Capillary Electrophoresis of Sialic Acid-Containing Glycoprotein. Effect

Capillary Electrophoresis of Sialic Acid-Containing. Glycoprotein. Effect of the Heterogeneity of. Carbohydrate Chains on Glycoform Separation. Using ...
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Anal. Chem. 2001, 73, 2640-2647

Capillary Electrophoresis of Sialic Acid-Containing Glycoprotein. Effect of the Heterogeneity of Carbohydrate Chains on Glycoform Separation Using an r1-Acid Glycoprotein as a Model Kazuaki Kakehi,* Mitsuhiro Kinoshita, Daisuke Kawakami, Junko Tanaka, Kazuo Sei, Kaori Endo, Yasuo Oda, Masahiro Iwaki, and Takashi Masuko

Faculty of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashi-Osaka 577-8502, Japan

r1-Acid glycoprotein (AGP) showed multiple peaks on separation using capillary electrophoresis in a chemically modified capillary with dimethylpolysiloxane at slightly acidic conditions. We analyzed glycoforms of AGP species after separation by ion-exchange chromatography, Con A affinity chromatography, and Cu(II)-chelating affinity chromatography. The AGP species thus obtained were digested with N-glycosidase F, and the released carbohydrate chains were analyzed by high-performance liquid chromatography after labeling with 3-aminobenzoic acid. The results afforded basic information on the contribution of carbohydrate chains to the separation mechanism of glycoforms of AGP by capillary electrophoresis. In addition, we describe an easy method for AGP analysis in serum samples using the electrokinetic injection. Glycoprotein drugs such as erythropoietin and tissue plasminogen activator produced by biotechnology are now commercially available, and evaluation of their quantitative and qualitative aspects has been an important target for their clinical use and industrial production. Carbohydrate chains of such glycoproteins often show variations during biosynthesis in the type of cell line1 and the culture conditions.2 Two strategies have been developed for the analysis of such variations. One is based on the separation of carbohydrate chains that are released from the protein core;3 the other is based on the direct separation of native glycoproteins, which is called glycoform analysis.4 There is little information on the relationship between carbohydrate chains and their contribution to glycoform separation, although some authors reported that the separation of glycoforms is mainly based on the number of sialic acid residues in the analysis by capillary electrophoresis.5 R1-Acid glycoprotein (orosomucoid, AGP) is a plasma glycoprotein of 41-43 kDa in molecular mass. Carbohydrates occupy * To whom correspondence should be addressed. Telephone: +81-6-67212332 (Ext 3822). Fax: +81-6-6721-2353. E-mail: [email protected]. (1) Parekh, R. B.; Dwek, R. A.; Thomas, J. R.; Opdenakker, G.; Rademacher, T. W.; Wittwer, A. J.; Howard, R.; Nelson, N. R.; Siegel, M. G. Biochemistry 1989, 28, 7644-7662. (2) Gawlitzek, M.; Valley, U.; Nimitz, M.; Wagner, R.; Conradt, H. S. J. Biotechnol. 1995, 42, 117-131. (3) Suzuki, S.; Honda, S. Electrophoresis 1998, 19, 2539-2560. (4) Kakehi, K.; Honda, S. J. Chromatogr.. A 1996, 720, 377-393. (5) Watson, E.; Yao, F. Anal. Biochem. 1993, 210, 389-393.

