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A Pyridylamination Method Aimed at Automatic Oligosaccharide Analysis of N-Linked Sugar Chains
Analytical Biochemistry 274, 229 –234 (1999) Article ID abio.1999.4263, available online at http://www.idealibrary.com on
A Pyridylamination Method Aimed at Automatic Oligosaccharide Analysis of N-Linked Sugar Chains 1 Kanta Yanagida, Shunji Natsuka, and Sumihiro Hase 2 Department of Chemistry, Graduate School of Science, Osaka University, Machikaneyama 1-1, Toyonaka, Osaka 560-0043, Japan
Received April 29, 1999
The procedure for preparation of pyridylaminated sugar chains from glycoproteins was improved with a view to its eventual automation. Following on the coupling reaction improvement already reported [N. Kuraya and S. Hase (1992) J. Biochem. 112, 122–126], two further aspects were improved in this study. Instead of sodium bicarbonate–acetic anhydride, volatile reagents were adopted for the re-N-acetylation of hexosamine residues after hydrazinolysis to give rapid removal of excess reagents. Subsequent to the pyridylamination reaction, excess reagents were removed by cation-exchange to isolate the pyridylaminated oligosaccharides in place of gel filtration. These alterations rendered a one-pot reaction possible and resulted in a large reduction in the amount of time needed compared with other methods so far reported. The procedure was successfully applied to the detection of sugar chains from Taka-amylase A and human erythrocyte membranes. © 1999 Academic Press
Cell-surface and soluble glycoconjugates are involved in many biological processes, including cell– cell and growth factor–receptor interactions and development-dependent alterations (1, 2). Because glycoconjugates exhibit great structural diversity and compounds that participate in specific interactions are usually present in only trace amounts, a highly sensitive yet convenient method is desired for their analysis. Pyridylamino derivatives of carbohydrates are useful for the sensitive analysis of sugar chain structures. Tag1 This work was supported in part by grants from the Shimadzu Science Foundation (to S.N.), the Japan Health Science Foundation (to S.H.), and the Future Program of the Japan Society for the Promotion of Science (to S.H.). 2 To whom correspondence should be addressed. Fax: 181-6-68505383.
0003-2697/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
ging with 2-aminopyridine has several advantages, including a high level of sensitivity, high stability, and excellent separation on reversed-phase HPLC (3). Following the introduction of this method (4) and subsequent improvement of the reductive amination reaction (5– 8), quantitative pyridylamination is now achievable using volatile reagents. The method has been applied to the analysis of carbohydrates in, for instance, methylation (4), partial acetolysis (9), glycosidase digestion (10), periodate degradation (11), mass spectrometry (12), NMR (13), two-dimensional mapping (14, 15), and partial acid hydrolysis (16). Since many samples are often processed at the same time, there is a strong demand for an automated pyridylamination procedure. However, certain features of the present method are difficult to mechanize—for example, a cation exchanger and gel filtration are respectively used to remove the N-acetylation and pyridylamination reagents. Here, we describe a one-pot pyridylamination procedure that is aimed at automation and its application to the detection of sugar chains from limited quantities of Taka-amylase A and human erythrocyte membranes. MATERIALS AND METHODS
Materials 2-Aminopyridine was purchased from Wako Pure Chemicals (Osaka) and recrystallized from hexane. A dimethylamine– borane complex was obtained from Wako; reversed-phase columns (Cosmosil 5C18-P, 0.46 3 15 cm and Cosmosil 5C18-P, 0.15 3 25 cm), 2,4,6-collidine, and a sialidase (Arthrobactor ureafacience) from Nacalai Tesque (Kyoto), Dowex 50W-X8 from Bio-Rad (Hercules, CA); and Palstation tubes from Takara Biomedicals (Kyoto). Human a 1-acid glycoprotein was purchased from Sigma (St. Louis, MO). 229
