TOF-MS of Oligosaccharides Labeled

MALDI-TOF-MS of 2-aminobenzamide-labeled N-glycans was shown to allow the analysis of sodium adducts and proton adducts in the positive-ion mode as ...
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Anal. Chem. 2005, 77, 6954-6959

Negative-Mode MALDI-TOF/TOF-MS of Oligosaccharides Labeled with 2-Aminobenzamide Manfred Wuhrer* and Andre´ M. Deelder

Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands

MALDI-TOF-MS of 2-aminobenzamide-labeled N-glycans was shown to allow the analysis of sodium adducts and proton adducts in the positive-ion mode as well as deprotonated species in the negative-ion mode from a single preparation spot, using N-glycans of adult worms of the human parasite Schistosoma mansoni as model substances. Fragment ion analysis of these species was performed by MALDI-TOF/TOF-MS. With laser-induced dissociation, sodium adducts and proton adducts mainly showed cleavage of glycosidic linkages. High-energy collision-induced dissociation of sodium adducts resulted in extensive cross-ring cleavages and provided information on linkage positions. Of particular value were the negativemode MALDI-TOF/TOF-MS analyses of the deprotonated N-glycans, which featured (1) various ring fragmentations giving linkage information, (2) extensive 1,3A cross-ring cleavage of mannoses carrying an antenna readily revealing the composition of the antenna, (3) D as well as [D 18]- ions providing specifically the composition of the 6-antenna, and (4) pronounced stability of fucose linkages resulting in detailed information on fucosylation positions. The outlined approach thus allows the acquisition of both heCID MS/MS spectra of sodium adducts and LID MS/ MS spectra of deprotonated species from a 2-aminobenzamide-labeled N-glycan prepared in 6-aza-2-thiothymine, resulting in a wealth of structural information. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has already been applied to carbohydrates during the development of this technique.1 It combines high sensitivity with tolerance toward salts and buffers, which has led to its frequent use in studies of carbohydrates and glycoconjugates.2-6 The method is particularly useful in profiling complex mixtures of oligosaccharides, and most of the analyses are performed in the positive-ion reflectron mode with detection of sodium adducts.2,3 The development of high-performance * To whom correspondence should be addressed. Tel: +31-71-5265077. Fax: +31-71-526-6907. E-mail: [email protected]. (1) Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1987, 78, 53-68. (2) Harvey, D. J. Mass Spectrom. Rev. 1999, 18, 349-450. (3) Harvey, D. J. Int. J. Mass Spectrom. 2003, 226, 1-35. (4) Zaia, J. Mass Spectrom. Rev. 2004, 23, 161-227. (5) Mechref, Y.; Novotny, M. V. Chem. Rev. 2002, 102, 321-369. (6) Morelle, W.; Michalski, J.-C. Curr. Anal. Chem. 2005, 29-57.

