General Microarray Technique for Immobilization and Screening of

9 Oct 2007 - Arjen R. de Boer,* Cornelis H. Hokke, Andre´ M. Deelder, and Manfred Wuhrer. Biomolecular Mass Spectrometry Unit, Department of ...
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Anal. Chem. 2007, 79, 8107-8113

General Microarray Technique for Immobilization and Screening of Natural Glycans Arjen R. de Boer,* Cornelis H. Hokke, Andre´ M. Deelder, and Manfred Wuhrer

Biomolecular Mass Spectrometry Unit, Department of Parasitology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands

We here present a printed covalent glycan microarray for protein-binding studies, using low-femtomole quantities of glycans. Glycans, either natural glycans, which were released from glycoproteins and glycolipids from natural sources, or synthetic glycans, were labeled with common fluorescent labels (e.g., 2-aminobenzamide or 2-aminobenzoic acid) by reductive amination and purified by HPLC. The purified glycoconjugates were covalently immobilized on commercial epoxide-activated glass slides via the secondary amine group that links the glycan moiety with the fluorescent tag. This immobilization procedure is generally applicable to reductively aminated glycans with different established fluorescent labels and allows the spatial arrangement of oligosaccharides. The microarray comprised a variety of natural glycans from various biological sources and synthetic glycans and provided informative binding fingerprints for the lectin concanavalin A as well as 14 monoclonal antibodies. Recognized glycans were characterized by tandem mass spectrometry revealing binding motifs. This natural glycan array allowed the characterization of the specificity of carbohydratebinding proteins for oligosaccharide ligands from sparse biological sources. Moreover, it was applied for the characterization of the microarray glycans by using known carbohydrate-binding proteins.

synthetic amine-functionalized glycans onto commercially available N-hydroxysuccinimide (NHS) slides.7 Often, however, target glycans will have to be isolated from natural sources. This is the case when chemical synthesis would be too tedious and time-consuming, but also when no prior knowledge about the structure of potential ligands of the biological target of interest (e.g., a specific glycoconjugate, cell type, or organism) is available. For the purification of natural glycans, derivatization with a fluorescent dye is a widely applied tool.8,9 The labeling is an effective way to improve both the detection and the separation selectivity in HPLC and CE.8,9 Derivatization is mostly performed by reductive amination of the aldehyde group at the reducing end of the oligosaccharide, which is easy to perform and fast. In 2005, Xia et al. reported a microarray suitable for the covalent immobilization of fluorescently labeled oligosaccharides onto NHS slides.10 Glycans were labeled with 2,6diaminopyridine, purified, and immobilized. This array showed a sensitivity of only the low-picomole level. Moreover, the introduction of 2,6-diaminopyridine as a reducing-end tag restricts possibilities for chromatography and mass spectrometric analysis. We here demonstrate a straightforward, femtomole-sensitive and robust glycan microarray technology that is suitable for the immobilization of fluorescently labeled oligosaccharides, either from natural or synthetic sources. The microarray is not restricted to one label or linker, but is compatible with widely used fluorescent labels including 2-aminobenzamide (2AB) and 2-aminobenzoic acid (AA). Subsequently, immobilization on the microarray is accomplished via the secondary aromatic amine group shared by most oligosaccharides labeled by reductive amination.

In the past decade, several glycan microarrays have been developed to study protein-carbohydrate interactions, which play an important role in many biological processes. The glycan microarrays mainly differ in the use of nonderivatized or derivatized oligosaccharides and in immobilization chemistry,1-5 as reviewed by Culf et al.6 Blixt et al. have developed a sensitive microarray based on the immobilization of 200, predominantly

EXPERIMENTAL SECTION Materials. Monoclonal antibodies (mAbs) were produced as described previously.11 Concanavalin A (Con A, from Canavalia ensiformis, Jack beans), Alexa Fluor 647 conjugate, rabbit antimouse IgG (H+L, Alexa Fluor 647 conjugate), and goat anti-mouse IgM (H, Alexa Fluor 555 conjugate) were from Invitrogen (Breda,

* To whom correspondence should be addressed. E-mail: [email protected]. (1) Feizi, T.; Chai, W. G. Nat. Rev. Mol. Cell Biol. 2004, 5, 582-588. (2) Grun, C. H.; van Vliet, S. J.; Schiphorst, W. E. C. M.; Bank, C. M. C.; Meyer, S.; van Die, I.; van Kooyk, Y. Anal. Biochem. 2006, 354, 54-63. (3) Houseman, B. T.; Mrksich, M. Chem. Biol. 2002, 9, 443-454. (4) Lee, M.; Shin, I. Org. Lett. 2005, 7, 4269-4272. (5) Ratner, D. M.; Adams, E. W.; Disney, M. D.; Seeberger, P. H. Chembiochem 2004, 5, 1375-1383. (6) Culf, A. S.; Cuperlovic-Culf, M.; Ouellette, R. J. OMICS 2006, 10, 289310.