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tion in a desiccator with sulfuric acid and coevaporated with toluene in vacuo. Hexosamine residues were reN-acetylated in a mixture consisting of 300 ml 0.1 M ammonium acetate buffer, pH 9.0, 15 ml 2,4,6-collidine, and 15 ml acetic anhydride (5-fold molar excess acetic anhydride against ammonium ion) at 0°C for 20 min. Reagents were evaporated using a SpeedVac concentrator (Savant, Holbrook, NY). Sugar chains released from the glycoproteins were heated with 15 ml of a coupling reagent (prepared by mixing 552 mg 2-aminopyridine and 200 ml acetic acid) at 90°C for 60 min. The Schiff base was reduced by heating with 55 ml of a reducing reagent (freshly prepared by mixing 200 mg dimethylamine– borane complex, 50 ml water, and 80 ml acetic acid) at 80°C for 35 min as previously reported (8). The reaction mixture was added to 150 ml water followed by extraction twice with 150 ml water-satuFIG. 1. Yields of pyridylaminated M5A prepared from Taka-amylase A using various reaction conditions for N-acetylation. Sugar chains released from Taka-amylase A were N-acetylated under various reaction conditions as described under Materials and Methods. (A) Using the conventional method (8); (B) using water:pyridine: acetic anhydride (10:50:3, v/v) at 0°C for 20 min; (C) using water: 2,4,6-collidine:acetic anhydride (40:10:3, v/v) at 0°C for 20 min; (D) using 0.1 M ammonium acetate buffer, pH 8.0:2,4,6-collidine:acetic anhydride (60:3:3, v/v) at 0°C for 20 min; (E) using 0.1 M ammonium acetate buffer, pH 9.0:2,4,6-collidine:acetic anhydride (60:3:3, v/v) at 0°C for 20 min. After N-acetylation, each sample was pyridylaminated. Yields of M5A-PA were quantified by reversed-phase HPLC. The yield of M5A-PA obtained under condition A was set as 100%.
M5A-PA 3 and BIBSF-PA were prepared from Takaamylase A and human IgG, respectively. Preparation of Erythrocyte Membranes Erythrocyte membranes were prepared from human blood according to the method of Ohsawa et al. (17). Human blood (200 ml) was centrifuged with the same volume of phosphate-buffered saline at 3000g for 15 min, and the erythrocytes precipitated were washed three times with phosphate-buffered saline. The erythrocytes were then lysed with a 20-fold volume of 10 mM Tris–HCl, pH 7.5, for 15 min at 0°C under gentle stirring. The membranes were collected by centrifugation at 14,000g for 40 min. The precipitate was washed three times with the buffer and then freeze-dried. Procedure Established A glycoprotein (less than 200 mg) was heated with 30 ml anhydrous hydrazine at 100°C for 10 h in a Palstation tube. Excess hydrazine was removed by evapora3 Abbreviations used: PA-, pyridylamino-; M5A, Mana1-6(Mana13)Mana1-6(Mana1-3)Manb1-4GlcNAcb1-4GlcNAc; BIBSF, Galb14GlcNAc b 1-2Man a 1-6(Gal b 1-4GlcNAc b 1-2Man a 1-3)(GlcNAc b 14)Manb1-4GlcNAcb1-4(Fuca1-6)GlcNAc.
FIG. 2. Elution profiles of PA-sugar chains prepared by the conventional method (A) and the method established in this work (B). PA-sugar chains were prepared from 200 mg Taka-amylase A by hydrazinolysis and N-acetylation. Excess pyridylamination reagents were removed by (A) chloroform extraction and a Toyopearl HW40-F column (1 3 25 cm) using 10 mM ammonium acetate buffer, pH 6.0, or (B) phenol– chloroform extraction and a Dowex 50W-X8 column (0.5 3 3 cm) using 20 mM ammonium acetate buffer, pH 8.5. Each sample was separated by reversed-phase HPLC under elution condition 1. The arrowhead indicates the elution position of M5A-PA. Two percent of the sample obtained was injected.
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231
Reversed-Phase HPLC Analysis
TABLE 1
Yields of M5A-PA Prepared from Taka-Amylase A by the Method Established in This Work Amount of Taka-amylase A (mg)
Relative yield (%) a
200 100 50 20 10 5 2 1
114 84 95 107 72 74 63 67
a Yield relative to that obtained from 200 mg Taka-amylase A using the conventional method.
rated phenol:chloroform (1:1, v/v) (18) and once with 150 ml chloroform. The aqueous phase was poured onto a small column of Dowex 50W-X8 (NH 41 form, 0.5 3 3 cm) (19), which was washed with 2 ml of 20 mM ammonium acetate buffer, pH 8.5. Sialidase Digestion of PA-Sugar Chains PA-sugar chains prepared from erythrocyte membrane were treated with 50 munits of the sialidase in 180 ml of 90 mM ammonium acetate buffer, pH 5.0, at 37°C for 16 h. The reaction was terminated when the solution was heated at 100°C for 5 min.
