Differentiation of Isomeric N-Glycan Structures by Normal-Phase

Anal. Chem. 2006, 78, 8491-8498

Differentiation of Isomeric N-Glycan Structures by Normal-Phase Liquid Chromatography-MALDI-TOF/ TOF Tandem Mass Spectrometry Sarah Maslen,† Pawel Sadowski,‡ Alex Adam,§ Kathryn Lilley,‡ and Elaine Stephens*,†

Chemistry Department, University of Cambridge, Lensfield Road, Cambridge, UK CB2 1EW, Biochemistry Department, University of Cambridge, Building O, Downing Site, Cambridge CB2 1QW, and Dionex UK Ltd., 4 Albany Court, Albany Industrial Estate, Camberley, Surrey, GU16 7QL, UK

The detailed characterization of protein N-glycosylation is very demanding given the many different glycoforms and structural isomers that can exist on glycoproteins. Here we report a fast and sensitive method for the extensive structure elucidation of reducing-end labeled N-glycan mixtures using a combination of capillary normal-phase HPLC coupled off-line to matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOFMS) and TOF/TOF-MS/MS. Using this method, isobaric N-glycans released from honey bee phospholipase A2 and Arabidopsis thaliana glycoproteins were separated by normal-phase chromatography and subsequently identified by key fragment ions in the MALDI-TOF/TOF tandem mass spectra. In addition, linkage and branching information were provided by abundant cross-ring and “elimination” fragment ions in the MALDI-CID spectra that gave extensive structural information. Furthermore, the fragmentation characteristics of N-glycans reductively aminated with 2-aminobenzoic acid and 2-aminobenzamide were compared. The identification of N-glycans containing 3-linked core fucose was facilitated by distinctive ions present only in the MALDI-CID spectra of 2-aminobenzoic acid-labeled oligosaccharides. To our knowledge, this is the first MS/MS-based technique that allows confident identification of N-glycans containing 3-linked core fucose, which is a major allergenic determinant on insect and plant glycoproteins. Glycosylated proteins are often very heterogeneous structures due to the great diversity in the glycan moieties (known as glycoforms). Many different glycoforms can exist on one glycoprotein, which are determined by the combination of glycosyltransferases and glycosidases present in a cell at any particular time.1 This heterogeneity is often increased further by the presence of structural isomers. Therefore, protein glycosylation structure elucidation is extremely challenging and multiple techniques are often required for complete characterization. * To whom correspondence should be addressed. E-mail: [email protected]. Tel: +44 1223 763126. Fax: +44 1223 336913. † Chemistry Department, University of Cambridge. ‡ Biochemistry Department, University of Cambridge. § Dionex UK Ltd. (1) Kornfeld, R.; Kornfeld, S. Annual. Rev. Biochem. 1985, 54, 631-664. 10.1021/ac0614137 CCC: $33.50 Published on Web 11/04/2006