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instruments during the last years has allowed fast measurement of samples with high mass accuracy and resolution. Determination of fragment ion spectra by MALDI postsource decay (PSD), however, has remained laborious and often suffers from low mass accuracy and resolution. The recently introduced MALDI-TOF/ TOF mass spectrometers allow the acquisition of high-quality fragment ion spectra from sodium adducts of oligosaccharides, either by laser-induced fragmentation (LID)7,8 or by high-energy collision-induced fragmentation (heCID).8-10 heCID MALDI-TOF/ TOF-MS of oligosaccharides in particular is characterized by extensive cross-ring fragmentation resulting in information on linkages and substitution positions. Besides the commonly observed Y and B ions, which arise from the cleavage of glycosidic linkages, these spectra exhibit informative A-type ring cleavages, which for substituted residues allow the assignment of substituents to specific hydroxyl groups.8-10 MALDI-TOF(/TOF)-MS offers more options for the characterization of oligosaccharides, including the analysis of proton adducts, or the analysis of deprotonated species. Analysis of the latter is almost exclusively performed for acidic glycans (for example, sialylated glycans)2-6 or glycan derivatives (for example, neutral glycans derivatized with 8-aminonaphthalene-1,3,6-trisulfonic acid).11 For MALDI-TOF-MS of oligosaccharides and derivatives thereof that are lacking an acidic group, analyses are generally performed in positive-ion mode with registration of sodium adducts, and the only reports measuring deprotonated oligosaccharides used β-carboline matrixes.12-14 PSD studies of deprotonated species13 or chloride adducts14 indicated the potential usefulness of this method for linkage analysis of oligosaccharides. We here describe negative-mode MALDI-TOF-MS of neutral glycans after labeling with 2-aminobenzamide (AB), using 6-aza2-thiothymine (ATT) as a matrix. AB-labeled oligosaccharides were registered in positive-ion mode as sodium adducts as well (7) Kurogochi, M.; Nishimura, S. Anal. Chem. 2004, 76, 6097-6101. (8) Lewandrowski, U.; Resemann, A.; Sickmann, A. Anal. Chem. 2005, 77, 3274-3283. (9) Spina, E.; Sturiale, L.; Romeo, D.; Impallomeni, G.; Garozzo, D.; Waidelich, D.; Glueckmann, M. Rapid Commun. Mass Spectrom. 2004, 18, 392-398. (10) Mechref, Y.; Novotny, M. V.; Krishnan, C. Anal. Chem. 2003, 75, 48954903. (11) Klein, A.; Lebreton, A.; Lemoine, J.; Perini, J. M.; Roussel, P.; Michalski, J. C. Clin. Chem. 1998, 44, 2422-2428. (12) Nonami, H.; Tanaka, K.; Fukuyama, Y.; Erra-Balsells, R. Rapid Commun. Mass Spectrom. 1998, 12, 285-296. (13) Nonami, H.; Wu, F.; Thummel, R. P.; Fukuyama, Y.; Yamaoka, H.; ErraBalsells, R. Rapid Commun. Mass Spectrom. 2001, 15, 2354-2373. (14) Yamagaki, T.; Suzuki, H.; Tachibana, K. Anal. Chem. 2005, 77, 1701-1707. 10.1021/ac051117e CCC: $30.25

© 2005 American Chemical Society Published on Web 09/23/2005

Figure 1. MALDI-TOF-MS detection of a AB-labeled, neutral oligosaccharide in positive-ion and negative-ion modes. A dilution series of a AB-labeled, core-(R1-6)-fucosylated trimannosyl N-glycan from S. mansoni prepared in 6-aza-2-thiothymine matrix (see box) on a stainless steel target plate was analyzed in both positive-ion (A-C) and negative-ion (D-F) modes. (G, H) Matrix regions for ATT. Red circle, mannose; blue square, N-acetylglucosamine; triangle, fucose; A, 2-aminobenzamide.

as in protonated form, while negative-mode analysis resulted in the detection of deprotonated species. We exploited the efficient negative-mode ionization for negative-mode MALDI-TOF/TOFMS of AB-labeled glycans. Negative-mode fragment ion analysis (1) regularly showed ring fragmentation giving information on linkage positions, (2) allowed the assignment of N-glycan antennas to specific branches, and (3) was characterized by stabile fucose linkages. Thus, using ATT as a matrix, MALDI-TOF(/TOF)-MS can be performed on sodiated and protonated as well as deprotonated AB-labeled oligosaccharides from a single sample spot, resulting in a wealth of structural information. EXPERIMENTAL SECTION Sample Preparation. N-Glycans were released by PNGase F treatment from Schistosoma mansoni adult worm glycoproteins and purified following the protocol outlined elsewhere.15 N-Glycans were reductively labeled with AB and fractionated by normal-phase HPLC as described before.15 Individual peak fractions were analyzed by MALDI-TOF(/TOF)-MS. MALDI-TOF(/TOF)-MS. Matrix (0.5 µL of a 5 mg/mL solution of 6-aza-2-thiothymine) was spotted and dried on a polished stainless steel target plate (Bruker). AB-labeled oligosaccharides (0.5 µL of a dilution in water) were added, and the droplet was dried in a stream of warm air. MALDI-TOF(/TOF)-MS data were obtained using an Ultraflex time-of-flight mass spectrometer (Bruker) equipped with a LIFT-MS/MS facility controlled by the FlexControl 2.0 software package. Spectra were acquired in the positive-ion or negative-ion mode at 50-Hz laser frequency, and 100-1000 individual spectra were averaged. For fragment ion analysis in the tandem time-of-flight mode, precursors were (15) Wuhrer, M.; Robijn, M. L. M.; Koeleman, C. A.; Balog, C. I. A.; Geyer, R.; Deelder, A. M.; Hokke, C. H. Biochem. J. 2004, 378, 625-632.