Two elution conditions were employed. Under elution condition 1, a Shiseido (Tokyo) Nanospace SI-1 HPLC system was used. A Cosmosil 5C18-P column (1.5 3 250 mm) was equilibrated with 20 mM ammonium acetate buffer, pH 4.0, containing 0.075% 1-butanol at a flow rate of 0.15 ml/min at 25°C. After injection of a sample, the concentration of 1-butanol was increased linearly to 0.4% in 90 min. Under elution condition 2, a liquid chromatograph (344 M; Beckman, Fullerton, CA) equipped with a fluorescence spectrometer (F-3000; Hitachi, Tokyo) was used. A Cosmosil 5C18-P column (4.6 3 150 mm) was equilibrated with 20 mM ammonium acetate buffer, pH 4.0, containing 0.03% 1-butanol at a flow rate of 1.5 ml/min at 25°C. After injection of a sample, the concentration of 1-butanol was increased linearly to 0.4% in 90 min. Elution was monitored by measurement of the fluorescence (excitation wavelength, 320 nm; emission wavelength, 400 nm). The amounts of PA-sugar chains were calculated by comparison of peak areas with a known amount of GlcNAc-PA. RESULTS AND DISCUSSION
Examination of Reaction Conditions for N-Acetylation Volatile reagents were adopted for N-acetylation so that excess reagent could be removed rapidly. We previously reported an N-acetylation procedure with
FIG. 3. Reversed-phase HPLC of PA-sugar chains prepared from 1 mg Taka-amylase A. PA-sugar chains were prepared by the method established in this work. The arrowhead indicates the elution position of M5A-PA. Elution condition 1 was used and 60% of the sample was injected.
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FIG. 4. Reversed-phase HPLC of PA-sugar chains prepared from 200 mg erythrocyte membrane. PA-sugar chains were prepared by the method established in this work. HPLC under elution condition 2 was used and 75% of the sample was injected. The arrowhead indicates the elution position of BIBSF-PA.
volatile reagents for component sugar analysis (20); however, this method was not suitable for the Nacetylation of hydrazynolysates since the reaction did not proceed well due to the hydrazine that remained. Therefore after sugar chains were released from Taka-amylase A by hydrazinolysis, N-acetylation was carried out under various reaction conditions. The sugar chains were then pyridylaminated and quantified by reversed-phase HPLC using a known amount of GlcNAc-PA as a standard. Four of the reaction conditions tested are shown in Fig. 1. When 2,4,6-collidine or pyridine in water was used as a base (reaction conditions B and C in Fig. 1), the yield was less than that obtained under the conventional condition. Under reaction conditions B and C, not only amino groups but also hydroxyl groups were acetylated judging from the fact that higher yields were observed when the products were treated with alkali. Among the reagents tested, 2,4,6-collidine in 0.1 M ammonium acetate buffer, pH 9.0 (condition E in Fig. 1) gave the best result. The reaction times of 10, 20, and 30 min were tested with the selected
reagents, and the same results were obtained (data not shown). The reaction time of 20 min was thus chosen. We therefore selected condition E for use in the subsequent experiments.