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∼45% of the whole protein6 and attach to the protein core as the form of five N-linked glycans.7 AGP is an acute-phase glycoprotein and exhibits large heterogeneity in structure based on the heterogeneity of carbohydrate chains and genetic variance of the protein portion. Carbohydrate chains of AGP are the N-linked oligosaccharides of complex type, and significant heterogeneity of the chains exists due to variation in the linkage of Nacetylneuraminic acid (NeuAc) to galactose (Gal) and due to the presence of fucose (Fuc) residues.8,9 Variations in the presence of di-, tri-, and tetraantennary carbohydrate chains are also the reasons for AGP heterogeneity. Micromole quantities of asialocarbohydrate chains were isolated from AGP and their structures confirmed using a combination of proton NMR and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS).10 Genetic variance of the protein portion of AGP is conveniently examined by chromatography on an immobilized copper(II) affinity adsorbent based on the complex formation between copper(II) and histidine residues in AGP.11 Binding specificity of each variant to various drugs has been attracting interest in the measurement of drug-protein interactions in blood.12 AGP and human serum albumin (HSA) play important roles in carrying drugs in plasma. HSA is the most abundant protein (40 mg/mL) in serum and largely responsible for the binding of acidic drugs, whereas AGP binds mainly basic and neutral drugs.13-14 AGP concentration in healthy human sera is 0.3-0.4 mg/mL, whereas during disease or injury such as neoplasia, rheumatoid arthritis, and burns, it increases 2-3 times.15 Serum AGP increases with age in the normal population, especially among women.16 (6) Schmid, K.; Emura, J.; Schmid, M. F.; Stevens, R. L.; Nimberg, R. B. Int. J. Pept. Protein Res. 1978, 11, 42-48. (7) Schmid, K.; Nimberg, R. B.; Kimura, A.; Yamaguchi, H.; Binette, J. P. Biochim. Biophys. Acta 1977, 492, 291-302. (8) Fournet, B.; Montreuil, J.; Strecker, G.; Dorland, L.; Haverkamp, J.; Vliegenthardt, J. F. G.; Binetter, J. P.; Schmid, K. Biochemistry 1978, 17, 5206-5214. (9) Treuheit, M. J.; Costello, C. E.; Halsall, H. B. Biochem. J. 1992, 283, 105112. (10) Stubbs, H. J.; Shia, M. A.; Rice, K. G. Anal. Biochem. 1997, 247, 357-365. (11) Herve, F.; Gomas, E.; Duche, J. C.; Tillement, J. P. J. Chromatogr. 1993, 615, 47-57. (12) Kuroda, Y.; Shibukawa, A.; Nakagawa, T. Anal. Biochem. 1999, 268, 9-14. (13) Routledge, P. A. Br. J. Clin. Pharmacol. 1986, 22, 499-506. (14) Kremer, J. M. H.; Wilting, J.; Janssen, L. M. H. Pharmacol. Rev. 1988, 40, 1-47. 10.1021/ac001382u CCC: $20.00

© 2001 American Chemical Society Published on Web 04/27/2001

Van Dijk et al. reported that the relative occurrence of AGP glycoforms is dependent on the pathological conditions determined by cytokines and hormones.17 Thus, the glycosylation of AGP in human sera is subject to marked changes during acute phase reaction. The changes comprise alterations in the type of branching of the carbohydrate structures as revealed by increased reactivity with concanavalin A (Con A) and the fucose-binding lectin. The expression of a sialyl Lewis X (sLex, NeuAcR23Galβ1-4(FucR1-3)GlcNAc-R) portion in the carbohydrate chains of AGP molecules has been a challengeable target in relation to the induction of inflammation. De Graaf et al. found a direct relation between the reactivity of AGP to the fucose-specific binding Aleuria auranita lectin and staining of AGP by anti-sLex monoclonal antibody under healthy and disease conditions.18 Thus, analysis of sialo- and asialocarbohydrate chains of AGP as well as its glycoforms is important for understanding the biological roles of AGP. In the previous paper, we reported comparative studies on the analysis of glycoforms of several sialic acid-containing glycoproteins such as fetuin and erythropoietin, as well as AGP from some animal sources,19 and showed that the method based on capillary electrophoresis was quite useful. In the present study, we developed a facile method for the glycoform separation of AGP in clinical samples using a combination of an easy desalting procedure and electrokinetic injection. We also analyzed sialo- and asialocarbohydrate chains released from AGP after digestion with N-glycosidase F followed by fluorescent labeling with 3-aminobenzoic acid using high-performance liquid chromatography20 and considered the effect of carbohydrate chains on glycoform separation of AGP by capillary electrophoresis. The effect of carbohydrate chains on glycoform separation (i.e., peak multiplicity) of sialic acid-containing glycoproteins by capillary electrophoresis was examined using the following strategies: (a) separation of AGP molecular species based on their negative charge using anion-exchange chromatography; (b) separation of AGP molecular species based on the heterogeneity of carbohydrate chains using Con A affinity chromatography; (c) separation of AGP molecular species based on the heterogeneity of the protein portion by Cu(II) metal affinity chromatography. Glycoforms of each fraction collected by these chromatographic techniques were examined by capillary electrophoresis. Carbohydrate chains of these fractions were also determined by highperformance liquid chromatography (HPLC) after releasing them from the protein core followed by labeling with 3-aminobenzoic acid (3-AB). The results described here will show a guide for the evaluation of glycoprotein drugs such as erythropoietin and tissue plasminogen activator. (15) Mackiewicz, A.; Pawlowski, T.; Powlowska, A. M.; Wiktrowicz, K.; Mackeiwicz, S. Clin. Chim. Acta 1987, 163, 185-190. (16) Young, R. C.; Patel, A.; Mayers, B. S.; Kakuma, T.; Alexopoulos, S. Am. J. Geriatr. Psychiatry 1999, 7, 331-334. (17) Van Dijk, W.; Brinkman, L.; Els, C. M.; Havenaar, E. C. Trends Glycosci. Glycotechnol. 1998, 10, 235-245. (18) De Graaf, T. W.; Van der Stelt, M. E.; Anbergen, M. G.; van Dijk, W. J. Exp. Med. 1993, 177, 657-666. (19) Kinoshita, M.; Murakami, E.; Oda, Y.; Funakubo, T.; Kawakami, D.; Kakehi, K.; Kawasaki, N.; Morimoto, K.; Hayakawa, T. J. Chromatogr., A 2000, 866, 261-271. (20) Kakehi, K.; Funakubo, T.; Suzuki, S.; Oda, Y.; Kitada, Y. J. Chromatogr., A 1999, 863, 205-218.