(7) Blixt, O.; Head, S.; Mondala, T.; Scanlan, C.; Huflejt, M. E.; Alvarez, R.; Bryan, M. C.; Fazio, F.; Calarese, D.; Stevens, J.; Razi, N.; Stevens, D. J.; Skehel, J. J.; van Die, I.; Burton, D. R.; Wilson, I. A.; Cummings, R.; Bovin, N.; Wong, C. H.; Paulson, J. C. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 17033-17038. (8) Shilova, N. V.; Bovin, N. V. Russ. J. Bioorg. Chem. 2003, 29, 309-324. (9) Anumula, K. R. Anal. Biochem. 2006, 350, 1-23. (10) Xia, B. Y.; Kawar, Z. S.; Ju, T. Z.; Alvarez, R. A.; Sachdev, G. P.; Cummings, R. D. Nat. Methods 2005, 2, 845-850. (11) Nibbeling, H. A. M.; Kahama, A. I.; Van Zeyl, R. J. M.; Deelder, A. M. Am. J. Trop. Med. Hyg. 1998, 58, 543-550.

10.1021/ac071187g CCC: $37.00 Published on Web 10/09/2007

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The Netherlands). RNase B, BSA, 1,2-epoxybutane, and 2,6diaminopyridine were from Sigma-Aldrich (Zwijndrecht, The Netherlands). Synthetic oligosaccharides were from Dextra Laboratories (Reading, UK) and Sigma-Aldrich. Natural glycans were isolated from RNase B, asialofetuin, fetuin, KLH (all from Sigma-Aldrich), Schistosoma mansoni adult worms and eggs (obtained from infected hamsters), and human serum (healthy volunteer). BSA- and NH2-oligosaccharide conjugates were prepared and purified as described previously.12 The polyvalency of the BSA neoglycoproteins was as follows: LeX, 8 glycans/BSA; LDN, 8 glycans/BSA; FLDN, 13 glycans/BSA; LDNF, 5 glycans/BSA. Glycan Release. N-Glycans from the biological sources RNase B, asialofetuin, fetuin, KLH, and human serum glycoproteins were released by PNGase F (peptide N-glycosidase F, Roche Diagnostics, Mannheim, Germany) after protein reduction and denaturation, as described previously.13 Released glycans were purified using sequentially RP- and graphitized carbon cartridges as described previously.13 The purification of N-glycans from S. mansoni adult worm glycoproteins has been described before.13 S. mansoni egg glycolipids were purified by organic solvent extraction,14 Folch partitioning,15 and desalting using an RP C18 cartridge.14 Glycan moieties were released from the glycolipids by recombinant ceramide glycanase (endoglycoceramidase II from Rhodococcus sp., Takara Shuzu, Otsu, Shiga, Japan) and purified on an RP C18 cartridge by elution with water.14 All glycan fractions were dried before labeling. Glycan Labeling and Purification. Natural and synthetic glycans were labeled with the fluorescence compound 2AB or AA (both from Sigma-Aldrich) by reductive amination with sodium cyanoborohydride.16 The labeled glycans were purified by HILIC (Amide-80 column, 4.6 mm × 25 cm, particle size 5 µm; Tosoh Bioscience, Stuttgart, Germany). Eluent A consisted of 50 mmol/L formic acid (pH 4.4); eluent B consisted of eluent A/acetonitrile 20:80. A linear gradient from 100 to 40% eluent B was applied at a flow rate of 1 mL/min. In general, fluorescence detection was performed at λex-λem 360-425 nm, but for large amounts of analyte, λex-λem 280-500 nm was used. Fractions were collected and analyzed by MALDI-TOF-MS. HILIC fractions 8, 10, and 15 of S. mansoni egg glycolipidderived glycans were fractionated in a second dimension, using RP HPLC on a Hypersil ODS column (2 mm × 25 cm; particle size 3 µm; Thermo Electron, Waltham, MA) at 0.2 mL/min. Eluent A consisted of 0.4% acetonitrile with 0.1% formic acid; eluent B consisted of 95% acetonitrile with 0.1% formic acid. Gradient conditions: 6.5 min constant at 5% eluens B, followed by a linear gradient to 50% eluent B in 25 min. Fractions were collected and analyzed by MALDI-TOF-MS. MALDI-TOF-MS(/MS). MALDI-TOF-MS(/MS) was performed on an Ultraflex II mass spectrometer (Bruker Daltonics, (12) Vermeer, H. J.; Halkes, K. M.; van Kuik, J. A.; Kamerling, J. P.; Vliegenthart, J. F. G. J. Chem. Soc., Perkin Trans. 2000, 2249-2263. (13) Wuhrer, M.; Koeleman, C. A. M.; Deelder, A. M.; Hokke, C. H. FEBS J. 2006, 273, 347-361. (14) Wuhrer, M.; Dennis, R. D.; Doenhoff, M. J.; Lochnit, G.; Geyer, R. Glycobiology 2000, 10, 89-101. (15) Folch, J.; Lees, M.; Stanley, G. H. S. J. Biol. Chem. 1957, 226, 497-509. (16) Bigge, J. C.; Patel, T. P.; Bruce, J. A.; Goulding, P. N.; Charles, S. M.; Parekh, R. B. Anal. Biochem. 1995, 230, 229-238.