Removal of Excess Pyridylamination Reagents Removal of excess reagents after the pyridylamination reaction is one of the key steps in improving both the sensitivity and the convenience of the procedure. Gel chromatography has mostly been employed, but it is not well suited to use with a large number of samples. Phenol– chloroform extraction and cation-exchange chromatography on a Dowex 50W-X8 column have also been reported by Tokugawa et al. (18) and Fan et al. (19), respectively. Using these two procedures in tandem, we found that phenol– chloroform extraction could remove a large part of the 2-aminopyridine and dimethylamine– borane complex, and the 2-aminopyridine still remaining was then removed by Dowex 50W-X8 chromatog-
PYRIDYLAMINATION FOR OLIGOSACCARIDE ANALYSIS
raphy. PA-oligosaccharides were recovered in 2 ml of the pass-through fraction with 20 mM ammonium acetate buffer, pH 8.5. The procedure was completed in about 10 min compared with about 5 h required for gel filtration. In Fig. 2, the method established in this work is compared with the conventional method consisting of a combination of chloroform extraction and gel filtration. Using our method, excess reagents and contamination appearing at 0 –15 min were removed as well as by the conventional method, but with a much shorter operation time. Application of Established Procedure to Glycoproteins The PA-sugar chain yield from glycoproteins was first examined using Taka-amylase A, which has Mana1-6(Mana1-3)Mana1-6(Mana1-3)Manb1-4GlcNAcb1-4GlcNAc (M5A) as the major sugar chain (21). The yield was slightly better than with the conventional method when 200 mg of the glycoprotein was used (Table 1). A M5A-PA peak was clearly identified by reversed-phase HPLC even when only 1 mg of Takaamylase A was used (Fig. 3). The present procedure was applied to a 1-acid glycoprotein, and PA-sialo sugar chains were obtained with the similar yield (data not shown). The newly established method was also employed with erythrocyte membranes to demonstrate its ability to detect sugar chains from a lipid-containing sample. The chromatogram obtained by reversedphase HPLC after sialidase digestion of the PAsugar chains is shown in Fig. 4. The major peak indicated by the arrowhead was Gal b1-4GlcNAcb12Mana1-6(Galb1-4GlcNAcb1-2Mana1-3)(GlcNAcb14)Manb1-4GlcNAcb1-4(Fuca1-6)GlcNAc-PA (BIBSFPA) judging from the elution position of authentic BIBSF-PA on a two-dimensional map. The structure was further confirmed by identification of b-galactosidase and a-fucosidase digestion (data not shown). Most of this sugar chain was from glycophorin A, which is one of the major glycoproteins on the erythrocyte membrane (22, 23). The amount of this major PA-sugar chain obtained was 45 pmol from 200 mg of dried erythrocyte membrane. The usefulness of the new method, which deals effectively with the problems of the N-acetylation procedure and the removal of excess reagents, was clearly proven judging from the results of the above two applications. As the reaction conditions for pyridylamination have already been improved, the reaction sequences for the preparation of PA-sugar chains from glycoproteins in a one-pot procedure suitable for developing an automatic device for PA-sugar chain preparation are now complete.
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REFERENCES 1. Varki, A. (1993) Biological roles of oligosaccharides: All of the theories are correct. Glycobiology 3, 97–130. 2. Nakakita, S., Natsuka, S., Ikenaka, K., and Hase, S. (1998) Development-dependent expression of complex-type sugar chains specific to mouse brain. J. Biochem. 123, 1164 – 1168. 3. Hase, S., Ikenaka, T., and Matsushima, Y. (1981) A highly sensitive method for analyses of sugar moieties of glycoproteins by fluorescence labeling. J. Biochem. 90, 407– 414. 4. Hase, S., Ikenaka, T., and Matsushima, Y. (1978) Structure analyses of oligosaccharides by tagging of the reducing end sugars with a fluorescent compound. Biochem. Biophys. Res. Commun. 85, 257–263. 5. Hase, S., Ibuki, T., and Ikenaka, T. (1984) Reexamination of the pyridylamination used for fluorescence labeling of oligosaccharides and its application to glycoproteins. J. Biochem. 95, 197– 203. 6. Yamamoto, S., Hase, S., Fukuda, S., Sano, O., and Ikenaka, T. (1989) Structures of the sugar chains of interferon-g produced by human myelomonocyte cell line HBL-38. J. Biochem. 105, 547– 555. 7. Kondo, A., Suzuki, J., Kuraya, N., Hase, S., Kato, I., and Ikenaka, T. (1990) Improved method for fluorescence labeling of sugar chains with sialic acid residues. Agric. Biol. Chem. 54, 2169 –2170. 8. Kuraya, N., and Hase, S. (1992) Release of O-linked sugar chains from glycoproteins with anhydrous hydrazine and pyridylamination of the sugar chains with improved reaction conditions. J. Biochem. 112, 122–126. 9. Natsuka, S., Hase, S., and Ikenaka, T. (1987) Fluorescence method for the structural analysis of oligomannose-type sugar chains by partial acetolysis. Anal. Biochem. 167, 154 –159. 10. Hase, S., Koyama, S., Daiyasu, H., Takemoto, H., Hara, S., Kobayashi, Y., Kyogoku, Y., and Ikenaka, T. (1986) Structure of a sugar chain of a protease inhibitor isolated from barbados pride (Caesalpinia pulcherrima Sw.) seeds. J. Biochem. 100, 1–10. 11. Hase, S., Kikuchi, N., Ikenaka, T., and Inoue, K. (1985) Structures of sugar chains of the third component of human complement. J. Biochem. 98, 863– 874. 12. Gu, J., Hiraga, T., and Wada, Y. (1994) Electrospray ionization mass spectrometry of pyridylaminated oligosaccharide derivatives: Sensitive and in-source fragmentation. Biol. Mass. Spectrom. 23, 212–217. 13. Koyama, S., Daiyasu, H., Hase, S., Kobayashi, Y., Kyogoku, Y., and Ikenaka, T. (1986) 1H-NMR analysis of the sugar structures of glycoproteins as their pyridylamino derivatives. FEBS Lett. 209, 265–268. 14. Hase, S., Natsuka, S., Oku, H., and Ikenaka, T. (1987) Identification method for twelve oligomannose-type sugar chains thought to be processing intermediates of glycoproteins. Anal. Biochem. 167, 321–326. 15. Tomiya, N., Awaya, J., Kurono, M., Endo, S., Arata, Y., and Takahashi, N. (1988) Analyses of N-linked oligosaccharides using a two-dimensional mapping technique. Anal. Biochem. 171, 73–90. 16. Makino, Y., Omichi, K., and Hase, S. (1998) Analysis of oligosaccharide structures from the reducing end terminal by combining partial acid hydrolysis and a two-dimensional sugar map. Anal. Biochem. 264, 172–179.