MATERIALS AND METHODS Study Participants. The study using human serum samples was conducted according to the Declaration of Helsinki and its amendments. The protocol was approved by the local Institutional Review Board and all participants signed informed consent prior to participating in the study. Materials. Samples of human AGP and bovine serum albumin (BSA) were obtained from Sigma (St. Louis, MO). Sephadex G-10 and G-25 (superfine grade) and chelating Sepharose were obtained from Pharmacia (Upsala, Sweden). 3-AB and sodium cyanoborohydride for fluorescent labeling of oligosaccharides were obtained from Tokyo Kasei (Chuo-ku, Tokyo, Japan). Peptide-N4-(acetylβ-D-glucosaminyl)asparagine amidase (N-glycosidase F, EC 3.2.2.18) was from Roche Molecular Biochemicals (Minato-ku, Tokyo, Japan). Neuraminidase (Arthrobacter ureafaciens) was a gift from Drs. Tsukada and Ohta (Marukin-Chuyu, Uji, Kyoto, Japan). Methyl R-glucoside (Me R-Glc) was from Sigma. All other samples and reagents were of the highest grade commercially available or of HPLC grade and used without further purification. A DB-1 capillary column for capillary gas chromatography was obtained from J&W Scientific (Folsom, CA). A POROS HQ column (4.6mm i.d., 100-mm length) was obtained from PE Biosystems. An octadecyl silica column (Cosmosil 5C18AR, 6-mm i.d., 150-mm length) for the separation of fluorescent-labeled carbohydrates was obtained from Nacalai Tesque (Nakagyo-ku, Kyoto, Japan). Con A Sepharose was prepared according to the method reported by Ito et al.21 Water purified with a Milli-Q purification system (Millipore) after double distillation of deionized water was used throughout the work. Capillary Electrophoresis. Capillary electrophoresis was performed with a P/ACE 5010 capillary electrophoresis system (Beckman, Fullerton, CA). The detection window was made at 7 cm from the outlet of the capillary by carefully removing the polyimide coating by burning, and the transparent portion was fixed on the detector block. On-line detection was performed with monitoring of the UV absorption at 200 nm. The composition and pH values of the electrolytes are given in the figure legends. Injections were performed automatically in the pressure mode for the standard samples or in the electrokinetic mode for serum samples. Data were collected and analyzed with a standard System Gold software (ver. 8.0) on an IBM PC. The peaks observed for the analysis of glycoforms of AGP in all electropherograms were numbered according to their migration times, and the peaks having the same peak number were observed at the same migration times when the mixture was analyzed. Analysis of AGP Glycoforms in Serum Samples. Serum samples were collected from normal male volunteers (23 years old) and from the patients who acquired methicillin-resistant Staphylococcus aureus (MRSA) during hospitalization. Desalting of serum samples was performed as follows. A small column made of polypropylene (available from Seikagaku Kogyo, 1-mL volume) packed with Sephadex G-25 (1 mL) was washed with water (10 mL) and then with water containing 0.1% BSA (3 mL) to reduce nonspecific adsorption of proteins in sera to the gel. The column was centrifuged at 2000 rpm for 5 min to remove excess water containing 0.1% BSA. A serum sample (100 µL) was applied to the column, the column again centrifuged for 5 min at 2000 rpm, (21) Ito, Y.; Seno, N.; Matsumoto, I. J. Biochem. 1985, 97, 1689-1694.