accelerated to 8 kV and selected in a timed ion gate. Fragment ions generated by LID of the precursor were further accelerated by 19 kV in the LIFT cell, and their masses were analyzed after the ion reflector passage. Measurements were in part performed using Post Lift Metastable Suppression, which allowed decimation of precursor and metastable ion signals produced after extraction out of the second ion source. Masses were annotated and processed with FlexAnalysis 2.0. External calibration of MALDITOF mass spectra was carried out using singly charged monoisotopic peaks of a mixture of human angiotensin I, bombesin, adrenocorticotropic hormone (ACTH 18-39), and somatostatin28 (Bruker). For MALDI-TOF/TOF-MS, calibrations were performed with fragment ion spectra obtained for the proton adducts of these peptides. For heCID, argon was used as collision gas and the pressure in the high-vacuum source was set to 5 × 10-6 mbar. RESULTS AND DISCUSSION Ionization Conditions. While efficient positive-mode ionization of oligosaccharides and their derivatives has often been demonstrated with various matrixes and additives, negative-mode ionization has been restricted to acidic oligosaccharides or acidic glycan derivatives.2-6 Negative-mode ionization of neutral glycans has hitherto only been described in a single report.12 Using ATT as matrix, we achieved efficient MALDI ionization of neutral ABlabeled glycans from the same sample spot in both (1) positiveion mode resulting in sodium and proton adducts and (2) negativeion mode giving deprotonated species. This is demonstrated for a 2-aminobenzamide-labeled, core-(R1-6)-fucosylated trimannosyl N-glycan obtained from glycoproteins of adult worms of the human parasite S. mansoni (Figure 1): using a preparation in ATT on a stainless steel target plate, low-femtomole amounts of the analyte Analytical Chemistry, Vol. 77, No. 21, November 1, 2005