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17. Ohsawa, F., Hirano, F., and Natori, S. (1990) Purification and properties of a b-galactoside-binding lectin from neonatal marmoset. J. Biochem. 107, 431– 434. 18. Tokugawa, K., Oguri, S., and Takeuchi, M. (1996) Large scale preparation of PA-oligosaccharides from glycoproteins using an improved extraction method. Glycoconjugate J. 13, 53–56. 19. Fan, J.-Q., Huynh, L. H., and Lee, Y. C. (1995) Purification of 2-aminopyridine derivatives of oligosaccharides and related compounds by cation-exchange chromatography. Anal. Biochem. 232, 65– 68. 20. Suzuki, J., Kondo, A., Kato, I., Hase, S., and Ikenaka, T. (1991) Analysis by high-performance anion-exchange chromatography
of component sugars as their fluorescent pyridylamino derivatives. Agric. Biol. Chem. 55, 283–284. 21. Minobe, S., Nakajima, H., Itoh, N., Funakoshi, I., and Yamashina, I. (1979) Structure of a major oligosaccharide of Takaamylase A. J. Biochem. 86, 1851–1854. 22. Yoshima, H., Furthmayr, H., and Kobata, A. (1980) Structures of the asparagine-linked sugar chains of glycophorin A. J. Biol. Chem. 255, 9713–9718. 23. Irimura, T., Tsuji, T., Tagami, S., Yamamoto, K., and Osawa, T. (1981) Structure of a complex-type sugar chain of human glycophorin A. Biochemistry 20, 560 –566.
A Pyridylamination Method Aimed at Automatic Oligosaccharide Analysis of N-Linked Sugar Chains 1 Kanta Yanagida, Shunji Natsuka, and Sumihiro Hase 2 Department of Chemistry, Graduate School of Science, Osaka University, Machikaneyama 1-1, Toyonaka, Osaka 560-0043, Japan
Received April 29, 1999
The procedure for preparation of pyridylaminated sugar chains from glycoproteins was improved with a view to its eventual automation. Following on the coupling reaction improvement already reported [N. Kuraya and S. Hase (1992) J. Biochem. 112, 122–126], two further aspects were improved in this study. Instead of sodium bicarbonate–acetic anhydride, volatile reagents were adopted for the re-N-acetylation of hexosamine residues after hydrazinolysis to give rapid removal of excess reagents. Subsequent to the pyridylamination reaction, excess reagents were removed by cation-exchange to isolate the pyridylaminated oligosaccharides in place of gel filtration. These alterations rendered a one-pot reaction possible and resulted in a large reduction in the amount of time needed compared with other methods so far reported. The procedure was successfully applied to the detection of sugar chains from Taka-amylase A and human erythrocyte membranes. © 1999 Academic Press
Cell-surface and soluble glycoconjugates are involved in many biological processes, including cell– cell and growth factor–receptor interactions and development-dependent alterations (1, 2). Because glycoconjugates exhibit great structural diversity and compounds that participate in specific interactions are usually present in only trace amounts, a highly sensitive yet convenient method is desired for their analysis. Pyridylamino derivatives of carbohydrates are useful for the sensitive analysis of sugar chain structures. Tag1 This work was supported in part by grants from the Shimadzu Science Foundation (to S.N.), the Japan Health Science Foundation (to S.H.), and the Future Program of the Japan Society for the Promotion of Science (to S.H.). 2 To whom correspondence should be addressed. Fax: 181-6-68505383.