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Figure 1. Glycoform analysis of AGP in a serum sample using the electrokineitc injection method: (a) an intact serum sample and (b) after desalting (see experimental procedure). Analytical conditions: capillary, a DB-1 capillary (20 cm, 50-µm i.d.); buffer, 20 mM sodium acetate (pH 4.5) containing 0.5% Tween 20; applied voltage, 15 kV; injection, 10 kV for 10 s; detection, UV absorption at 200 nm. Peaks are numbered according to their migration times. The peaks having the same peak number were observed at the same migration times (see also Figures 2 and 5-7).

and the effluent collected. The concentration of inorganic salts in the effluent was reduced by this procedure. A portion of the effluent was injected to CE using the electrokinetic mode at 5 kV for 10 s and analyzed using a capillary of which inner surface is chemically modified with dimethylpolysiloxane using the conditions described in the legends of Figure 1. Anion-Exchange Chromatography of AGP. A POROS column (type HQ) was previously equilibrated with 50 mM Tris-HCl buffer (pH 7.5). An AGP sample (1 mg) was dissolved in the same buffer (250 µL), and a portion (50 µL) was injected into the column and separated in a linear gradient elution. The gradient elution was performed from the equilibrating buffer to the same buffer containing NaCl to a concentration of 0.5 M at a flow rate of 1.0 mL/min over 40 min. Although the separation was not good, the effluent from the column was collected. The above procedures were repeated three times. The fractions collected were combined and dialyzed against distilled water followed by lyophilization to dryness. Con A Affinity Chromatography of AGP. AGP (1 mg) was dissolved in 50 mM Tris-HCl buffer (pH 7.5, 1 mL) containing 0.15 M NaCl, 1 mM CaCl2, 1 mM MgCl2, and 1 mM MnCl2. The solution was applied to a small column (0.6-cm i.d., 10-cm length) packed with Con A Sepharose previously equilibrated with the same buffer. The column was eluted with the same buffer (60 mL) and then with the buffer (60 mL) containing 10 mM Me R-Glc, collecting 3-mL fractions. The effluent was monitored, observing the absorbance at 280 nm. The fractions collected were dialyzed against water for 48 h at 4 °C and lyophilized to dryness. Cu(II)-Metal Affinity Chromatography of AGP. The equilibration of chelating Sepharose with Cu(II) ion was performed according to the method of Herve et al. with slight modification.22 The chelating Sepharose (3 mL) was washed with water and then packed into a column (0.7-cm i.d., 10-cm length). The column was washed with five column volumes of 0.2 M copper chloride in distilled water. Excess metal ions were removed by washing the gel with five column volumes of 0.1 M acetate buffer (pH 3.8) containing 0.5 M NaCl. Finally, the column was equilibrated with (22) Herve, F.; Duche, J. C.; Barre, J.; Millot, M. C.; Tillement, J. P. J. Chromatogr. 1992, 577, 43-59.

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20 mM phosphate buffer (pH 7.0) containing 0.5 M NaCl (equilibrating buffer). A sample of AGP (1 mg) dissolved in the equilibrating buffer (1 mL) was applied to the column. The column was washed with the equilibrating buffer (20 mL) and then with the same buffer containing 20 mM imidazole. Fractions (1.5 mL) were collected, monitoring the absorbance at 280 nm. The peak fractions were collected, dialyzed against water, and lyophilized to dryness. Analysis of Carbohydrate Chains Released from AGP with N-Glycosidase F. Carbohydrate chains of AGP were analyzed according to the method reported previously.20 The method is based on HPLC of fluorescence-labeled carbohydrate chains released from AGP with N-glycosidase F, and the procedure is as follows. A sample of AGP (50 µg, ∼1.2 nmol as calculated from the molecular mass of 40 kDa) was dissolved in 50 µL of water and mixed with 20 mM phosphate buffer (pH 7.5, 50 µL) in a screwcapped polypropylene tube (1.5-mL volume). One unit (5 µL) of N-glycosidase F was added to the solution. The mixture was incubated for 24 h at 37 °C and then kept for 3 min at 100 °C to denature the enzyme. The mixture was evaporated to dryness with a centrifugal evaporator (SpeedVac, Savant, Farmingdale, NY). The dried mixture was mixed with a solution (0.7 M, 5 µL) of 3-AB in a mixture of dimethyl sulfoxide and acetic acid (7:3, by volume). A solution (5 µL) of freshly prepared 2 M sodium cyanoborohydride in the same solution was added to the mixture. The mixture was kept for 1 h at 50 °C and then diluted with water (200 µL) after cooling to room temperature. Ethyl acetate (200 µL) was added to the mixture, and the mixture shaken vigorously. The organic phase was removed carefully and the aqueous phase evaporated to dryness. The residue was dissolved in a small volume of water (200 µL) containing 20% methanol and 0.2% acetic acid and applied to a small column (1.0-cm i.d., 30-cm length) of Sephadex G-10 previously equilibrated with the same solvent. The fractions containing the oligosaccharides labeled with 3-AB were eluted earlier, collected with monitoring the fluorescence at 405 nm and with irradiating at 305 nm, and evaporated to dryness. The residue was dissolved in a small volume of water (500 µL), and a portion (10 µL) was analyzed by HPLC using an ODS column on a Jasco HPLC PU-980 apparatus installed with a FP920 fluorescent detector. Separation was performed in linear gradient elution using (a) 50 mM ammonium formate buffer (pH 4.4) to (b) the same buffer containing acetonitrile at a concentration of 20% at a flow rate of 0.8 mL/min. The gradient elution was from 8 to 12% of solvent B over 70 min. Digestion of 3-AB-Labeled Sialocarbohydrate Chains with Neuraminidase. A half of the mixture of sialocarbohydrate chains labeled with 3-AB obtained by the procedure as described above was dissolved in 20 mM acetate buffer (pH 5.3, 40 µL). Neuraminidase (10 munits/10 µL) was added to the solution and the mixture incubated at 37 °C overnight. The mixture was kept in a boiling water bath for 3 min and centrifuged. A portion (5 µL) of the supernatant was analyzed by HPLC in the same linear gradient elution as described for the analysis of sialooligosaccharides (see above). MALDI-TOF MS. The N-linked sialo- and asialooligosaccharides as their 3-AB derivatives were analyzed by MALDI-TOF MS, which was performed on a Voyager DE PRO (PE Biosystems,