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could be detected as sodium adducts (positive-ion mode; Figure 1A-C). Furthermore, proton adducts of the analyte could be detected in positive-ion mode (A). Analysis of a dilution series in the negative-ion mode (Figure 1D-F) resulted in the detection of the deprotonated species at the 50-fmol level. Under the conditions applied, positive-mode detection appeared to be ∼1 order of magnitude more sensitive than negative-mode detection (C, F). Besides the deprotonated species, several other variants and adducts of the analyte were detected in negative-ion mode: the ion at m/z 1459 could be interpreted as an adduct of a prominent matrix ion (m/z 283 in negative-ion mode (Figure 1H), m/z 285 in positive-ion mode (Figure 1G); interpreted as a dehydrogenated, deprotonated dimer of matrix molecules) to the analyte. The ion at m/z 1316 would then be interpreted as containing one matrix molecule less, thus representing the adduct of a dehydrogenated, deprotonated matrix molecule to the analyte. Dehydrogenation mechanisms associated with matrix ionization have been described for 2,5-dihydroxybenzoic acid.16 MALDI-TOF/TOF-MS of Detected Species. The MALDITOF/TOF-MS analyses of oligosaccharides, which are hitherto described in the literature, were performed with underivatized,8 2-aminopyridine-labeled,7,17 AB-labeled,15 or permethylated18-19 oligosaccharides and have been restricted to alkali adducts or proton adducts. As MALDI-TOF-MS of AB-labeled neutral oligosaccharides under the conditions described above allowed the detection of [M - H]- ions in addition to [M + Na]+ and [M + H]+ species measured in the positive-ion mode, we decided to perform a comparative MALDI-TOF/TOF-MS study including negative-mode analyses using individual AB-labeled S. mansoni N-glycans of known structure20-22 as model compounds. Particular attention will be paid to the effect of argon as high-energy collision gas and the observation of diagnostic ring fragments. MALDI-TOF/TOF-MS of Sodium Adducts. AB-labeled oligosaccharides were analyzed by MALDI-TOF/TOF-MS using LID (Figure 2A, C) and heCID (Figure 2B, D). The LID spectra showed the fragmentation of glycosidic linkages with mainly Y and B ions,23 with the cleavage of the chitobiose core as well as loss of the core fucose dominating. The heCID spectrum additionally exhibited C and Z ions as well as various ring cleavages providing linkage information, in accordance with literature data.8-10 The unassigned fragments at m/z 978 (Figure 2A, B) and 1384 (C, D) are interpreted as arising from 0,2A6 ring fragmentation of N-glycan N-glycosides with closed-ring conformation of the innermost GlcNAc. These N-glycosides of 2-aminobenzamide with 2-aminobenzamide are suppposed to be byproducts of the derivatization step (reductive amination reaction) and were detected by (16) Karas, M.; Gluckmann, M.; Schafer, J. J. Mass Spectrom. 2000, 35, 1-12. (17) Geyer, H.; Wuhrer, M.; Kurokawa, T.; Geyer, R. Micron 2004, 35, 105106. (18) Stephens, E.; Sugars, J.; Maslen, S. L.; Williams, D. H.; Packman, L. C.; Ellar, D. J. Eur. J. Biochem. 2004, 271, 4241-4258. (19) Stephens, E.; Maslen, S. L.; Green, L. G.; Williams, D. H. Anal. Chem. 2004, 76, 2343-2354. (20) Cummings, R. D.; Nyame, A. K. Biochim. Biophys. Acta 1999, 1455, 363374. (21) Khoo, K. H. Trends Glycosci. Glycotechnol. 2001, 31, 493-506. (22) Hokke, C. H.; Deelder, A. M. Glycoconjugate J. 2001, 18, 573-587. (23) Domon, B.; Costello, C. Glycoconjugate J. 1988, 5, 253-257.