0003-2697/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
ging with 2-aminopyridine has several advantages, including a high level of sensitivity, high stability, and excellent separation on reversed-phase HPLC (3). Following the introduction of this method (4) and subsequent improvement of the reductive amination reaction (5– 8), quantitative pyridylamination is now achievable using volatile reagents. The method has been applied to the analysis of carbohydrates in, for instance, methylation (4), partial acetolysis (9), glycosidase digestion (10), periodate degradation (11), mass spectrometry (12), NMR (13), two-dimensional mapping (14, 15), and partial acid hydrolysis (16). Since many samples are often processed at the same time, there is a strong demand for an automated pyridylamination procedure. However, certain features of the present method are difficult to mechanize—for example, a cation exchanger and gel filtration are respectively used to remove the N-acetylation and pyridylamination reagents. Here, we describe a one-pot pyridylamination procedure that is aimed at automation and its application to the detection of sugar chains from limited quantities of Taka-amylase A and human erythrocyte membranes. MATERIALS AND METHODS
Materials 2-Aminopyridine was purchased from Wako Pure Chemicals (Osaka) and recrystallized from hexane. A dimethylamine– borane complex was obtained from Wako; reversed-phase columns (Cosmosil 5C18-P, 0.46 3 15 cm and Cosmosil 5C18-P, 0.15 3 25 cm), 2,4,6-collidine, and a sialidase (Arthrobactor ureafacience) from Nacalai Tesque (Kyoto), Dowex 50W-X8 from Bio-Rad (Hercules, CA); and Palstation tubes from Takara Biomedicals (Kyoto). Human a 1-acid glycoprotein was purchased from Sigma (St. Louis, MO). 229
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YANAGIDA, NATSUKA, AND HASE
tion in a desiccator with sulfuric acid and coevaporated with toluene in vacuo. Hexosamine residues were reN-acetylated in a mixture consisting of 300 ml 0.1 M ammonium acetate buffer, pH 9.0, 15 ml 2,4,6-collidine, and 15 ml acetic anhydride (5-fold molar excess acetic anhydride against ammonium ion) at 0°C for 20 min. Reagents were evaporated using a SpeedVac concentrator (Savant, Holbrook, NY). Sugar chains released from the glycoproteins were heated with 15 ml of a coupling reagent (prepared by mixing 552 mg 2-aminopyridine and 200 ml acetic acid) at 90°C for 60 min. The Schiff base was reduced by heating with 55 ml of a reducing reagent (freshly prepared by mixing 200 mg dimethylamine– borane complex, 50 ml water, and 80 ml acetic acid) at 80°C for 35 min as previously reported (8). The reaction mixture was added to 150 ml water followed by extraction twice with 150 ml water-satuFIG. 1. Yields of pyridylaminated M5A prepared from Taka-amylase A using various reaction conditions for N-acetylation. Sugar chains released from Taka-amylase A were N-acetylated under various reaction conditions as described under Materials and Methods. (A) Using the conventional method (8); (B) using water:pyridine: acetic anhydride (10:50:3, v/v) at 0°C for 20 min; (C) using water: 2,4,6-collidine:acetic anhydride (40:10:3, v/v) at 0°C for 20 min; (D) using 0.1 M ammonium acetate buffer, pH 8.0:2,4,6-collidine:acetic anhydride (60:3:3, v/v) at 0°C for 20 min; (E) using 0.1 M ammonium acetate buffer, pH 9.0:2,4,6-collidine:acetic anhydride (60:3:3, v/v) at 0°C for 20 min. After N-acetylation, each sample was pyridylaminated. Yields of M5A-PA were quantified by reversed-phase HPLC. The yield of M5A-PA obtained under condition A was set as 100%.