Framingham, MA). A nitrogen laser was used to irradiate samples with ultraviolet light (337 nm), and an average of 100 shots was taken. The instrument was operated in linear operation using negative polarity. An accelerating voltage of 18 kV was used. Samples (0.5 µL) were applied to a polished stainless steel target, to which added a solution (0.3 µL) of 2,5-dihydroxybenzoic acid (DHB) in a mixture of acetonitrile-water (1:1). The mixture was dried in atmosphere by keeping it for several hours at room temperature. RESULTS AND DISCUSSION Easy Separation of AGP Glycoforms Using Electrokinetic Injection. In the analysis of biological samples such as blood or urine by capillary electrophoresis, direct injection using the pressure mode is not a good strategy, because a large amount of proteins often deteriorate separation efficiency due to nonspecific adsorption to the capillary surface. Furthermore, inorganic salts sometimes make it difficult to keep good reproducibility. On the contrary, injection by the electrokinetic mode is applied to selective introduction of the target component by choosing appropriate injection conditions. When an intact serum sample was injected in the electrokinetic mode, no peaks were observed as shown in Figure 1a. In the electrokinetic injection of an intact serum sample, protein constituents were not introduced effectively into the capillary, because a large amount of inorganic ions hampered the effective introduction of proteins. Therefore, serum samples were previously desalted by passing them through a small column of Sephadex G-25. By inserting a desalting procedure, we could observe glycoform peaks of AGP clearly, as shown in Figure 1b. Furthermore, we achieved efficient concentration of the protein zone at the injection point by using desalted serum samples, as predicted by the Kohlraush principle as reviewed by Hjerten in detail.23 A similar concentration effect of the analyte ions was also reported using a simple amino acid as a model.24 In the present study, separation was performed in 20 mM acetate buffer at pH 4.5. Almost all the proteins other than AGP in serum have pI values higher than 4.5, but AGP still has negative charge at this pH. Therefore, electrokinetic injection at this pH allowed selective introduction of AGP. The present method is completed within 30 min per sample including the desalting procedure and will permit routine analysis of clinical samples. Serum samples obtained from a few patients infected with MRSA were analyzed according to the method described above. The results are shown in Figure 2. In these analyses, a DB-1 capillary of 75-µm i. d. was used at the applied voltage of 20 kV. The separation was completed within 12 min, and 10 peaks were observed on each electropherogram. Although we attempted to find some characteristics in the patient sera, a conclusive relationship was not observed between relative abundance of each glycoform and clinical data such as the concentrations of AGP or C-reactive protein, which is known as the inflammation-sensitive protein (data not shown). Glycoforms of AGP in plasma or serum are often analyzed by crossed affinity immnoelectrophoresis, with lectins as the affinity component in the first-dimension gel and polyclonal anti-AGP IgG in the second-