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MALDI-TOF-MS at a 2-Da lower molecular mass than reductively aminated species (m/z 1197 in Figure 1A). While the detection limit for sodium adducts in the MS-mode was ∼1 fmol, these rather stable alkali adducts were only inefficiently fragmented by heCID, resulting in a minimal sample need of ∼1 pmol for high-quality heCID spectra. MALDI-TOF/TOF-MS of Protonated Species. LID fragment spectra of proton adducts of AB-labeled neutral oligosaccharides were dominated by B and Y ions (Figure 3A, C), which is in accordance with previous observations.15 Misleading ions arising from fucose rearrangements were observed for the core-fucosylated analytes (m/z 836 in Figure 3A and m/z 1242 in Figure 3C). Fucose rearrangement or internal residue loss represents a known phenomenon in MS/MS analyses of protonated glycans,24-27 making fragment ion analysis of protonated species a deceitful source of information for the characterization of glycans and glycoconjugates. When argon was used as a collision gas, no changes in fragmentation spectra were observed compared to the LID spectra. Thus, no ring fragmentations of protonated species were observed under heCID conditions, in contrast to the findings for gas-on experiments with sodium adducts (Figure 2B). Though proton adducts constituted only minor species in positive-mode MS (Figure 1A-C), they could sensitively be analyzed by MALDI-TOF/TOF-MS. In contrast to sodium adducts, which with both LID and heCID exhibited a poor fragment ion yield, proton adducts exhibited very labile glycosidic bonds and were very efficiently decomposed with elevated laser energy, resulting in high-quality MS/MS spectra of subpicomole quantities of analyte. MALDI-TOF/TOF-MS of Deprotonated Species. Unlike the positive-mode LID fragment ion spectra of sodium and proton adducts (Figures 2A, 3A, and 3C), which were dominated by B and Y ions, LID of deprotonated AB-labeled neutral glycans additionally exhibited intense C and Z ions as well as various ring cleavages (Figure 3B and D, Figure 4). As in the case of proton adducts, gas-on and gas-off spectra were found to be identical. Fragmentation in negative-mode LID resembled that described by Yamagaki et al. using negative-mode MALDI-TOF-MS with norharman matrix and PSD of chloride adducts 14 and reminded one very much of the recent findings of Harvey28-30 with negativemode electrospray ionization of neutral oligosaccharides with lowenergy CID. The following features became apparent: (a) Complex type structures exhibited intense 1,3A fragment ions arising from ring cleavage of the antenna-bearing mannose residues. The observation of a 1,3A ring fragmentation is in accordance with the 2-substitution of the mannose by GlcNAc in complex-type N-glycan structures (Figure 3D, Figure 4). This ion was found to be the dominant fragment ion of the lower mass range of both the LacDiNAc-containing N-glycan (m/z 465; Figure 3D) and the Lewis X-bearing N-glycan (m/z 570; Figure 4). (24) Harvey, D. J.; Mattu, T. S.; Wormald, M. R.; Royle, L.; Dwek, R. A.; Rudd, P. M. Anal. Chem. 2002, 74, 734-740. (25) Franz, A. H.; Lebrilla, C. B. J. Am. Soc. Mass Spectrom. 2002, 13, 325-337. (26) Tadano-Aritomi, K.; Hikita, T.; Kubota, M.; Kasama, T.; Toma, K.; Hakomori, S. I.; Ishizuka, I. J. Mass Spectrom. 2003, 38, 715-722. (27) Brull, L. P.; Kovacik, V.; Thomas-Oates, J. E.; Heerma, W.; Haverkamp, J. Rapid Commun. Mass Spectrom. 1998, 12, 1520-1532. (28) Harvey, D. J. J. Am. Soc. Mass Spectrom. 2005, 16, 647-659. (29) Harvey, D. J. J. Am. Soc. Mass Spectrom. 2005, 16, 631-646. (30) Harvey, D. J. J. Am. Soc. Mass Spectrom. 2005, 16, 622-630.

Figure 2. MALDI-TOF/TOF-MS of a AB-labeled N-glycans in sodiated form. An AB-labeled, core-(R1-6)-fucosylated trimannosyl N-glycan from S. mansoni (A, B; see Figure 1) and an N-glycan carrying additionally a LacDiNAc antenna (C, D)sboth obtained from S. mansoniswere analyzed by MALDI-TOF/TOF-MS in sodiated form either by LID (gas-off; A, C) or heCID (gas-on; B, D). Fragment ion nomenclature follows Domon and Costello.23 Red circle, mannose; blue square, N-acetylglucosamine; triangle, fucose; A, 2-aminobenzamide; F, fucose; H, hexose.

