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Carbohydrate-Protein Interactions Investigated on Plastic Chips Statically Coated with Hydrophobically Modified Hydroxyethylcellulose Fuquan Dang,*,† Eiki Maeda,‡ Tomo Osafune,‡ Kazuki Nakajima,§ Kazuaki Kakehi,§ Mitsuru Ishikawa,† and Yoshinobu Baba†,‡ Health Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Hayashi-cho 2217-14, Takamatsu 761-0395, Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, and Faculty of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashi-Osaka 577-850, Japan We developed a novel method for rapid screening of carbohydrate-protein interactions using poly(methyl methacrylate) (PMMA) channels statically coated with hydrophobically modified hydroxyethylcellulose (HM-HEC). We found that a self-assembled monolayer (SAM) of HM-HEC on a PMMA surface intact by water allows rapid and reproducible separations of glycan samples using a 20 mM phosphate without HM-HEC. The underlying mechanism for dynamic and static coatings on the PMMA surface is discussed. Simultaneous analysis of the molecular interaction between a complex mixture of carbohydrates from r1-acid glycoprotein and proteins has been successfully achieved in PMMA channels statically coated with a SAM of HM-HEC. Glycosylation is one of the most common post-translational modifications of proteins to modulate protein functions both on the cellular surfaces and within cells. Carbohydrate-protein interactions play a vital role in many biological processes such as trafficking and clearing of glycoproteins, immune defense, malignancy, and cell-cell recognization.1,2 Because significant alterations in carbohydrate structure or site occupancy occur in glycosylation of proteins in response to cellular signals or stages, the characterization of the carbohydrate chains that carbohydratebinding proteins recognize remains greatly challenging.3 The methods that allow high-speed and high-throughput screening of carbohydrate-protein interactions are therefore crucial to better understanding the biological roles of carbohydrate alternations in glycosylation and the relationship between their structures and functions. Carbohydrate-protein interactions have been investigated using various techniques, such as calorimetry,4 surface plasmon reso* To whom correspondence should be addressed. E-mail: fuquan-dang@ aist.go.jp. Phone: +81-87-869-4104. Fax: +81-87-869-4113. † AIST. ‡ Nagoya University. § Kinki University. (1) Dwek, R. A. Chem. Rev. 1996, 96, 683–720. (2) Roth, J. Chem. Rev. 2002, 102, 285–303. (3) Qiu, R.; Regnier, F. E. Anal. Chem. 2005, 77, 7225–7231. (4) Dam, T. K.; Brewer, C. F. Chem. Rev. 2002, 102, 387–429. 10.1021/ac902014c CCC: $40.75 2009 American Chemical Society Published on Web 11/10/2009
nance,5 frontal affinity chromatography (FAC),6 affinity capillary electrophoresis (ACE),7,8 and carbohydrate microarray.9 The bioaffinity assays based on modern separation techniques such as ACE have received considerable attention because of their ability to allow simultaneous identification and evaluation of interactions between multiple carbohydrate chains and proteins in a single run. The performance of such bioaffinity assays greatly depends on the characteristics of separation techniques. Because of the instinct characteristics of high speed, high resolution, high throughput, and great integration potential, microchip electrophoresis (µ-CE) has become one of the most powerful techniques in separation science. ACE in microchip format (µ-ACE) has been exploited for rapid screening of antibody-antigen and cyclodextrin-drug interactions and glycoform analysis of glycoproteins.10-13 Separation of a complex mixture of glycans in the presence of a specific protein at various concentrations in the running buffer is a basis for µ-ACE to monitor the carbohydrate-protein interactions (Figure 1A). Individual glycans in the mixture are brought into equilibrium with the protein added in the running buffer during microchip electrophoresis. The binding specificities and constants of individual glycans with a protein are simultaneously evaluated from the corresponding shift in the electrophoretic mobility. The band broadening and distortion further evidence the binding behavior. It is thus of utmost importance to minimize the nonspecific interactions of analytes with the channel surface; otherwise, the results may not reflect intrinsic affinities of individual glycans with the protein but artifacts of nonspecific interactions with the surface. (5) Duverger, E.; Frison, N.; Roche, A.