© 2006 American Chemical Society

Mass spectrometry (MS) is one of the most sensitive and widely used methods for detailed N-glycan characterization.2 It is capable of providing glycan composition as well as sequence, branching, and sometimes linkage information by tandem MS. Structural isomers can also be identified by some mass spectrometric tandem methods, although, in many cases these are overlooked and left uncharacterized. The need to identify and separate isobaric oligosaccharides has become increasing important recently since the introduction of quantitative glycan profiling methods. In these cases, the comparison of isotope-labeled N-glycans from different systems, using oligosaccharide composition only, can be complicated by the existence of various structures with the same molecular weight. Therefore, a sensitive and highthroughput method is desired that is capable of differentiating oligosaccharide isomers, as well as defining structure in terms of sequence, linkage positions and antennae substitutions. High-performance liquid chromatography (HPLC) methods can be employed to separate isobaric glycan structures. In particular, high-pH anion-exchange chromatography (HPAEC) gives extremely high chromatographic resolution.3 However, the resulting oligosaccharide-containing fractions contain high salt, thus making this method difficult to directly interface with mass spectrometric techniques and an on-line desalting device is required.4,5 Normal-phase (NP) HPLC has less chromatographic resolution than HPAEC but is still capable of resolving some isobaric oligosaccharides.6-8 Importantly, this method uses volatile buffer salts that are directly compatible with mass spectrometric techniques. In NP-HPLC, retention is caused by polar interactions, which generally increases with elongation of the glycan chains and often allows some prediction of elution positions. This chromatographic technique is known to be very useful in the analysis of fluorescently labeled glycans and has frequently been (2) Dell, A.; Morris, H. R. Science 2001, 291, 2351-2356. (3) Hardy, M. R.; Townsend, R. R. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 32893293. (4) Bruggink, C. W. M.; Koeleman, C. A.; Barreto, V.; Liu, Y.; Pohl, C.; Ingendoh, A.; Hokke, C. H.; Deelder, A. M. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2005, 829, 136-143. (5) Torto, N.; Hofte, A.; van der Hoeven, R.; Tjaden, U.; Gorton, L.; MarkoVarga, G.; Bruggink, C.; van der Greef, J. J. Mass Spectrom. 1998, 33, 334341. (6) Wuhrer, M.; Koeleman, C. A. M.; Hokke, C. H.; Deelder, A. M. Int. J. Mass Spectrom. 2004, 232, 51-57. (7) Wuhrer, M.; Koeleman, C. A.; Deelder, A. M.; Hokke, C. H. Anal. Chem. 2004, 76, 833-838. (8) Anumula, K. R.; Dhume, S. T. Glycobiology 1998, 8, 685-694.

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employed on an analytical or microbore scale to separate labeled glycans with fluorescence detection followed by analysis by mass spectrometry.9-11 The mass spectrometric techniques which have the capability for extensive oligosaccharide characterization in positive ion mode include matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS/MS) and ion trap mass spectrometry (IT-MS). These methods give sugar fragment ions in MALDI high-energy-CID and MSn spectra, respectively, which have been shown to facilitate isomer identification.12-15 In addition, both these mass spectrometric methods can also provide glycan sequence, branching, and linkage (from cross-ring fragments) information.12,13,16,17 Consequently, the coupling of these techniques to NP-HPLC should allow more comprehensive structure elucidation of complex oligosaccharide mixtures. Recently, nanoscale NP-HPLC was coupled to IT-MS for the characterization of native and 2-aminobenzamide-labeled N-glycans.6,7 No linkage information was obtained, but separation and identification of structural isomers was achieved at low-femtomole sensitivity. In this study, capillary NP-HPLC is employed in an automated fashion with MALDI-TOF/TOF tandem mass spectrometry for the structural analyses of N-glycan mixtures labeled with reducingend UV-absorbing labels. The N-glycans from honey bee phospholipase A2 (PLA2) were chosen for initial evaluation of this technique, as they have been well characterized.18 To test this method’s applicability to a more complex in vivo sample, a Golgirich fraction from Arabidopsis thaliana was chosen. This sample contained numerous glycoproteins with a high degree of heterogeneity as the Golgi is the site of glycoprotein maturation. Using these samples, we show that NP-HPLC is capable of resolving many isomers that are easily identified by specific fragment ions in the MALDI-CID spectra. In addition, the cross-ring fragments and “elimination” ions in the high-energy spectra provide detailed information on sequence, branching, and linkage positions. Furthermore, the fragmentation characteristics of glycans reductively aminated with 2-aminobenzoic acid (2-AA) and 2-aminobenzamide (2-AB) were compared to determine which reducing-end derivative provides the most sensitive and structurally informative fragmentation spectra. (9) Butler, M. Quelhas, D.; Critchley, A. J.; Carchon, H.; Hebestreit, H. F.; Hibbert, R. G.; Vilarinho, L.; Teles, E.; Matthijs, G.; Schollen, E.; Argibay, P.; Harvey, D. J.; Dwek, R. A.; Jaeken, J.; Rudd, P. M. Glycobiology 2003, 13, 601-622. (10) Charlwood, J.; Birrell, H.; Camilleri, P. J. Chromatogr., B: Biomed. Sci. Appl. 1999, 734, 169-174. (11) Royle, L.; Roos, A.; Harvey, D. J.; Wormald, M. R.; van Gijlswijk-Janssen, D.; Redwan, E.-R. M.; Wilson, I. A.; Daha, M.; Dwek, R. A.; Rudd, P. M. J. Biol. Chem. 2003, 278, 20140-20153. (12) Stephens, E.; Maslen, S. L.; Green, L. G.; Williams, D. H. Anal. Chem. 2004, 76, 2343-2354. (13) Stephens, E.; Sugars, J.; Maslen, S. L.; Williams, D. H.; Packman, L. C.; Ellar, D. J. Eur. J. Biochem. 2004, 271, 4241-4258. (14) Suzuki, Y.; Suzuki, M.; Ito, E.; Ishii, H.; Miseki, K.; Suzuki, A. Glycoconjugate J. 2005, 22, 427-431. (15) Matamoros Fernandez, L. E.; Obel, N.; Scheller, H. V.; Roepstorff, P. Carbohydrate Res. 2004, 339, 655-664. (16) Mechref, Y.; Novotny, M. V.; Krishnan, C. Anal. Chem. 2003, 75, 48954903. (17) Sheeley, D. M.; Reinhold, V. N. Anal. Chem. 1998, 70, 3053-3059. (18) Kubelka, V.; Altmann, F.; Staudacher, E.; Tretter, V.; Marz, L.; Hard, K.; Kamerling, J. P.; Vliegenthart, J. F. G. Eur. J. Biochem. 1993, 213, 11931204.