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Figure 3. MALDI-TOF/TOF-MS of AB-labeled N-glycans in protonated and deprotonated form. An AB-labeled, core-(R1-6)-fucosylated trimannosyl N-glycan (A, B) and an N-glycan carrying additionally a LacDiNAc antenna (C, D)sboth obtained from S. mansoniswere analyzed by LID-MALDI-TOF/TOF-MS in protonated (A, C) and deprotonated (B, D) forms. Fragment ion nomenclature follows Domon and Costello.23 Red circle, mannose; green square, N-acetylgalactosamine; blue square, N-acetylglucosamine; triangle, fucose; A, 2-aminobenzamide. 6958 Analytical Chemistry, Vol. 77, No. 21, November 1, 2005

Figure 4. Negative-mode MALDI-TOF/TOF-MS of a AB-labeled N-glycan with two Lewis X antennas. A AB-labeled, core-(R1-6)-fucosylated diantennary N-glycan with two terminal Lewis X motifs was analyzed by LID-MALDI-TOF/TOF/MS in its deprotonated form. As the antennas are symmetrical, they give rise to identical fragment ions. For several of the observed fragments, various explanations are possible, of which only one is given in the figure. Fragment ion nomenclature follows Domon and Costello.23 Red circle, mannose; green square, N-acetylgalactosamine; blue square, N-acetylglucosamine; triangle, fucose; A, 2-aminobenzamide.

Composition of antennas can thus be readily deduced from these prominent ions, similar to negative-mode ESI-CID-MS of oligosaccharides.28 (b) For the LacDiNAc- and Lewis X-containing N-glycans (Figure 3D, Figure 4), D ions, their dehydrated form [D - 18]-, or both were observed, which are specific for the 6-antenna. These types of ions have been described by Harvey for fragmentation of N-glycans in negative-mode electrospray MS with low-energy CID.28 For the LacDiNAc containing N-glycan analyzed (Figure 3D), this pair of ions at m/z 711 and 729 was thus interpreted as GalNAc(β1-4)GlcNAc(β1-2)Man(R1-6)Man (representing a C4RZ3β fragment, which may eliminate a water molecule) and indicated a 6-antenna position of the LacDiNAc moiety. For the diantennary Lewis X-containing N-glycan, the corresponding [D - 18]- fragment was detected at m/z 816 (Figure 4), while for unknown reasons, the D ion was not detected (expected at m/z 834). (c) Fucose linkages were found to be very stable. In negativemode LID, the glycosidic bond between fucose and other monosaccharides appeared to be similarly stable as other glycosidic linkages in the oligosaccharides, in contrast to the findings in positive-ion mode (Figures 2A, 3A, 3C). This reveals negativemode MS/MS as a suitable technique for the allocation of fucoses (Figure 3D, Figure 4).28 (d) Linkage-specific ring fragmentations were observed that obeyed rules similar to those set up for positive-mode heCID of sodiated oligosaccharides,8-10 which can, for example, be seen in Figure 3D; the LacDiNAc antenna structure is characterized by two C-type ions (m/z 220 and 423; Figure 3D), as well as 2,4A and 0,2A ring fragmentation of the penultimate HexNAc (m/z 262 and

322, respectively), which was indicative of a 4-substitution of this HexNAc in accordance with the GalNAc(β1-4)GlcNAc- terminal motif. Similarly to proton adducts, deprotonated species could also be decomposed efficiently in MALDI-TOF/TOF-MS analyses. The sample amounts that had to be applied to the target plate for these experiments were ∼1 pmol. CONCLUSIONS The finding that AB-labeled N-glycans could readily be analyzed as deprotonated species by negative-mode MALDI-TOFMS using ATT allowed the efficient analysis of these species by negative-mode MALDI-TOF/TOF-MS. The obtained fragmentation patterns differed considerably from those obtained in positive mode and provided detailed structural information. Fragmentation generally followed the rules established by Harvey for electrospray low-energy CID anion adducts of oligosaccharides. The here outlined possibility to obtain a second valuable data set by negative-mode MALDI-TOF/TOF-MS of oligosaccharides next to high-energy CID of sodium adducts makes MALDI-TOF/TOFMS a versatile and useful method in glycosylation analysis. ACKNOWLEDGMENT We thank Dr. C. H. Hokke for discussions. We acknowledge the expert technical assistance of Carolien A. M. Koeleman.

Received for review June 23, 2005. Accepted August 22, 2005. AC051117E

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