-C.; Monsigny, M. Biochimie 2003, 85, 167–179. (6) Schriemer, D. C.; Bundle, D. R.; Li, L.; Hindsgaul, O. Angew. Chem., Int. Ed. 1998, 37, 3383–3387. (7) Nakajima, K.; Oda, Y.; Kinoshita, M.; Kakehi, K. J. Proteome Res. 2003, 2, 81–88. (8) Shimura, K.; Kasai, K. Anal. Biochem. 1997, 251, 1–16. (9) Fukui, S.; Feizi, T.; Galustian, C.; Lawson, A. M.; Chai, W. Nat. Biotechnol. 2002, 20, 1011–1017. (10) Cheng, S. B.; Skinner, C. D.; Taylor, J.; Attiya, S.; Lee, W. E.; Picelli, G.; Harrison, D. J. Anal. Chem. 2001, 73, 1472–1479. (11) Guijt, R. M.; Baltussen, E.; van Dederm, G. W. Electrophoresis 2002, 23, 823–835. (12) Stettler, A. R.; Schwarz, M. A. J. Chromatogr., A 2005, 1063, 217–225. (13) Mao, X.; Luo, Y.; Dai, Z.; Wang, K.; Du, Y.; Lin, B. Anal. Chem. 2004, 76, 6941–6947.
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Figure 1. Schematic illustration of (A) µ-ACE in a PMMA channel statically coated with a SAM of HM-HEC and (B) the structure of HMHEC. The degree of substitution (DS) of HM-HEC is 0.01 relative to the average number of n-C16H33 groups per glucose unit.
So far, dynamic coating is one of the most widely used methods for surface modification of disposable plastic chips, wherein nonspecific interactions of analytes with the surface are minimized by a coating layer of surface modifiers, such as surfactants and water-soluble polymers, physically adsorbed on the channel wall.14-18 Generally, surface modifiers at high concentrations must be kept in the running buffer to achieve efficient coating and thus good separation. The surface modifiers often greatly increase the viscosity or conductivity of the buffer solution, causing difficulties in buffer loading and rinsing of microchannels. Furthermore, the surface modifiers, especially those at high concentrations, are detrimental to many applications such as online coupling of mass spectrometry (MS), enzymatic assays, and µ-ACE. It has been observed that some water-soluble polymers can form static coating where polymers need not be in the running buffer.19,20 Static coating is extremely attractive to the applications sensitive to surface modifiers in the running buffer because of the elimination of the nondesirable effects of surface modifiers; however, the mechanism for static coating on a solid surface still remains largely unknown. In the current work, we report a new µ-ACE method for rapid screening of the carbohydrate-protein interactions on a poly(methyl methacrylate) (PMMA) channel statically coated with hydrophobically modified hydroxyethylcellulose (HM-HEC). We found that a self-assembled monolayer (SAM) of HM-HEC on a PMMA surface allows rapid and reproducible separations of glycans using a 20 mM phosphate without HM-HEC, whereas that of hydroxyethylcellulose (HEC) allows the same good separations in a 20 mM phosphate only with HEC at high concentrations. The great difference in the coating performance between HM-HEC (14) Dang, F.; Zhang, L.; Hagiwara, H.; Mishina, Y.; Baba, Y. Electrophoresis 2003, 24, 714–721. (15) Belder, D.; Ludwig, M. Electrophoresis 2003, 24, 3595–3606. (16) Dolnı´k, V. Electrophoresis 2004, 25, 3589–3601. (17) Tran, N. T.; Taverna, M.; Miccoli, L.; Angulo, J. F. Electrophoresis 2005, 26, 3105–3112. (18) Dang, F.; Kakehi, K.; Cheng, J.; Tabata, O.; Kurokawa, M.; Nakajima, K.; Ishikawa, M.; Baba, Y. Anal. Chem. 2006, 78, 1452–1458. (19) Doherty, E. A. S.; Meagher, R. J.; Albarghouthi, M. N.; Barron, A. E. Electrophoresis 2003, 24, 34–54. (20) Lucy, C. A.; MacDonald, A. M.; Gulcev, M. D. J. Chromatogr., A 2008, 1184, 81–105.