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EXPERIMENTAL SECTION Preparation of Glycan Samples. The trimannosyl core standard, Dextran 1000 and honey bee PLA2 were purchased from Sigma. A dextran ladder was prepared by incubating Dextran in 200 µL of 0.1 M trifluoroacetic acid for 60 min at 100 °C followed by lyophilization. N-Glycans from PLA2 were released using PNGase A or PNGase F from glycopeptides produced from a tryptic digest as described previously.13 A. thaliana N-glycans were prepared from proteins in Golgi-rich fractions prepared from callus cultures.19 The Arabidopsis proteins were precipitated with acetone, resolubilized in 2% Triton-X-100 in 50 mM ammonium acetate buffer (pH 7.8), and reduced and alkylated with dithiothreitol and iodoacetamide, respectively, using standard protocols. The sample was diluted 10 times with 50 mM ammonium acetate buffer (pH 7.8) and digested with trypsin (Promega) overnight. Trypsin was inactivated by incubation at 100 °C for 5 min, and the N-glycans were released from the resulting glycopeptides using 0.3 mU of PNGase A (Roche), after reducing the pH of the solution to 5.0 with acetic acid. Released N-glycans were separated from the peptide mixture using a C18 Sep-Pak cartridge (Waters), and the glycans were purified further by using HyperSep Hypercarb cartridges (ThermoHypersil-Keystone). Purified N-glycan mixtures were reductively aminated using 2-AB or 2-AA using optimized labeling conditions described previously.20 The N-glycans were purified from the reductive amination reagents using a GlycoClean S cartridge (Glyko, Prozyme) and dried down prior to NP-LCMALDI. Capillary Normal-Phase Chromatography: MALDI-TOF/ TOF-MS/MS. Normal-phase capillary HPLC was carried out using a LC-Packings Ultimate System (LC Packings/Dionex), which was used to generate the gradient that flowed at 3 µL min-1. Solvent A was 50 mM ammonium formate adjusted to pH 4.4 with formic acid. Solvent B was 20% solvent A in acetonitrile. The labeled oligosaccharides, dissolved in 80% acetonitrile, were loaded onto an amide-80 column (300 um × 15 cm; LC Packings/Dionex) and eluted with increasing aqueous concentration. For elution of PLA2 N-glycans, the NP column was equilibrated at 5% A and the gradient was initiated 5 min after injection and increased linearly to 52% A over 96 min. For elution of A. thaliana N-glycans, the NP column was equilibrated at 5% A and the aqueous gradient was initiated 5 min after injection and increased linearly to 52% A over 131 min. The column effluent passed through a capillary UV detector (set at 254 nm) directly to a Probot sample fraction system (Dionex), which spotted onto a MALDI plate at 20-s intervals for the PLA2 N-glycans. For better chromatographic resolution, spotting was carried out every 15 s for the Arabidopsis N-glycans. The sample spots were overlaid with 0.6 µL of 2,5DHB (dissolved in 50% methanol) and rapidly dried in a vacuum desiccator in order to produce small crystals for easy automated spectral acquisition. All mass spectra were recorded on a 4700 Proteomics Analyzer (Applied Biosystems). This MALDI tandem mass spectrometer uses a 200-Hz frequency-triple Nd:YAG laser operating at a wavelength of 355 nm. Averages of 1500 shots were (19) Dunkley, T. P.; Hester, S.; Shadforth, I. P.; Runions, J.; Weimar, T.; Hanton, S. L.; Griffin, J. L.; Bessant, C.; Brandizzi, F.; Hawes, C.; Watson, R. B.; Dupree, P.; Lilley, K. S. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 65186523. (20) Bigge, J. C. Patel, T. P.; Bruce, J. A.; Goulding, P. N.; Charles, S. M.; Parekh, R. B. Anal. Biochem. 1995, 230, 229-238.