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Bremen, Germany). Fragment analysis was achieved by laserinduced decomposition by using the LIFT-TOF/TOF-MS/MS facility. Samples were spotted with 6-aza-2-thiothymine (SigmaAldrich) or 2,5-dihydroxybenzoic acid (Bruker Daltonics) matrix and analyzed in the positive or negative (reflectron) mode. Glycan Microarray Design. Printing and immobilization were performed following the manufacturer’s instructions (Schott Nexterion, Jena, Germany). Analytes were dissolved in 20 µL of 50: 50 H2O/spotting buffer (Nexterion Spot, Schott Nexterion) in 384well V-bottom plates (Genetix, New Milton, UK) at 10 µmol/L unless stated otherwise (Figure 1 and Figure S-1 (Supporting Information); structures were drawn using GlycoWorkbench software, http://www.eurocarbdb.org). In total, 240 samples were printed on epoxide-modified glass slides (Slide E, Schott Nexterion) by contact printing (surface contact 25 ms) using the Omnigrid 100 microarrayer (Genomic Solutions, Ann Arbor, MI). The microarray was equipped with SMP3 pins with uptake channels (Telechem International, Sunnyvale, CA), which deposed ∼0.7 nL at each contact. Each sample was printed in triplicate per array, and each array was printed five times per glass slide (3600 spots/slide). Dot spacing was 250 (X) and 300 µm (Y), and array spacing was 7500 µm. Printed slides were incubated overnight at room temperature at sufficient humidity to prevent drying of the spots. Thin-filmlike hydrophobic barriers were created by a PAP-PEN (RPI, Mount Prospect, IL) between the printed arrays present on a glass slide. The barriers created a proper surface tension to hold solutions within the array area. Compounds that did not bind to the glass slide were removed by rinsing with PBS containing 0.05% Tween 20. Remaining active epoxide groups were blocked by 4% BSA in PBS-0.05% Tween 20 for 90 min at room temperature. Subsequently, the slides were rinsed with PBS. Binding Assay. Each microarray on the glass slides was incubated with a mAb or lectin in PBS-0.01% Tween 20 with 1% BSA for 90 min at room temperature. Slides were then washed with successive rinses in PBS-0.05% Tween 20 and PBS. In twostep binding assays, slides were in addition incubated with antiIgG or anti-IgM (10 µg/mL, in PBS-0.01% Tween 20 with 1% BSA) for 45 min at room temperature. During incubation, the slides were protected from light. Subsequently, slides were rinsed by PBS-0.05% Tween 20, PBS, and H2O and dried by N2. Data Analysis. Slides were scanned for fluorescence by the G2565BA scanner (Agilent Technologies, Santa Clara, CA) using two lasers (532 and 633 nm). At these wavelengths, AA and 2AB do not fluoresce. Data and image analyses were performed with GenePix Pro 6.0 (Molecular Devices, Sunnyvale, CA). Spots were integrated using irregular alignment without background subtraction. Total intensities of the replicates were averaged and plotted against the sample numbers. RESULTS Coupling Technique. The secondary aromatic amine present on glycans after reductive amination with fluorescent dyes such as AA, 2AB, and 2,6-diaminopyridine (DAP) reacts readily with epoxy-activated reagents at room temperature. This was demonstrated by reacting fluorescently labeled glycans with epoxybutane and characterizing the reaction products by MALDI-TOF/TOFMS/MS, which confirmed the allocation of the epoxybutane