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and HEC is attributed to the different orientations of glucose rings in the SAMs. The perpendicular arrangement of glucose rings with HEC results in a SAM composed of both H-bonded sugar chains and non-H-bonded sugar chains, whereas the flat orientation of glucose rings with HM-HEC leads to a SAM composed of only H-bonded sugar chains. The separation performance with the SAMs of HEC and HM-HEC evidence that H-bonded sugar chains that are tightly anchored on the surface intact by water result in irreversible adsorption, whereas non-H-bonded sugar chains that are loosely attached on the surface lead to reversible adsorption. The current work provides new insights into the underlying mechanism for dynamic and static coatings on the PMMA surface. The simultaneous analysis of the molecular interactions between a complex mixture of N-linked asialoglycans from human R1-acid glycoprotein (AGP) and various carbohydratebinding proteins, lectins, is successfully performed in PMMA channels statically coated with a SAM of HM-HEC. EXPERIMENTAL SECTION Chemicals and Solutions. Concanavalin A (ConA) and Tulipa gesneriana agglutinin (TGA) were obtained from Seikagaku Kogyo (Nihon-bashi, Tokyo). Wheat germ agglutinin (WGA) and Aleuria aurantia lectin (AAL) were purchased from Vector Laboratories (Burlingame, CA). Peptide N-glycosidase (PNGase F, EC.3.6.1.52 recombinant) with 5× reaction buffer (250 mM sodium phosphate, pH 7.5), denaturation solution (2% SDS, 1 M β-mercaptoethanol), and Triton X-100 (15% solution) were obtained from ProZyme (San Leandro, CA). Highly purified 8-aminopyrene-1,3,6-trisulfonate (APTS) was acquired from Molecular Probes (Eugene, OR). All other chemicals and reagents were obtained from Sigma (St. Louis, MO). HM-HEC was synthesized from HEC (Mw ≈ 100 000) and glycidyl hexadecyl ether, purified, and characterized as described.21 Coating solutions with 0.5 wt % HM-HEC or 1.0 wt % HEC were prepared by adding the appropriate amount of HM-HEC or HEC to a 20 mM phosphate buffer (pH 6.98) and stirring until the solution appeared homogeneous. Running buffers with various concentrations of lectins were prepared by stepwise dilution of lectin stock solutions of 1 mM using 20 mM phosphate (pH 6.98). Release and Derivatization of N-Linked Asialoglycans from AGP. N-linked asialoglycans from human AGP were released with peptide N-glycosidase and labeled with APTS as described.7,22 Briefly, 200 µg of human AGP (Sigma) was mixed with 10 µL of 5× reaction buffer, 2.5 µL of denaturation solution, and 35 µL of water and heated at 100 °C for 5 min to denature the proteins. After cooling, the solution was mixed with 2.5 µL of Triton X-100 and 2 µL of PNGase F and incubated for 24 h at 37 °C. After incubation, the mixture was mixed with 150 µL of cold ethanol to precipitate protein, followed by centrifugation at 14000 rpm at 4 °C for 3 min. The supernatant was collected and evaporated to dryness by a centrifugal vacuum evaporator at room temperature. The residue was dissolved in 50 µL of 2 M aqueous acetic acid and kept at 80 °C for 2 h to remove sialic acids from (21) Beheshti, N.; Bu, H.; Zhu, K.; Kjoniksen, A.-L.; Knudsen, K. D.; Pamies, R.; Cifre, J. G. H.; Torre, J. G.; Nystro¨m, B. J. Phys. Chem. B 2006, 110, 6601–6608. (22) Dang, F.; Zhang, L.; Jabashini, M.; Kaji, N.; Baba, Y. Anal. Chem. 2003, 75, 2433–2439.