Figure 1. Normal-phase capillary-LC-MALDI-TOF-MS of a dextran ladder labeled with 2-aminobenzamide. (A) UV chromatogram monitored at 254 nm and (B) base peak chromatogram (m/z 600-2000). Peaks are marked with the glucose oligomers Hex3-Hex9.

used to obtain the MS spectra in automatic mode. Glycan molecular ions [M + Na]+ were identified in the MALDI data, and their elution positions in NP-LC were determined by carrying out an extracted ion chromatogram. Molecular ion signals [M + Na]+ in every MALDI spot were selected for MALDI-CID in search of structural isomers. High-energy CID spectra were acquired manually with an average of 5000 laser shots/spectrum. The collision energy was set at 1 kV, and the oligosaccharide ions were collided in the CID cell with argon at a pressure of 2 × 10-6 Torr. RESULTS AND DISCUSSION Normal-Phase LC-MALDI-TOF/TOF Analyses of Standard Oligosaccharides. Automated NP-LC-MALDI-TOF-MS was first applied for the separation and detection of oligomeric glucose units in a dextran ladder (labeled with 2-AB), which are frequently used for standardizing the retention times of glycans and assessing the chromatography. The UV and MALDI base peak chromatogram (Figure 1) showed that the system was working effectively. An N-glycan trimannosyl standard was labeled with two different UV-absorbing tags (2-AB and 2-AA), and the sensitivity of each derivative in NP-LC-MALDI was compared. A wide range of sample concentrations could be detected using this method, down to the femtomole level. The 2-AA label proved to be slightly more sensitive at low concentrations, where a molecular ion signal [M + Na]+ could be observed after injection of 100 fmol of standard (data not shown). In addition, the fragmentation characteristics of an N-glycan standard (Man3GlcNAc2), labeled with either 2-AA or 2-AB, were compared upon high-energy CID of the [M + Na]+ molecular ions (data not shown). The fragment ions observed from MALDI-CID of the 2-AB-labeled glycan were similar to those observed previously on a TOF/TOF tandem mass spectrometer,21 where a range of cross-ring fragments were observed in addition to dominant glycosidic cleavages (B and Y ions). The 2-AA-labeled oligosaccharide gave the same types of fragment ions for this paucimannosidic glycan as observed for the 2-AB derivative. However, the 2-AA derivative appeared to give more intense high-energy cross-ring cleavages (A and X ions) and elimination ions (D ions22) as compared to glycosidic Y ions. These (21) Morelle, W., Slomianny, M. C., Diemer, H., Schaeffer, C., van Dorsselaer, A.; Michalski, J. C. Rapid Commun. Mass Spectrom. 2005, 19, 2075-2084.