M5A-PA 3 and BIBSF-PA were prepared from Takaamylase A and human IgG, respectively. Preparation of Erythrocyte Membranes Erythrocyte membranes were prepared from human blood according to the method of Ohsawa et al. (17). Human blood (200 ml) was centrifuged with the same volume of phosphate-buffered saline at 3000g for 15 min, and the erythrocytes precipitated were washed three times with phosphate-buffered saline. The erythrocytes were then lysed with a 20-fold volume of 10 mM Tris–HCl, pH 7.5, for 15 min at 0°C under gentle stirring. The membranes were collected by centrifugation at 14,000g for 40 min. The precipitate was washed three times with the buffer and then freeze-dried. Procedure Established A glycoprotein (less than 200 mg) was heated with 30 ml anhydrous hydrazine at 100°C for 10 h in a Palstation tube. Excess hydrazine was removed by evapora3 Abbreviations used: PA-, pyridylamino-; M5A, Mana1-6(Mana13)Mana1-6(Mana1-3)Manb1-4GlcNAcb1-4GlcNAc; BIBSF, Galb14GlcNAc b 1-2Man a 1-6(Gal b 1-4GlcNAc b 1-2Man a 1-3)(GlcNAc b 14)Manb1-4GlcNAcb1-4(Fuca1-6)GlcNAc.
FIG. 2. Elution profiles of PA-sugar chains prepared by the conventional method (A) and the method established in this work (B). PA-sugar chains were prepared from 200 mg Taka-amylase A by hydrazinolysis and N-acetylation. Excess pyridylamination reagents were removed by (A) chloroform extraction and a Toyopearl HW40-F column (1 3 25 cm) using 10 mM ammonium acetate buffer, pH 6.0, or (B) phenol– chloroform extraction and a Dowex 50W-X8 column (0.5 3 3 cm) using 20 mM ammonium acetate buffer, pH 8.5. Each sample was separated by reversed-phase HPLC under elution condition 1. The arrowhead indicates the elution position of M5A-PA. Two percent of the sample obtained was injected.
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Reversed-Phase HPLC Analysis
TABLE 1
Yields of M5A-PA Prepared from Taka-Amylase A by the Method Established in This Work Amount of Taka-amylase A (mg)
Relative yield (%) a
200 100 50 20 10 5 2 1
114 84 95 107 72 74 63 67
a Yield relative to that obtained from 200 mg Taka-amylase A using the conventional method.
rated phenol:chloroform (1:1, v/v) (18) and once with 150 ml chloroform. The aqueous phase was poured onto a small column of Dowex 50W-X8 (NH 41 form, 0.5 3 3 cm) (19), which was washed with 2 ml of 20 mM ammonium acetate buffer, pH 8.5. Sialidase Digestion of PA-Sugar Chains PA-sugar chains prepared from erythrocyte membrane were treated with 50 munits of the sialidase in 180 ml of 90 mM ammonium acetate buffer, pH 5.0, at 37°C for 16 h. The reaction was terminated when the solution was heated at 100°C for 5 min.
Two elution conditions were employed. Under elution condition 1, a Shiseido (Tokyo) Nanospace SI-1 HPLC system was used. A Cosmosil 5C18-P column (1.5 3 250 mm) was equilibrated with 20 mM ammonium acetate buffer, pH 4.0, containing 0.075% 1-butanol at a flow rate of 0.15 ml/min at 25°C. After injection of a sample, the concentration of 1-butanol was increased linearly to 0.4% in 90 min. Under elution condition 2, a liquid chromatograph (344 M; Beckman, Fullerton, CA) equipped with a fluorescence spectrometer (F-3000; Hitachi, Tokyo) was used. A Cosmosil 5C18-P column (4.6 3 150 mm) was equilibrated with 20 mM ammonium acetate buffer, pH 4.0, containing 0.03% 1-butanol at a flow rate of 1.5 ml/min at 25°C. After injection of a sample, the concentration of 1-butanol was increased linearly to 0.4% in 90 min. Elution was monitored by measurement of the fluorescence (excitation wavelength, 320 nm; emission wavelength, 400 nm). The amounts of PA-sugar chains were calculated by comparison of peak areas with a known amount of GlcNAc-PA. RESULTS AND DISCUSSION
Examination of Reaction Conditions for N-Acetylation Volatile reagents were adopted for N-acetylation so that excess reagent could be removed rapidly. We previously reported an N-acetylation procedure with
FIG. 3. Reversed-phase HPLC of PA-sugar chains prepared from 1 mg Taka-amylase A. PA-sugar chains were prepared by the method established in this work. The arrowhead indicates the elution position of M5A-PA. Elution condition 1 was used and 60% of the sample was injected.