glycans. After lyophilization, the resulting residue was mixed with 5 µL of 0.1 M APTS in 15% acetic acid solution and 10 µL of 0.5 M NaBH3CN in tetrahydrofuran. The mixture was kept at 55 °C for 1.5 h. The reaction solution was diluted with 200 µL of water. The fluorescent yellow aqueous phase was collected and applied on a column of Sephadex G-25 (1 cm, 50 cm length) equilibrated with water. The earlier eluted fluorescent fractions were collected and evaporated to dryness. The residue was dissolved in 100 µL of water and stored at -20 °C. An aliquot of the above solution was diluted to the desired concentrations with water prior to analysis. Static Coating of PMMA Channels. The microchannels of a PMMA chip (Hitachi Chemical, Tokyo) were incubated with 0.5 wt % HM-HEC or 1.0 wt % HEC solution for 2 min at room temperature (26 °C), then rinsed copiously with water, and finally dried with N2 gas. The channel walls were thus statically coated with a SAM of HM-HEC or HEC intact by water. The formation and molecular arrangement of SAMs on the PMMA surface are identified by the measurements of atomic force microscopy (AFM), infrared external-reflection (IR-ER) spectroscopy, and water contact angle.23 Affinity Microchip Electrophoresis. The µ-ACE experiments were carried on the stage of an inverted fluorescence microscope (Olympus IX70, Olympus, Tokyo) with laser-induced fluorescence (LIF) detection, as described previously.24 A running buffer that contains various concentrations of lectins was introduced into the microchannels of a PMMA chip using a syringe. All reservoirs on the microchip were filled with a running buffer or a sample using a pipet before analysis; then the separations were performed at a field strength of 300 V/cm. RESULTS AND DISCUSSION Static Coating with a SAM of HM-HEC on a PMMA Channel. Static coating is extremely attractive among the surface modification methods in separation science due to its simplicity of coating formation and regeneration. However, the research and development of static coating are greatly hindered by the lack of a detailed understanding of the governing force and mechanism for spontaneous adsorption from solution onto a solid surface. Recently, we found that cellulose and surfactants adsorb from an aqueous solution onto the PMMA surface in an ordered and cooperative way governed by hydrogen bonding.23 HEC and HMHEC are found to form SAMs with different molecular arrangements on the PMMA surface at 1.0% HEC and 0.5% HM-HEC, respectively. The glucose rings of HEC stand perpendicular on the surface, H-bond to the surface COOMe groups with their CdO and Me-O bonds parallel to the surface, and self-assemble into a tight monolayer by accommodating additional sugar chains between H-bonded sugar chains. In contrast, the glucose rings of HM-HEC lie flat with their side hydrocarbon chains perpendicular on the surface and H-bond to the perpendicularly oriented CdO groups on the PMMA surface. The great difference in the orientation of glucose rings between HEC and HM-HEC is due to the long hydrocarbon chains (C16) in HM-HEC (Figure 1B). The perpendicular orientation of glucose rings in HEC is attributed (23) Dang, F.; Hasegawa, T.; Biju, V.; Ishikawa, M.; Kaji, N.; Yasui, T.; Baba, Y. Langmuir 2009, 25, 9296–9301. (24) Dang, F.; Shinohara, S.; Tabata, O.; Yamaoka, Y.; Kurokawa, M.; Shinohara, Y.; Ishikawa, M.; Baba, Y. Lab Chip 2005, 5, 472–478.