Figure 2. Normal-phase capillary-LC-MALDI-TOF-MS of the released N-glycans from honey bee PLA2, labeled with 2-aminobenzoic acid. (A) Base peak chromatogram of PNGaseA-released glycans (m/z 800-3000). The elution positions of most of the N-glycans are indicated. (B, C) Superimposed extracted-ion chromatograms of Hex2HexNAc2Fuc, Hex3HexNAc2Fuc, and Hex3HexNAc4Fuc2, released by (B) PNGase A and (C) PNGase F. Symbol representation of glycan structure is as follows: GlcNAc, black square; GalNAc, white square; mannose, white circle; fucose, white diamond; xylose, filled diamond; 1-6 linkage, [/]; 1-4 linkage, -; 1-3 linkage, \; 1-2 linkage, |. Linkage not determined is shown by a dotted line.

former ions (cross-rings and D ions) give important information about linkage positions. Normal-Phase LC-MALDI-TOF/TOF Analyses of N-Glycans from PLA2. The potential for direct identification of glycan structural isomers by automated normal-phase MALDI-TOF/TOF tandem mass spectrometry was evaluated by analysis of the glycans from PLA2. Honey bee PLA2 has been shown previously to contain four sets of isomeric glycan structures, three of which differ by the substitution of R1-3 or R1-6 fucose in the core.18 The 2-AA and 2-AB derivatives of the PNGaseA-released glycans from PLA2 were analyzed using this normal-phase MALDI method. The base peak chromatogram revealed that these moieties were separated by the capillary amide column over a range of ∼30 min (Figure 2A). In addition, both 2-AA and 2-AB reducing-end derivatives showed similar base peak chromatograms and elution profiles (data not shown). From the observed masses in the MALDI-MS spectra, the compositions of the glycans could be deduced. Subsequent MALDI-CID of all the molecular ion signals [M + Na]+ revealed the presence of 15 different glycan structures. In addition to the dominant glycans (Hex2HexNAc2 and Hex3HexNAc2), other minor species were observed that are composed of core-(R1-3; R1-6) fucosylated structures, components with fucosylated lacdiNAc chains (GalNAcGlcNAcFuc), and oligomannosidic glycans. These data are in accordance with previous structural studies of these N-glycans.18 (22) Harvey, D. Mass Spectrom. Rev. 1999, 18, 349-450.

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Figure 3. (A, B) NP-LC-MALDI-CID spectra of a 2-AA-labeled paucimannosidic structure from PLA2 (Hex3HexNAc2Fuc) substituted with (A) 3-linked core fucose and (B) 6-linked core fucose. (C) NP-LC-MALDI-CID spectrum of a paucimannosidic structure from PLA 2 containing 3-linked fucose and labeled with 2-aminobenzamide. The identities of the ions labeled a and b (which indicate the presence of 3-linked core fucose) in (A) are shown in Scheme 1. Other fragment ions are labeled according to the nomenclature proposed by Domon and Costello27 and Spina et al.26 Symbol representation of glycan structure is summarized in the legend to Figure 2.

In all cases, the R1-3 and R1-6 core fucosylated isomers were resolved by normal-phase chromatography (Figure 2B). This is expected as on reversed-phase HPLC, R1-3-fucosylation causes a significant decrease, and R1-6-fucosylation a considerable increase, in elution time.23 However, the direct identification of these core fucosylated structural isomers could only be achieved by high-energy CID of glycans reductively aminated with 2-AA. Significant signals in the MALDI-CID spectra of the R1-3 core fucosylated structures derivatized with 2-AA were assigned to ions produced from cleavage and elimination of the fucose from the 3-position of reducing-end GlcNAc (Figure 3A and Scheme 1). These signals were absent from all corresponding glycans containing 6-linked core fucose (Figure 3B) and also the R1-3 fucosylated glycan labeled with 2-AB (Figure 3C). Therefore, the carboxylic acid of the 2-AA derivative is likely to interact with the carbohydrate to produce these diagnostic fragments for 3-linked fucose on reducing-end GlcNAc. Corroboration of these R1-3 and R1-6 core fucose assignments was achieved by the analysis of PLA2 glycans released by PNGaseF, which only contain 6-linked fucosylated cores (Figure 2C). (23) Hase, S.; Koyama, S.; Daiyasu, H.; Takemoto, H.; Hara, S.; Kobayashi, Y.; Kyogoku, Y.; Ikenaka, T. J. Biochem. (Tokyo) 1986, 100, 1-10.