Figure 1. Structural schemes of part of the glycan structures that were spotted on the microarray. Nature of the depicted glycan structures: 2-34, isolated from the glycoprotein RNase B; 44, isolated from S. mansoni adult worms; 62-65, synthetic NH2-oligosaccharide conjugates; 102-111, isolated from S. mansoni egg glycolipids, two-dimensional HPLC fractions; 153, isolated from S. mansoni egg glycolipids, onedimensional HPLC fraction; 167-186, isolated from the glycoprotein KLH; 193-196, synthetic BSA-oligosaccharide conjugates; 218-230, isolated from RNase B; and 239, synthetic BSA-oligosaccharide conjugate. A complete list of structures can be found in Figure S-1.

substituent to the fluorescent tag (see Figure S-2 Supporting Information). This finding prompted us to test epoxy-activated glass slides for the immobilization of fluorescently labeled natural glycans. Array Design. Glycoproteins and glycolipids were extracted from various natural sources. Oligosaccharide moieties were enzymatically released, purified, and labeled with AA and 2AB (Figure 2A). Labeled glycans were fractionated by one-dimensional or two-dimensional HPLC (HILIC and RP; Figure 2B and see Figure S-3 Supporting Information). MALDI-TOF-MS analyses of the fractionated glycans were carried out to assess purity and composition (Figure 2C), and further structural characterization was performed by MALDI-TOF/TOF-MS/MS. The fluorescently labeled glycans were printed onto epoxide slides (Figure 2D). The immobilization was performed at room temperature. The resulting spot sizes were between 50 and 100 µm. The list of structures present in the array can be found in Figure 1 and Figure S-1. Arrays were overlaid with carbohydrate-binding proteins (CBPs), which were Con A 10 µg/mL as well as 14 mAbs, either IgG or IgM, followed by fluorescently labeled secondary antibodies (Figure 2E). Fluorescence signals were integrated and plotted against the sample numbers (Figure 2F), resulting in unique

fingerprints (Figure 3 and Figure S-4 Supporting Information). For each of the CBPs, recognized natural glycans present on the array were characterized by MALDI-TOF/TOF-MS/MS, identifying shared structural motifs of the binding glycan species. In the following, the results of the validation of the glycan array by the analysis of five previously characterized CBPs, namely, Con A and four monoclonal antibodies, are described. The binding specificity determined with the natural glycan array is compared to the specificity reported in the literature, demonstrating the potential of this printed microarray for the characterization of protein-carbohydrate interactions. Analysis of Con A. Con A is known for binding terminal nonreducing R-D-mannose and R-D-glucose residues with the trimannosyl moiety Man(R1-6)[Man(R1-3)]Man as primary epitope.17,18 On our microarray, Con A bound to the oligomannosetype structures 14, 28, 44, and 218-230 (Figure 3A,B). Other positive samples were 166-188, with fractions 167, 168, 173, 177, and 181 being the most intense. These fractions also contain oligomannose glycans with the Man(R1-6)[Man(R1-3)]Manβ1(17) Goldstein, I. J.; Poretz, R. D. The Lectins; Academic Press: London, 1986. (18) Gupta, D.; Oscarson, S.; Raju, T. S.; Stanley, P.; Toone, E. J.; Brewer, C. F. Eur. J. Biochem. 1996, 242, 320-326.

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Figure 2. Scheme of the microarray approach as presented in this paper. (A) Glycans are derivatized, (B) fractionated by HPLC, (C) analyzed by MALDI-TOF-MS(/MS), (D) immobilized on microarray epoxide slides, (E) assayed for protein interaction, and (F) obtained data are interpreted.