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FIG. 4. Reversed-phase HPLC of PA-sugar chains prepared from 200 mg erythrocyte membrane. PA-sugar chains were prepared by the method established in this work. HPLC under elution condition 2 was used and 75% of the sample was injected. The arrowhead indicates the elution position of BIBSF-PA.
volatile reagents for component sugar analysis (20); however, this method was not suitable for the Nacetylation of hydrazynolysates since the reaction did not proceed well due to the hydrazine that remained. Therefore after sugar chains were released from Taka-amylase A by hydrazinolysis, N-acetylation was carried out under various reaction conditions. The sugar chains were then pyridylaminated and quantified by reversed-phase HPLC using a known amount of GlcNAc-PA as a standard. Four of the reaction conditions tested are shown in Fig. 1. When 2,4,6-collidine or pyridine in water was used as a base (reaction conditions B and C in Fig. 1), the yield was less than that obtained under the conventional condition. Under reaction conditions B and C, not only amino groups but also hydroxyl groups were acetylated judging from the fact that higher yields were observed when the products were treated with alkali. Among the reagents tested, 2,4,6-collidine in 0.1 M ammonium acetate buffer, pH 9.0 (condition E in Fig. 1) gave the best result. The reaction times of 10, 20, and 30 min were tested with the selected
reagents, and the same results were obtained (data not shown). The reaction time of 20 min was thus chosen. We therefore selected condition E for use in the subsequent experiments.
Removal of Excess Pyridylamination Reagents Removal of excess reagents after the pyridylamination reaction is one of the key steps in improving both the sensitivity and the convenience of the procedure. Gel chromatography has mostly been employed, but it is not well suited to use with a large number of samples. Phenol– chloroform extraction and cation-exchange chromatography on a Dowex 50W-X8 column have also been reported by Tokugawa et al. (18) and Fan et al. (19), respectively. Using these two procedures in tandem, we found that phenol– chloroform extraction could remove a large part of the 2-aminopyridine and dimethylamine– borane complex, and the 2-aminopyridine still remaining was then removed by Dowex 50W-X8 chromatog-
PYRIDYLAMINATION FOR OLIGOSACCARIDE ANALYSIS
raphy. PA-oligosaccharides were recovered in 2 ml of the pass-through fraction with 20 mM ammonium acetate buffer, pH 8.5. The procedure was completed in about 10 min compared with about 5 h required for gel filtration. In Fig. 2, the method established in this work is compared with the conventional method consisting of a combination of chloroform extraction and gel filtration. Using our method, excess reagents and contamination appearing at 0 –15 min were removed as well as by the conventional method, but with a much shorter operation time. Application of Established Procedure to Glycoproteins The PA-sugar chain yield from glycoproteins was first examined using Taka-amylase A, which has Mana1-6(Mana1-3)Mana1-6(Mana1-3)Manb1-4GlcNAcb1-4GlcNAc (M5A) as the major sugar chain (21). The yield was slightly better than with the conventional method when 200 mg of the glycoprotein was used (Table 1). A M5A-PA peak was clearly identified by reversed-phase HPLC even when only 1 mg of Takaamylase A was used (Fig. 3). The present procedure was applied to a 1-acid glycoprotein, and PA-sialo sugar chains were obtained with the similar yield (data not shown). The newly established method was also employed with erythrocyte membranes to demonstrate its ability to detect sugar chains from a lipid-containing sample. The chromatogram obtained by reversedphase HPLC after sialidase digestion of the PAsugar chains is shown in Fig. 4. The major peak indicated by the arrowhead was Gal b1-4GlcNAcb12Mana1-6(Galb1-4GlcNAcb1-2Mana1-3)(GlcNAcb14)Manb1-4GlcNAcb1-4(Fuca1-6)GlcNAc-PA (BIBSFPA) judging from the elution position of authentic BIBSF-PA on a two-dimensional map. The structure was further confirmed by identification of b-galactosidase and a-fucosidase digestion (data not shown). Most of this sugar chain was from glycophorin A, which is one of the major glycoproteins on the erythrocyte membrane (22, 23). The amount of this major PA-sugar chain obtained was 45 pmol from 200 mg of dried erythrocyte membrane. The usefulness of the new method, which deals effectively with the problems of the N-acetylation procedure and the removal of excess reagents, was clearly proven judging from the results of the above two applications. As the reaction conditions for pyridylamination have already been improved, the reaction sequences for the preparation of PA-sugar chains from glycoproteins in a one-pot procedure suitable for developing an automatic device for PA-sugar chain preparation are now complete.
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