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The remaining two structural isomers expected in PLA2 N-glycans18 that are composed of Hex3HexNAc2 (m/z 1054 [M + Na]+) were not completely separated by the normal-phase column (Figure 4A). However, MALDI-CID of m/z 1054 in all MALDI spots containing this molecular ion revealed the presence of both structures (Figure 4B and C). The minor isomer (Figure 4C) (which eluted slightly earlier than its more abundant isobaric counterpart) was identified by signals at m/z 758 (1,5X3) and 509 (D3) that were not detected in the NP-MALDI-CID spectrum of the corresponding major paucimannosidic glycan (Figure 4B). In particular, the D3 ion indicates that mannose at the 3-position of core β-mannose is absent in this oligosaccharide. This assignment is supported by the presence of a minor signal at m/z 421, which corresponds to the3,5A3 cross-ring fragment of core β-mannose and indicates that two mannose residues are linked to the upper (6linked) arm. Importantly, the fragment ions for this minor isomer were not present at detectable levels in the MALDI-CID spectrum of the Hex3HexNAc2 isobaric mixture due to the overwhelming dominance of the major paucimannosidic structure (Figure 4D). Thus, the partial separation of these isobaric glycans by NP-HPLC facilitated both identifications.

Scheme 1. Proposed Mechanisms for the Formation of Ions Labeled (A) a and (B) b Present in the CID Spectra of N-Glycans Containing 3-Linked Core Fucose That Were Reductively Aminated with 2-Aminobenzoic Acid (See Figures 3A and 5C)a

a There may be hydrogen bonding between the carboxylic acid of 2-AA and the acetamido group of reducing-end GlcNAc in (A). For the PLA2 N-glycan in Figure 3A, R ) Man3HexNAc. For the A. thaliana N-glycan in Figure 5C, R ) Man3PentHexNAc2.

Interestingly, one minor high-mannose structure that has not been previously identified on PLA2 was observed in these NPLC-MALDI data. The fragment ions in the MALDI-CID spectrum of this oligosaccharide (Man5GlcNAc2) (Figure 4E), did not indicate the presence of the conventional (Man(R1-6) [Man(R13) ]Man(R1-6) [ManR1-3]Man(β1-4)GlcNAc(β1-4)GlcNAc N-glycan that was expected. Instead, the D3 ion (at m/z 509.3), which is derived from a C3 cleavage followed by the elimination of two mannose residues from the 3-position of core β-mannose, indicates that the Man5GlcNAc2 structure (depicted in Figure 4E) is present. This is confirmed by the3,5A3 ion at m/z 421.2, which shows that only two mannose residues are linked via C-6 of core mannose. Automated Normal-Phase LC-MALDI-TOF/TOF Tandem Mass Spectrometry of N-Glycans from A. thaliana. The total N-glycans released from A. thaliana glycoproteins by PNGase A were reductively aminated with 2-AA and analyzed by NP-LCMALDI. This reducing-end derivative was chosen due to its ability to promote the formation of MALDI-CID fragment ions that indicate the presence of 3-linked core fucose (see Scheme 1). Glycans with core R1,3-fucose residues are expected to be present in these samples as previous studies have shown these complextype structures, also substituted with core β(1-2)-xylose, are abundant in this organism.24 The resulting MALDI base peak chromatogram (shown in Figure 5A) indicates that a range of structures are present that elute over ∼45 min. All molecular ion signals [M + Na]+ observed in the NP-LC-MALDI experiment were subjected to high-energy CID for detailed glycan structure elucidation and isomer identification. Sixteen different components were identified, which consist of small complex-type N-glycans (substituted with 3-fucose and/or 2-xylose) and oligomannosidic structures, all of which have (24) Lerouge, P.; Cabanes-Macheteau, M.; Rayon, C.; Fischette-Laine, A. C.; Gomord, V.; Faye, L. Plant Mol. Biol. 1998, 38, 31-48.