to inter-glucose-ring interactions between the adjacent chains, whereas the flat orientation of glucose rings in HM-HEC is due to the perpendicular aggregation of the long hydrocarbon chains on the surface. Because no H-bonds are observed between the sugar sheets in native cellulose,25 the additional sugar chains should be held in the SAM of HEC primarily by hydrophobic, van der Waals, and other weak forces. Thus, the SAM of HEC consists of H-bonded and non-H-bonded sugar chains with a ratio of ∼1:1, whereas the SAM of HM-HEC is composed of only H-bonded sugar chains. We have observed that H-bonded sugar chains are tightly anchored on the PMMA surface, resulting in irreversible adsorption intact by water, whereas non-H-bonded sugar chains are loosely attached on the PMMA surface, resulting in dynamic and reversible adsorption that can be readily removed by rinsing with water.23 If this observation is correct, HEC at high concentrations must be kept in the running buffer to maintain a complete SAM on the PMMA surface, whereas HM-HEC need not be in the running buffer. In short, HEC allows only dynamic coating, whereas HM-HEC permits both dynamic and static coatings. Indeed, the separation efficiency and reproducibility of APTSlabeled N-linked asialoglycans from AGP using PMMA channels dynamically coated with 1.0% HEC are the same as those using PMMA channels dynamically coated with 0.5% HMHEC.23 However, the separation of APTS-labeled N-linked glycans from AGP was poor in a PMMA channel statically coated with a SAM of HEC using a 20 mM phosphate buffer without HEC (the top red line in Figure 2A). The obvious peak broadening and increase in migration time for all components including APTS clearly indicate the weak and reversible adsorption of analytes on the surface. This observation is consistent with expectation with an incomplete SAM of HEC wherein non-H-bonded sugar chains are readily removed by rinsing with water. On the other hand, the separation of the same sample was good and reproducible in a PMMA channel statically coated with a SAM of HM-HEC using a 20 mM phosphate buffer without HM-HEC (the bottom black line in Figure 2A). The RSD values of the migration times of all five glycans were smaller than 1.12% (data obtained in independent separations on six channels, n ) 6). The resolutions of two triantennary glycans (AII and AIII) and two tetraantennary glycans (AIV and AV) with only one fucose residue different in their structures (Figure 2B) were 1.10 and 1.04 (n ) 6), respectively. Keeping 0.5 wt % HM-HEC in the running buffer increased the separation time by ∼10 s but no evident improvement was found in separation efficiency and reproducibility. Note that the assignment of µ-CE peaks to particular glycan structures is made according to our previous work.26,27 These results provide valuable insights into the potential mechanism for dynamic and static coatings on the PMMA surface. The perpendicular arrangement of glucose rings on the surface is preferred for most cellulose derivatives such as HEC because the sugar chains can be highly packed to form a tight SAM, wherein the interactions of glucose rings between the adjacent (25) Kondo, T. Hydrogen Bonds in Cellulose and Cellulose Derivatives. In Polysaccharides: Structural Diversity and Functional Versatility; Severian, D., Ed.; Marcel Dekker: New York, 2005; pp 69-98. (26) Kakehi, K.; Kinoshita, M.; Kawakami, D.; Tanaka, J.; Sei, K.; Endo, K.; Oda, Y.; Iwaki, M.; Masuko, T. Anal. Chem. 2001, 73, 2640–2647. (27) Sei, K.; Nakano, M.; Kinoshita, M.; Masuko, T.; Kakehi, K. J. Chromatogr., A 2002, 958, 273–281.
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Figure 2. (A) Microchip electropherogram of APTS-labeled N-linked glycans derived from AGP (10 ng/mL) on a PMMA channel statically coated with SAMs of HEC (red) and HM-HEC (black). Conditions: 20 mM phosphate, pH 6.98, Esep ) 300 V/cm. The effective separation length is 30 mm. (B) Structures of N-linked asialoglycans derived from human AGP.
chains are optimized. The distance between two adjacent Hbonded chains estimated at 8.89 Å28 is large enough to accommodate another sugar chain between them because the distance between the sugar sheets in native cellulose is found to be at 4.35 Å.29 Thus, the non-H-bonded chains can be embedded between the H-bonded chains to form a tight SAM on the PMMA surface. Because the adsorption of non-H-bonded chains into the SAM is dynamic and reversible, cellulose at high concentrations must be kept in the solution to push the adsorption-desorption equilibrium of non-H-bonded sugar chains toward the adsorption side. The presence of non-H-bonded chains with a perpendicular arrangement of glucose rings on the surface is the fundamental reason why most cellulose derivatives primarily allow dynamic coating rather than static coating. On the other hand, the long hydrocarbon (C16) chains in HM-HEC completely alter the molecular arrangement of HM-HEC on the surface despite its extremely small amount of 1 C16 chain in every 100 glucose residues. The perpendicular orientation of the hydrocarbon chains in HMHEC is highly favorable for their molecular aggregation and organizes the glucose rings flat on the surface. The flat-lying glucose rings of HM-HEC form H-bonds with the perpendicularly oriented CdO groups on the PMMA surface. In such a molecular arrangement, the hydrophobic, van der Waals, and short-range steric forces among the side hydrocarbon chains and those between the glucose rings and the PMMA surface are maximized. As a result, the SAM of HM-HEC composed (28) The distance is calculated using a molecular model of cellobiose H-bonded to the parallel-oriented CdO groups in a syndiotactic triad of PMMA built in ChemBio3D Ultra 11.0 (CambridgeSoft, Cambridge, MA). (29) Vietor, R. J.; Mazeau, K.; Lakin, M.; Pe´rez, S. Biopolymers 2000, 54, 342– 354.
