the molecule (Fig. 4b). In the same manner, component C (see structure in Fig. 5a) with two modified sugars was analyzed. The MS 2 spectrum from [M ⫹ Na] ⫹ ions at m/z 1221.3 (Fig. 5a) shows an intense fragment ion at m/z 1098.2, i.e., the same mass as observed for component A in Fig. 2a. Further structural analysis of this fragment ion yielded the same spectra as depicted above in Figs. 2 and 3; thus, it could be concluded that the structures are identical and that component C comprises a second modification with a mass increment of 123 Da. The mass differences of 43 and 123 Da to m/z 1162.3 [M ⫹ Na ⫺ 59] ⫹ (Fig. 5b) agree well with the loss of the second modification, phosphoethanolamine. In both spectra intense fragment ions from cleavages of 1 and

2 H 2O molecules are registered; their losses seem to be promoted by the PE group. Since these fragment ions are not used for structural interpretation, they will not be discussed here in detail. The location of the PE group can be elucidated from the mass increment between B 4 (m/z 982.1) and B 3 fragments (m/z 697.1), which is 285 Da, thus demonstrating that the second hexose contains the PE group. The last point to clarify is the linkage position of PE. As is already known from component A, the respective sugar moiety is (1–3)-linked. As this linkage hinders ring fragmentation, it cannot be predicted whether the PE group is attached to C-4 or C-6 of this sugar residue. MS 2 of the pseudomolecular ion [M ⫺ H] ⫺ (m/z 1197.7) in the negative-ion mode (Fig. 6a) reveals a very weak signal at m/z 464.2, which can be explained as a Y 2 fragment, the disaccharide from the reducing end of the molecule. MS 3 of this fragment ion (Fig. 6b) showed, besides the (1– 4) linkage between the sugar

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Negative-ion MS n spectra of component C. (a) MS 2 of the deprotonated molecule at m/z 1197.7; (b) MS 3 of the Y 2 fragment in (a).

units, the C fragment (m/z 301.8) followed by losses of 60 and 90 Da for the 0,2A 1 (m/z 242.0) and 0,3A 1 fragments (m/z 211.9). The latter fragment could only appear when the PE group was (1– 6)-linked to the sugar. CONCLUSION

The results show that nano-ESI MS n yields valuable information concerning the structure of the sugar core of glycosphingolipids containing one or two noncarbohydrate substituents. Derivatization of the compounds prior to mass analysis is not necessary. Intact glycolipids have not been subjected to this type of analysis so far. The results obtained from positive-ion and negative-ion spectra of neutral, underivatized oligosaccharides can be transferred to zwitterionic compounds: positive-ion spectra provide information concerning the monosaccharide sequence of the sugar core; negative-ion spectra reveal, additionally, all linkage positions. Moreover, the position of the substituents PC

and PE can be easily determined. The fact that the negative charge remains attached to the phosphate group upon fragmentation makes additional fragmentation steps necessary. Sequential MS/MS up to MS 4 is needed to obtain the desired information. In this context, the outstanding feature of the ion-trap mass analyzer in producing a fragmentation spectrum from very low abundant ion signals has to be emphasized. An example for this is the analysis of the Y 2 fragment of component C in Fig. 6, which has only 0.2% of the intensity of the base peak. Only in the case of PC as substituent were chemical cleavage and removal of this group by HF necessary in order to determine the last linkage site. For the described structural analysis, MS 1–MS 4 from positive and negative ions, approximately 150 –250 ng of sample was necessary. The time for this analysis, provided that the fragmentation protocol is known, is only 10 –15 min. Although no information is obtained about the isomeric structures of the sugar units (Glc vs Man) and their anomeric configu-

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rations, the nano-ESI MS n method can be regarded as an effective tool which makes the complex and timeconsuming structural elucidation of glycosphingolipids easier and/or can be used for a simple and quick confirmation of certain elements in a proposed structure. ACKNOWLEDGMENTS We are grateful to Dr. Roger D. Dennis, Institute of Biochemistry, University of Giessen, for critical reading of the manuscript. This study was supported by the German Research Council (SFB 535 and Graduiertenkolleg “Molecular Biology and Pharmacology”). This paper is in partial fulfilment of the requirements of C.H.F. for the degree of Dr. rer. nat. at Giessen University.

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