dimension gel.25 Although the resolution of AGP glycoforms by this two-dimensional gel format is excellent, the procedure is timeconsuming and cumbersome. As described in the introduction, carbohydrate chains of AGP were closely related to various biological events such as inflammation and neoplasia, although the biological role of AGP is not clear. It is important to establish a facile method to estimate the molecular species of AGP to understand the biological role. On the basis of the consideration above, we examined the effect of carbohydrate chains of AGP on the separation of molecular species (i.e., glycoforms) of AGP by capillary electrophoresis. For the analysis of carbohydrate chains of AGP, we released carbohydrate chains by digestion with N-glycosidase F. Release of Carbohydrate Chains from the Protein Core and Derivatization with 3-AB. We employed N-glycosidase F for the release of carbohydrate chains without prior digestion of the protein portion with a protease. In the present study, we focus the target on the analysis of the released carbohydrate chains from the intact AGP sample, even if N-glycosidase F had only limited access to the carbohydrate chains, because it seems important to analyze the carbohydrate chains that are exposed on the surface of native AGP and easily accessible by other proteins. Such carbohydrate chains may play more important roles in biological and clinical events. The carbohydrate chains thus released were fluorescently labeled with 3-AB. Labeling reaction with 3-AB proceeds under mild conditions and does not cause the loss of N-acetylneuraminic acid residues.20

(23) Hjerten, S. Electrophoresis 1990, 11, 665-690. (24) Chien, R.-L.; Burgi, D. S. J. Chromatogr. 1991, 559, 141-152.

(25) Bierhuizen, M. F.; De Wit, M.; Govers, C. A.; Ferwerda, W.; Koeleman, C.; Pos, O.; Van Dijk, W. Eur. J. Biochem. 1988, 175, 387-394.

Figure 2. Glycoform analysis of a few serum samples obtained from the patients with MRSA. Analytical conditions: capillary, a DB-1 capillary (20 cm, 75-mm i.d.); buffer, 20 mM sodium acetate (pH 4.5) containing 0.5% Tween 20; applied voltage, 20 kV; injection, 5 kV for 10 s; detection, UV absorption at 200 nm.

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Figure 3. Negative-ion MALDI-TOF mass spectrum of carbohydrate chains released from AGP after derivatization with 3-AB: a, sialocarbohydrate chains; b, asialocarbohydrate chains. The numbers are defined in Table 1.

MALDI-TOF MS of the Mixture of 3-AB-Labeled Carbohydrate Chains. We examined the mixture of sialocarbohydrate chains released from AGP by MALDI-TOF MS as their 3-AB derivatives and also the mixture of asialocarbohydrate chains after digestion of sialocarbohydrate chains with neuraminidase. The asialocarbohydrate chains showed six major ions as shown in Figure 3b, and the peaks were easily assigned to the structures as shown in Table 1 from their molecular ions. However, the sialocarbohydrate chains showed molecular ions as well as the ions that cleaved sialic acid residue(s). The sialobiantennary carbohydrate chain showed two peaks at m/z 2343 and 2052. These ions correspond to (M - H)- and (M NeuAc)- ions, respectively. Sialotriantennary carbohydrate chains showed three ions at m/z 3000 (M - H)-, 2709 (M - NeuAc)-, and 2418 (M - NeuAc2)-, respectively. The sialotriantennary carbohydrate chain containing Fuc residue, VI, also showed a clear molecular ion at m/z 3146 (M - H)- as well as the ions of (M NeuAc)- and (M - NeuAc2)- at m/z 2855 and 2564, respectively. Sialocarbohydrate chains of other multiantennary types did not show obvious molecular ions and related fragment ions. Several papers reported that N-acetylneuraminic acid residues were easily cleaved during MALDI-TOF MS.26-28 We attempted to detect clear molecular ions of sialic acid-containing carbohydrates without loss of sialic acids by fragamention by using other matrix materials such as 6-aza-2-thiothymine or 2′,4′,6′-trihydroxyacetophenone, but could not obtain good results. Analysis of Carbohydrate Chains of Native AGP by HPLC. The mixture of the fluorescent-labeled asialocarbohydrate chains was analyzed by HPLC. The results are shown in Figure 4b. The peaks were assigned to the carbohydrate chains in Table 1 by observing the MALDI-TOF MS after collection of the peaks. We could not confirm the peaks marked with asterisks, because they did not show any ions. The results obtained for the analysis of sialocarbohydrate chains are also shown in Figure 4a. Although we could not identify all the sialocarbohydrate chains, some of (26) Yamagaki, T.; Nakanishi, H. Glycoconjugate J. 1999, 16, 385-389. (27) Papac, D. I.; Wong, A.; Jones, A. J. S. Anal. Chem. 1996, 68, 3215-3223. (28) Harvey, D. Mass Spectrom. Rev. 1999, 18, 349-451.