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Figure 3. Representative electropherograms of APTS-labeled Nlinked glycans derived from AGP with various concentrations of (A) ConA and (B) AAL on PMMA channels statically coated with a SAM of HM-HEC. The conditions are the same as in Figure 2A.
of only H-bonded sugar chains intact by water allows static coating. Thus, a SAM primarily composed of H-bonded sugar chains with a flat arrangement of glucose rings on the surface is the key to achieving static coating. Because the SAM of HMHEC allows rapid and reproducible separation of APTS-labeled glycans, the following µ-ACE experiments were performed in PMMA channels statically coated with HM-HEC. Characterization of Carbohydrate-Protein Interactions by µ-ACE. Figure 3A shows representative microchip electropherograms of APTS-labeled N-linked asialoglycans from AGP in the presence of various concentrations of ConA, which is one of the most characterized lectins with the binding specificity for either mannose or glucose residues.4 Addition of ConA to the running buffer led to significant changes in the peak AI. The peak intensity of AI gradually decreased with increasing ConA, and nearly disappeared at 2.0 µM ConA. On the other hand, the peak profile and migration time of the other four glycans (AII to AV) remained unchanged with increasing ConA. This result clearly indicated that ConA specifically binds to glycan AI, forming a stable glycanprotein complex.8 As shown in Figure 2B, the five glycans derived from AGP share a common core trimannoside. The structure of AI includes two GlcNAcβ(1,4)Gal residues attached to the core trimannoside, while AII-AV have more than two GlcNAcβ(1,4)Gal
bulbs of tulips. TGA is found to specifically bind to mouse erythrocytes, but its binding specificity is not fully characterized.30 Addition of TGA to the running buffer caused a discernible increase in migration time for AI, AII, and AIII but an observable decrease in migration time for AIV and AV. As a result, AII and AIII began to fuse at 2.0 µM TGA and further merged with AIV and AV into a broad peak at 4.0 µM. Finally, AII and AIII exchanged migration order with AIV and AV and migrated as a broad peak at 8.0 µM. Note that the peak profile of AIV and AV was kept nearly the same with increasing TGA, indicating negligible interactions of AIV and AV with both TGA and the surface. The unexpected decrease in the migration time of AIV and AV should result from an anodic EOF induced by the addition of TGA, because the migration time of APTS also gradually decreased from 20.69 s without TGA to 18.94 s with 8.0 µM TGA. No observable change in the peak shape of APTS with increasing TGA further evidences the negligible nonspecific interactions of analytes with the surface. Taking into account an anodic EOF, increasing TGA caused detectable migration shifts with peak broadening of AI, AII, and AIII, indicating the weak affinities of AI, AII, and AIII for TGA with relatively rapid on-and-off kinetics. The value of Ka for low-affinity systems was evaluated from mobility shifts using an equation proposed by Taga et al.:31 1 1 A +B ) t - t1 Ka [P]
Figure 4. Representative electropherograms of APTS-labeled Nlinked glycans derived from AGP with various concentrations of (A) TGA and (B) WGA on PMMA channels statically coated with a SAM of HM-HEC. The conditions are the same as in Figure 2A.