Figure 3. Microarray results for Con A and four mAbs. (A) Microarray image after incubation with Con A. Samples were spotted in triplicate. The numbers correspond with the compounds on the sample list. The original red color of the spots was changed to white for better visualization. (B) Glycan binding of Con A, (C) glycan binding of mAb 100-4G11, (D) 128-1E7-C, (E) 114-4D12-A, and (F) 291-2G3-A.

4GlcNAc epitope. In addition, Con A bound to RNase B, which contains a single N-glycosylation site occupied with oligomannose glycans. These results are fully in accordance to the previously described specificity of Con A.17,18 8110

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The detection limit for oligomannose structures after incubation with Con A was 1 fmol. The linearity was at least 2 orders of magnitude. Replicates of the oligomannose structure Man6GlcNAc2-AA (218-230) were printed to investigate the repeat-

Figure 4. MALDI-TOF-MS (A) and MALDI-TOF/TOF-MS/MS (B) spectra of 167. Spectra were acquired in the negative ion mode.

ability, which was 14.9% RSD (n ) 39). The nonspecific binding was minimal as investigated by printing blanks (spotting buffer and BSA). The results demonstrated that both labels (AA and 2AB) were suitable for immobilization. The labels provided sufficient linker length and glycan density to be successful for screening CBP interactions. Moreover, the labels did not interfere with the final microarray scanning because of the low emission wavelengths of AA and 2AB. In addition, DAP-labeled glycans were likewise analyzed (data not shown). Analysis of mAb 100-4G11. The mAb 100-4G11 bound most intensely to RNase B (206) as well as to the KLH-derived AAlabeled N-glycans (fractions 167, 168, and 177, Figure 3C). MALDITOF-MS analysis of fraction 167 showed one major peak with m/z 1030.6 ([M - H]-), which corresponds to Hex3HexNAc2-AA (Figure 4A). The fragment spectrum of this glycan obtained by MALDI-TOF/TOF-MS/MS was consistent with a terminal trimannosyl core, thereby identifying the N-glycan as Man(R1-6)[Man(R1-3)]Man(β1-4)GlcNAc(β1-4)GlcNAc- (Figure 4B), which has been described previously.19 The glycans 168 and 177 likewise contained the trimannosyl structural motif (MS/MS data not shown). Moreover, mAb 100-4G11 showed (less intensive) binding to other N-glycans exhibiting terminal trimannosyl structures (2, 14, 22, 44, 172, and 173). The preferred binding of 100-4G11 to the trimannosyl epitope was in accordance with expectations based on the literature.20 The binding characteristics of mAb 100-4G11 are similar to that of Con A, with the difference for the highmannose-type structures 16, 18, 20, 30, 32, and 34, which gave rise to intense signals with Con A but only weak signals with 1004G11. Analysis of mAb 128-1E7-C. The mAb 128-1E7-C bound to N-glycans from KLH (180-189) (Figure 3D). MALDI-TOF/TOFMS/MS of these oligosaccharides revealed a common Fuc(R13)GalNAc(β1-4)[Fuc(R1-3)]GlcNAc (FLDNF) structural motif. For example, the MS spectrum of fraction 186 indicated a signal of m/z 2184.8 ([M - H]-, Figure 5A), consistent with an N-glycan of composition Hex5HecNAc4Fuc2Xyl1. MALDI-TOF/TOF-MS/MS (19) Geyer, H.; Wuhrer, M.; Resemann, A.; Geyer, R. J. Biol. Chem. 2005, 280, 40731-40748. (20) van Remoortere, A.; Bank, C. M. C.; Nyame, A. K.; Cummings, R. D.; Deelder, A. M.; van Die, I. Glycobiology 2003, 13, 217-225.