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Figure 4. HPLC profile of the carbohydrate chains released from AGP after digestion with N-glycosidase F and labeling with 3-AB: (a) sialocarbohydrate chains; (b) asialocarbohydrate chains. The numbers are defined in Table 1.

the peaks were assigned by converting the component peak to asialocarbohydrate by digestion with neuraminidase. Some sialocarbohydrate chains showed multiple peaks (see I, II, and VI), although the reason is not clear at present. The apparent ratios of the asssigned carbohydrate chains were different from those reported by Stubbs et al.10 This is probably due to the heterogeneity of AGP preparations (see later section, Table 2). As indicated in the previous section, we performed the

Table 1. List of the Asialocarbohydrate Chains Released from AGPa

a Abbreviations: Gal, galactose; GlcNAc, N-acetylglucosamine; Man, mannose; Fuc, fucose; 3-AB, 3-aminobenzoic acid. The structures are based on those reported by Stubbs et al.10

digestion with N-glycosidase F without prior digestion of the protein portion with a protease in the present study. Therefore, it was plausible that N-glycosidase F could not access all the carbohydrate chains of AGP molecules. This may be another reason why relative abundance of each carbohydrate chain was different from the reported data. The established method described above for the analysis of carbohydrate chains was employed to understand the mechanism on glycoform separation of AGP by capillary electrophoresis. We also employed three chromatography techniques for the separation of molecular species, and the fractions obtained by these conventional chromatography techniques were examined by capillary electrophoresis. Further, their carbohydrate chains were also analyzed by HPLC as their 3-AB derivatives. Analysis of the Fractions Separated by Ion-Exchange Chromatography. Although we could not resolve the glycoform

Table 2. Compositions of Carbohydrate Chains of AGP Subfractions Separated by Three Chromatographic Techniques As Examined by HPLC method for separation anion exchange chromatography Con A affinity chromatography Cu(II)-affinity chromatography native AGP (human) a

% ratios to total peak areaa fraction

I

II

III

IV

V

VI

1 2 3 1 2 1 2

5.4 4.6 5.1 5.5 7.0 3.5 11.0 6.7

8.5 9.6 14.1 13.6 9.2 8.6 23.4 14.0

2.1 1.7 1.8 1.6 2.2 1.1 3.5 2.8

35.9 30.5 22.9 2.3 60.0 37.8 18.0 26.0

18.9 16.0 13.6 24.7 6.5 16.4 12.1 15.0

29.2 37.6 42.5 52.3 15.1 32.6 32.0 35.5

The numbers I-VI are defined in Table 1.

sof AGP by anion-exchange chromatography, we obtained three fractions by collecting the peak component as shown in Figure Analytical Chemistry, Vol. 73, No. 11, June 1, 2001

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Figure 5. Analysis of AGP subfractions separated by anionexchange chromatography. (a) Fractionation of AGP by anionexchange chromatography. (b) Capillary electrophoresis of AGP subfractions separated by anion-exchange chromatography.

5a. The earlier eluting component contains less negative charges and the later eluting component more negative charges on anionexchange chromatography. We examined these fractions by capillary electrophoresis and showed the results in Figure 5b. As predicted from the elution in anion-exchange chromatography, component 1 in Figure 5a was resolved into six peaks and observed at later migration times. The later eluting component (component 3 in Figure 5a) was observed at earlier migration times. Electroosmotic flow is negligible at the conditions using a DB-1 capillary in the buffer containing 0.5% Tween 20. Therefore, these data indicated that the resolution of glycoforms was mainly based on the negative charges. Analysis of the Fractions Separated by Affinity Chromatography Using a Con A-Sepharose Column. Nicollet et al. reported that AGP glycoforms were separated based on their carbohydrate chains by Con A affinity chromatography.29 AGP from human was separated into two fractions: nonbound fractions and those eluted with 10 mM Me R-Glc (bound fractions) as shown in Figure 6a. Two-fifths of the total AGP were adsorbed on a Con A column and eluted with 10 mM Me R-Glc. The bound fractions contained slower migrating peaks predominantly as shown in Figure 6b. On the other hand, nonbound fractions contained components that migrated faster. This indicates that nonbound fractions contain more negative charges than the bound fractions, as shown in the results obtained by anion-exchange chromatography. Analysis of the Fractions Separated by Affinity Chromatography on an Immobilized Copper(II) Affinity Adsorbent. Affinity chromatography of AGP on an immobilized copper(II) affinity adsorbent allowed fractionation of the genetic variants of human AGP.11 AGP was separated into two fractions, nonbound and bound fractions using 20 mM phosphate buffer (pH 7.0) containing imidazole at a concentration of 20 mM. The separation was done based on the number of histidine residues. Figure 7b shows that slower migrating fractions are abundant in the bound fractions, although the difference is not obvious between nonbound and bound fractions. Effect of Carbohydrate Chains on the Separation of Glycoforms of AGP. We analyzed carbohydrate chains of all the (29) Nicollet, I.; Lebreton, J. P.; Fontaine, M.; Hiron, M. Biochim. Biophys. Acta 1981, 28, 235-245.