been observed previously on Arabidopsis glycoproteins.25 The abundant high-mannose oligosaccharides (Man5GlcNAc2-Man9GlcNAc2) were also accompanied by their truncated counterparts containing only one GlcNAc in the chitobiose core (Man5GlcNAcMan9GlcNAc). These data indicate that the PNGaseA enzyme preparation contained contaminating endoglycosidase H that is able to cleave high-mannose glycans between the two GlcNAc residues in the core. Interestingly, the extracted ion chromatograms and MALDI fragmentation data indicate that all complex-type N-glycans consisted of one main structural isomer. For example, structures composed of Man3GlcNAc3Xyl (m/z 1389 [M + Na]+) and Man3GlcNAc3XylFuc (m/z 1535 [M + Na]+) are substituted with one GlcNAc-containing antenna that is found on the upper arm of the trimannosyl core (see extracted ion chromatogram in Figure 5B). The corresponding isomers with GlcNAc on the lower arm were not present at detectable levels. This is shown by the presence of dominant nonreducing D3 ions (at m/z 682) in all fragment ion spectra acquired from MALDI spots spanning each peak (see Figure 5C for Man3GlcNAc3XylFuc). The cross-ring fragments 3,5A3 and 0,4A3 ions (m/z 462 and 448, respectively) confirm these assignments and show that GlcNAc-Man is attached to the 6-position of core β-mannose. Other interesting ions in these spectra include the E3 ion,26 which is produced from elimination of xylose at the 2-position of the B3 ion. The presence of 3-linked core fucose is also verified by the a and b ions (at m/z 1167 and 1122, respectively) that are observed when glycans containing this epitope are reductively aminated with 2-AA (see Scheme 1). Structural isomers were identified and partially separated for the high-mannose (Man7) N-glycans. This is best exemplified by (25) Rayon, C.; Cabanes-Macheteau, M.; Loutelier-Bourhis, C.; Salliot-Maire, I.; Lemoine, J.; Reiter, W. D.; Lerouge, P.; Faye, L. Plant Physiol. 1999, 119, 725-733. (26) Spina, E.; Sturiale, L.; Romeo, D.; Impallomeni, G.; Garozzo, D.; Waidelich, D.; Glueckmann, M. Rapid Commun. Mass Spectrom. 2004, 18, 392-398. (27) Domon, B.; Costello, C. E. Glycoconjugate J. 1988, 5, 397-409.

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Figure 4. (A) Superimposed extracted-ion chromatograms of PLA2 N-glycans composed of Hex3HexNAc2 and Hex5HexNAc2. (B, C) NP-LCMALDI-CID spectra of Hex3HexNAc2 isobaric glycans found in MALDI spot numbers (B) 77 and (C) 74. (D) MALDI-CID spectrum of the 2-AAlabeled Hex3HexNAc2 (m/z 1054 [M + Na]+) isobaric mixture acquired without separation by NP-HPLC. (E) Accumulated NP-MALDI-CID spectrum of the PLA2 high-mannose N-glycan (composed of Hex5HexNAc2) found in DHB spots spanning the peak labeled with a curly bracket in (A). All fragment ions are assigned according to the nomenclature proposed by Domon and Costello27 and Spina et al.26 Symbol representation of glycan structure is summarized in the legend to Figure 2.

the truncated Man7GlcNAc oligosaccharides (at m/z 1499 [M + Na]+), whose extracted ion chromatogram is shown in Figure 6A. This separation is not surprising, as previous studies have shown 8496 Analytical Chemistry, Vol. 78, No. 24, December 15, 2006

that isomers of 2-AA-labeled high-mannose N-glycans can be partially resolved by normal-phase HPLC.8 Furthermore, two highmannose structures, composed of Man7GlcNAc2, have also been