showed intense peaks of m/z 1486.8 and 1835.9 fragments (Y4R and Y5R′ fragments, Figure 5A) indicating an FLDNF structural motif that is in accordance with a previous study.19 The FLDNF structural motif was likewise detected in the KLH N-glycan fractions 180, 181, and 183-189, which were recognized by mAb 128-1E7-C. Moreover, this mAb also bound to the glycan moieties obtained from S. mansoni egg glycolipids. Also these binding glycans (102, 105, 111, 154, 156, and 161; Figure S-5A Supporting Information) were shown to contain the FLDNF structural motif, thereby establishing this structural determinant as a natural target structure of the mAb 128-1E7-C. This mAb also bound to the chemically synthesized FLDN (Fuc(R1-3)GalNAc(β1-4)GlcNAcβ1), which was immobilized via an amine-containing tether (65) and as an FLDN-BSA neoglycoprotein (195, 196, and 239). The recognition of both FLDNF and FLDN by mAb 128-1E7-C is in agreement with Biacore findings of van Roon et al.21 Analysis of mAb 114-4D12-A. This mAb bound to S. mansoni egg glycolipid-derived glycans 106, 109, 158, and 163. Analysis of the recognized structures by MALDI-TOF/TOF-MS/ MS readily indicated a shared terminal Fuc(R1-2)Fuc(R1-3)GalNAc(β1-4)[Fuc(R1-2)Fuc(R1-3)]GlcNAc (DF-LDN-DF) binding motif (Figure 3E, MS/MS data in Figure S-5B). This result is in agreement with the analysis of the 114-4D12-A binding characteristics using affinity chromatography, which revealed that DF-LDN-DF is 114-4D12-A target structure on S. mansoni egg O-glycans.22 Analysis of mAb 291-2G3-A. Biacore analysis and crystallization studies have shown that 291-2G3-A binds to the Lewis X (LeX) (Gal(β1-4)[Fuc(R1-3)]GlcNAc) trisaccharide.23 In accordance with this study, this mAb bound intensely to the synthetic LeX structures immobilized directly via an aminecontaining tether (62, 63) or as LeX-BSA neoglycoprotein (193, (21) van Roon, A. M.; Aguilera, B.; Cuenca, F.; van, R. A.; van der Marel, G. A.; Deelder, A. M.; Overkleeft, H. S.; Hokke, C. H. Bioorg. Med. Chem. 2005, 13, 3553-3564. (22) Robijn, M. L. M.; Koeleman, C. A. M.; Wuhrer, M.; Royle, L.; Geyer, R.; Dwek, R. A.; Rudd, P. M.; Deelder, A. M.; Hokke, C. H. Mol. Biochem. Parasitol. 2007, 151, 148-161. (23) van Roon, A. M. M.; Pannu, N. S.; de Vrind, J. P. M.; van der Marel, G. A.; van Boom, J. H.; Hokke, C. H.; Deelder, A. M.; Abrahams, J. P. Structure 2004, 12, 1227-1236.

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Figure 5. MALDI-TOF-MS (A) and MALDI-TOF/TOF-MS/MS (B) spectra of 186. Spectra were acquired in the negative ion mode.

194) (Figure 3F). In addition, 291-2G3-A bound also to 153, which comprises a LeX-containing glycan next to a glycan exhibiting pseudo-LeY (Fuc(R1-3)Gal(β1-4)[Fuc(R1-3)]GlcNAc). This establishes mAb 291-2G3-A as a mAb with affinity for LeX and possibly also for pseudo-LeY. In conclusion, the binding motifs found for Con A and the four mAbs with the natural glycan array were in agreement with the specificity described in the literature. Fingerprinting using this natural glycan array built from relevant biological material is therefore suitable for the characterization of protein-carbohydrate interactions. DISCUSSION A variety of fluorescent labels are available for reductive amination of glycans.8,9 During reductive amination, the primary amine on the label reacts with the aldehyde of the oligosaccharide forming an imine (Schiff base), which is subsequently reduced to a secondary amine by hydride reagents. For many fluorescent labels, this secondary amine group is aromatic. Since secondary aromatic amines are reactive with epoxide groups (Figure 2), we have chosen to use epoxide glass slides for the construction of the presented natural glycan microarray. The amine group shared by all reductive amination labels is therefore suitable for both glycan labeling and subsequent immobilization of the labeled glycan on the glass slide. Therefore, the coupling procedure presented here is broadly applicable for a variety of established fluorescent labels. The microarray of glycans labeled with AA and 2AB shows a low-femtomole detection limit with Con A and various mAbs, comparable to the NHS-based microarray of Blixt et al.7 Overall, the results show that this approach for screening of both natural and synthetic glycans using standard protocols is facile and straightforward. The array is not limited to either synthetic or natural glycans, nor to pure samples. The ability to combine all kind of glycans just increases the potential of the array in CBP characterization. We have analyzed the binding properties of various previously characterized anti-carbohydrate mAbs to validate an array of 240 samples, which comprise a heterogeneous 8112