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Figure 6. Separation of AGP by Con A affinity chromatography. (a) Con A affinity chromatography. The eluent was changed to that containing 10 mM Me R-Glc at the arrow. (b) Capillary electrophoresis of the nonbound (1) and bound (2) fractions.

Figure 7. Separation of AGP by Cu(II) metal affinity chromatography. (a) Cu(II) metal affinity chromatography. The eluent was changed to that containing 20 mM imidazole at the arrow. (b) Capillary electrophoresis of the nonbound (1) and bound (2) fractions.

fractions obtained by anion-exchange chromatography, Con A affinity chromatography, and Cu(II) affinity chromatography and found that ratios of the asialooligosaccharides in each fraction showed interesting profiles (Table 2). Although some data in the table were reported by several groups separately, the archives may be useful for understanding the clinical and biological significance of AGP. Proteins having highly negative charges are obviously retained on an anion-exchange column. The early eluting fraction (fraction 1) in anion-exchange chromatography contains a biantennary carbohydrate chain (IV in Table 2) most abundantly (35.9%). The ratio of IV decreased to 22.9% in the later eluting fraction (fraction 3). On the other hand, the ratios of triantennary carbohydrate chain (VI) and tetraantennary carbohydrate chain (II) increased from 29.2 and 8.5% to 42.5 and 14.1%, respectively. As indicated in MALDI-TOF MS of the sialocarbohydrate chains (Figure 3a), these carbohydrates are substituted with N-acetylneuraminic acid. Therefore, AGP molecular species containing multiantennary carbohydrate chains were observed at ealier migration times by capillary electrophoresis, but eluted later by anion-exchange chromatography. The relative ratios of I and V that have the Lex structure did not change obviously. The nonbound fraction on

Con A affinity chromatography contains a large amount of triantennary carbohydrate chain VI (52.3%). V (24.7%), which has the Lex structure in its molecule, was also present predominantly in this fraction. Kuroda et al. reported that Con A could exclusively recognize the biantennary carbohydrate chain of AGP.12 The present results supported their data, and the biantennary carbohydrate chain (IV) was hardly detected in the nonbound fraction (2.3%). The relative ratios of IV also had an obvious effect on glycoform separation of AGP by capillary electrophoresis. The fractions including a larger amount of IV (i.e., bound fractions) were observed later by capillary electrophoresis. This effect may also be due to less negative charges of IV, because one biantennary carbohydrate chain carries only two N-acetylneuraminic acid residues. Copper(II)-immobilized metal ion affinity chromatography on iminodiacetate Sepharose can fractionate AGP into the A genetic variant and a mixture of F1 and S variants.11 The separation that occurred was mainly based on the number of histidine residues. The bound fraction contains the A variant, and the unbound fraction consists of F1 and S variants. Interestingly, the bound fraction (fraction 2) contained a larger amount of tetraantennary carbohydrate chains (I and II), although there is no obvious defference between nonbound and bound fractions by capillary electrophoresis. This seems to afford a piece of evidence that AGP polymorphism based on the presence of two different genes affects the bioformation of nascent glycoprotein.

CONCLUSIONS In the present paper, we described an easy method for the analysis of AGP in serum samples by capillary electrophoresis using the electrokinetic injection method. AGP, a unique protein, which has a low pI value, was selectively introduced to the capillary and separated into multiple peaks. We applied the method to analyze the serum samples from the patients infected with MRSA and attempted to find some relationship about the peak multiplicity of AGP in normal and patient sera. Further, three conventional chromatography techniques (anion exchange chromatography, Con A affinity chromatography, Cu(II)-metal affinity chromatography) were used to separate molecular species of AGP. We analyzed the glycoforms and the distributions of carbohydrate chains of the fractions thus obtained by capillary electrophoresis and HPLC, respectively. We found that capillary electrophoresis is a powerful technique for the glycoform analysis of sialic acid-containing glycoprotein. However, it is difficult to predict the changes of the ratios of respective carbohydrate chains during biological and clinical events. Thus, we have to examine both glycoforms and carbohydrate chains for accurate evaluation of the sialic acid-containing glycoproteins such as glycoprotein pharmaceuticals. Received for review November 27, 2000. Accepted March 11, 2001. AC001382U

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