Figure 5. Normal-phase capillary-LC-MALDI-TOF-MS of the PNGase A-released N-glycans from A. thaliana, labeled with 2-AA. (A) Base peak chromatogram of released glycans (m/z 900-3000). The elution positions of some of the N-glycans are indicated. (B) Superimposed extracted-ion chromatograms of the complex-type N-glycans Hex3HexNAc3Pent and Hex3HexNAc3PentFuc. (C) Accumulated MALDI-CID spectrum of Hex3HexNAc3PentFuc acquired from the region labeled with a curly bracket in (B). Symbol representation of glycan structure is summarized in the legend to Figure 2.

shown to exist in Arabidopsis by HPAEC analyses, but their structures were not defined.25 However, in the current study, highenergy CID of these partly separated Man7 isomers gave fragment ions that identified these components. The MALDI-CID spectrum of the early-eluting Man7GlcNAc component is shown in Figure 6B. This spectrum is dominated by a signal at m/z 671.4 (D4), which indicates that three mannose residues are linked to the 3-position of β-mannose. This assignment is corroborated by the presence of the nonreducing cross-ring 0,4A4 and3,5A4 ions, which show that three mannose residues are also attached to upper arm of the core. Many dominant signals for this early-eluting isomer are also found in the MALDI-CID spectrum of its isobaric counterpart (e.g., m/z 527.4, 1013.4, and 1041.4) because the structures are not completely resolved. However, the antenna compositions of the latter isomer are revealed by the significant appearance of the D4 (m/z 833.4) ion and the cross-ring fragment

ions 3,5A4 (m/z 745.5) and 0,4A4 (m/z 731.5) (Figure 6C). These structurally informative ions are accompanied concomitantly with Y2R and1,5X2R fragments, which confirm that four mannose residues are attached to the 6-arm of core β-mannose. Furthermore, the increase in abundance of fragments at m/z 509.4 (D3R) and 493.4 (D3R - 16) suggests that the extra nonreducing-end mannose is attached to the uppermost antenna. However, the other isomeric structure with mannose linked to the middle antenna cannot be completely ruled out and may also be present. CONCLUSION Normal-phase capillary LC coupled to MALDI-TOF/TOF tandem mass spectrometry is suitable for the extensive characterization of N-glycan mixtures, as shown by the analysis of N-glycans released from honey bee phospholipase A2 and A. thaliana glycoproteins. Structural isomers containing R1-3 and Analytical Chemistry, Vol. 78, No. 24, December 15, 2006

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Figure 6. Normal-phase capillary-LC-MALDI-TOF-MS of the PNGase A-released N-glycans from A. thaliana, labeled with 2-AA. (A) Extractedion chromatogram showing the elution positions of the isobaric glycans composed of Hex7HexNAc. (B, C) Accumulated MALDI-CID spectra of Hex7HexNAc found in MALDI spots across the regions indicated with the curly brackets labeled (B) I and (C) II. Symbol representation of glycan structure is summarized in the legend to Figure 2.

R1-6 linked core fucose are clearly separated by the normal-phase column, and subsequent MALDI-CID of the 2-AA derivatives provide fragment ions diagnostic for 3-linked fucose on reducingend GlcNAc. Interpretation of the NP-MALDI-CID spectra of partially separated oligomannosidic isomers was complicated by contaminating signals from neighboring isobaric components. However, despite this limitation, these isobaric structures could still be defined by key fragments that reveal antenna compositions. Therefore, due to its capability of providing extensive structural information (e.g., antenna compositions, branching, sequence, and linkage positions) and its high-chromatographic resolution, this

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NP-MALDI-TOF/TOF method is a valuable tool for the detailed characterization of N-glycan complex mixtures. ACKNOWLEDGMENT The authors thank Prof. C. V. Robinson for her guidance and support. Thanks also to Dr. Graham Taylor and Chris Barton for useful discussions. Received for review July 31, 2006. Accepted September 21, 2006. AC0614137