residues attached to the core trimannoside. Thus, the binding of ConA to the core trimannoside was strong but sensitive to steric hindrance. The recognition capacity of ConA to the core trimannoside is completely inhibited by more than two GlcNAcβ(1,4)Gal residues attached to the core trimannoside as observed for AII-AV. AAL shows a binding behavior similar to that of ConA with peak disappearance (Figure 3B). A fucose-specific nonglycosylated protein, AAL is isolated from the fruits of a mushroom, A. aurantia. A trace amount, 0.25 µM, of AAL resulted in observable peak broadening of AIII and AV with a fucose residue in the structure. An increased concentration, 0.50 µM, of AAL caused more drastic decreases in the peak intensity and migration time of AIII and AV. As a result, AIII was merged into AIV to cause the fronting of AIV, whereas AV was turned into the tailing of AIV as indicated by arrows. At 1.0 µM AAL, both AIII and AV disappeared completely, whereas AI, AII, and AIV remained unchanged. The peak disappearance combined with evident peak shifting and distortion of AIII and AV revealed that the specific binding of AAL to the fucose residue is a high-affinity system with a relatively slow on-and-off kinetics of the complex. Figure 4A shows representative microchip electropherograms with increasing concentration of TGA, a lectin isolated from the
(1)
where t is the migration time of the ligand (APTS-labeled glycans in this case) in the presence of a protein (TGA in this case), t1 is the migration time of the ligand in the absence of a protein, [P] is the concentration of a protein, and A and B are constants. From a linear regression of a plot between [t - t1]-1 and [P]-1, the Ka value of AI with TGA was estimated to be 3.06 × 104 M-1. Note that the migration times of AI were calibrated using the migration times of APTS for variations caused by EOF. Figure 4B shows representative microchip electropherograms with increasing concentration of WGA. At 1.0 µM and below, increasing WGA gradually decreased the migration velocities of AI, AII, and AIV. Because no changes in migration velocity were observed for AIII and AV, resolutions of AII and AIII, and AIV and AV, were accordingly decreased with increasing WGA. These observations definitely indicate the weak binding affinities of AI, AII, and AIV for WGA. Given the fact that all five glycans share the same WGA binding units, β(1,4) oligomers of GlcNAc,32 in the structure (Figure 2B), the fucose residue of AIII and AV should be responsible for their absence of binding affinities for WGA. Again, the recognition capacity of a protein can be totally inhibited by a subtle change in the substrate structure. At 2.0 µM or more WGA, AII and AIII merged into a single peak, and so did AIV and AV. Evident peak broadening and increased migration times were also observed for APTS at 2.0 µM or more WGA, indicating serious nonspecific analyte-surface interactions. Note that no reproducible separations of glycans were obtained with (30) Oda, Y.; Tatsumi, Y.; Aonuma, S. Chem. Pharm. Bull. 1991, 39, 3350– 3352. (31) Taga, A.; Uegaki, K.; Yabusako, Y.; Kitano, A; Honda, S. J. Chromatogr., A 1999, 837, 221–229. (32) Chandrasekaran, E. V.; Chawda, R.; Rhodes, J. M.; Locke, R. D.; Piskorz, C. F.; Matta, K. L. Carbohydr. Res. 2003, 338, 887–901.
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WGA higher than 2.0 µM. Thus, WGA should be kept at concentrations lower than 2.0 µM for static coating with HM-HEC. CONCLUSIONS We for the first time demonstrated the rapid screening of molecular interactions between carbohydrate chains and proteins in PMMA channels statically coated with HM-HEC and discussed the underlying mechanism for dynamic and static coatings on the PMMA surface. Binding of lectins caused various changes in electrophoretic behaviors of glycans, including mobility shift, peak broadening, and peak decrease and disappearance, and more often a combination of these changes, revealing the diversity of carbohydrate-protein interactions with respect to the binding kinetics and thermodynamics. From the features of high speed, high reproducibility, and ease of operation, we proposed that the current method might be a promising alternative for simultaneous analysis of interactions between a complex mixture of carbohydrates and proteins in biological samples. Static coating with HM-
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HEC is remarkably stable and can minimize the nonspecific adsorption of most analytes on the PMMA surface but not for some proteins with a high surface affinity such as WGA. Further efforts in development of new static coating materials that can minimize nonspecific adsorption of proteins are necessary to improve applicability of the current method, and such studies are under way in this laboratory. ACKNOWLEDGMENT The present work is supported in part by the CREST program of the Japan Science and Technology Corporation (JST) and a grant from the New Energy and Industrial Technology Development Organization (NEDO) of the Ministry of Economy, Trade and Industry, Japan. Received for review September 7, 2009. Accepted October 28, 2009. AC902014C