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set of purified glycans and glycan mixtures. CBP specificities were readily revealed from the array results and were in agreement with literature reports, demonstrating the potential of this microarray for CBP screening. We have applied one-dimensional and two-dimensional HPLC fractionation to the glycans obtained from complex biological sources. The analytical power of the natural glycan microarray may be increased by applying additional HPLC separation techniques (high-pH anion-exchange chromatography and graphitized carbon HPLC), capillary electrophoresis (for example, after derivatization of glycans with 1-aminopyrene-3,6,8-trisulfonate (APTS)), and affinity purification using lectins and antibodies. For this purpose, the free choice between a variety of fluorescent labels appears to be highly advantageous, as it allows the use of a range of separation techniques.8,9 In conclusion, our approach links modern glycoanalytical techniques based on separation techniques and MS to an array platform for the study of protein-carbohydrate interactions. The glycan microarray may be used to answer a variety of questions: (1) In this study, we have shown that the array can be used to reveal the structural element(s) (epitope) that is recognized by CBPs. (2) When using well-characterized CBPs, the array can be used to identify glycans or glycan fractions from complex biological samples that carry the CBP binding motif, thereby providing structural information on glycans and glycan fractions. (3) Another opportunity is to use the glycan microarray for profiling; i.e., glycans are isolated from healthy and diseased tissue, spotted on the microarray, and screened by patient body fluids to obtain clinical profiles, which may provide biomarker candidates indicative of a certain diagnosis or prognosis. Future applications of the array will be the analysis of novel protein-carbohydrate interactions for bacterial adhesins, viral agglutinins, human lectins, and serum anti-carbohydrate antibodies with various target glycans, isolated from biological samples of interest. These biological samples may be glycoproteins and glycolipids from various human tissues and tumors and from

bacterial polysaccharides and glycoconjugates, as well as glycans from parasites and virus particles. Moreover, libraries of hundreds of well-characterized fluorescently labeled glycans generated during structural studies are stored in many glycoanalytical laboratories, and these may now be used for the preparation of tailor-made natural glycan arrays. With the future growth and specialization of such natural glycan arrays, their analytical potential will increase, and it should be possible to obtain very detailed pictures of the fine specificity of CBPs for their authentic glycan ligands. Both the mass spectrometric techniques and the array feature low-femtomole sensitivity and are, therefore, applicable to small quantities of biological materials. For large sets of glycans, structural information and information on protein binding can be directly linked, providing detailed insights into protein-carbohydrate interactions.

performance liquid chromatography; KLH, keyhole limpet hemocyanin; LeX, Lewis X, Gal(β1-4)[Fuc(R1-3)]GlcNAc; LDN, GalNAc(β1-4)GlcNAc; LDNF, GalNAc(β1-4)[Fuc(R1-3)]GlcNAc; mAb, monoclonal antibody; MALDI-TOF-MS, matrix-assisted laser desorption/ionization-time of flight-mass spectrometry; Man, mannose; RNase, ribonuclease; RP, reversed phase; Xyl, xylose.

ABBREVIATIONS 2AB, 2-aminobenzamide; AA, 2-aminobenzoic acid; BSA, bovine serum albumin; CBP, carbohydrate binding protein; Con A, concanavalin A; FLDN, Fuc(R1-3)GalNAc(β1-4)GlcNAc; FLDNF, Fuc(R1-3)GalNAc(β1-4)[Fuc(R1-3)]GlcNAc; Fuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; Hex, hexose; HexNAc, N-acetylhexosamine; HILIC, hydrophilic interaction liquid chromatography; HPLC, high-

SUPPORTING INFORMATION AVAILABLE Figures S-1-S-5 as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org/ac.

ACKNOWLEDGMENT This project was financed by the Netherlands Genomics Initiative (Horizon Breakthrough Project 050-71-302). We thank Carolien A.M. Koeleman for excellent technical assistance and Alexandra van Remoortere for critically reading the manuscript, and we appreciate the support of Dr. Jord C. Stam (Utrecht University, The Netherlands) and of the Leiden Genome Technology Center (Leiden, The Netherlands).

Received for review June 5, 2007. Accepted August 3, 2007. AC071187G

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