ANALYTICAL
BIOCHEMISTRY
197,132-136
(19%)
Sugar Analysis of Glycoproteins and Glycolipids after Methanolysis by High-Performance Liquid Chromatography with Pulsed Amperometric Detection’ Anja Lampio and Jukka Finne* Department of Biochemistry, University of Helsinki, Unioninkatu 35, SF-001 70, Helsinki, Finland; and *Department of Medical Biochemistry, University of Turku, Kiinamyllynkatu 10, SF-20520 Turky Finland
Received
January
30,199l
A procedure for the analysis of the monosaccharide composition of glycoproteins and glycolipids by methanolysis and high-performance liquid chromatography with pulsed amperometric detection is described. The advantage over previous methods is the analysis of underivatized methyl glycosides of all glycoconjugate monosaccharides including sialic acid and uranic acid in a single chromatographic step at the subnanomolar level.
0 1991
Academic
Press,
Inc.
Anion-exchange chromatography with PAD’ is a novel and sensitive method for analyzing underivatized monosaccharides and oligosaccharides (l-3). In most applications, carbohydrates are separated on the anionexchange column at alkaline pH, which is also a prerequisite for the electrochemical detection of sugars. When an eluant of low alkalinity is used, or in applications using lower pH with other types of columns (4), postcolumn alkalinization becomes necessary. As reported by Hardy et al. (5) the monosaccharide composition of glycoconjugates can be conveniently determined at high sensitivity by high-performance anion-exchange chromatography and PAD after acid hydrolysis. However, sialic acid, an important component of glycoproteins and glycolipids, was destroyed under the conditions used for the analysis and was therefore not quantified. In gas-liquid chromatography, methanolysis has been widely used for the monosaccharide analysis of glycoconjugates (6-8). The advantages are the high yields of sugars including sialic acid and a more
reliable peak identification due to the multiple peaks obtained for each monosaccharide. Analysis of underivatized methyl glycosides of monosaccharides by high-performance liquid chromatography on reversed-phase columns has been reported (912), but these studies do not include sialic acid. Moreover, the detection methods used do not seem to have the same sensitivity as PAD. In the present paper we report on the analysis at the subnanomolar level of all constituent sugars of glycoproteins and glycolipids, including sialic acid and uranic acid, after methanolysis using high-performance liquid chromatography and PAD. MATERIALS
AND
METHODS
Reagents and Materials Thirty percent (w/w) NaOH solution was purchased from Merck (Darmstadt,. Germany). Glucoheptose, 2deoxygalactose, and 3-0-methylglucose were obtained from Pfanstiehl Laboratories (Waukegan, IL), and other monosaccharides and fetuin were from Sigma (St. Louis, MO). Glycopeptides were prepared from fetuin and from the marine sponge Microciona prolifera (13) after pronase digestion (14). Human glycophorin A was isolated by the method of Hamaguchi and Cleve (15): Neutral glycolipids were purified from mammalian redcell membranes by the method of Saito and Hakomori (16). Gangliosides were isolated from pig brain (17) and purified by anion-exchange chromatography followed by chromatography on latrobeads 6RS-8060 silica gel (18). Ultrapure water (Mill;-&, Millipore) was used in the preparation and analysis of the samples. Methanolysis
1 This study was supported by grants from Foundation and the Finnish Academy. ’ Abbreviation used: PAD, pulsed amperometric
the
Sigrid detection.
Jus6lius
Methanolysis of carbohydrates was performed by the method of Chaplin (8). All samples (0.5-200 nmol) were
132 All
Copyright 0 1991 rights of reproduction
0003-2697/91$3.00 by Academic Press, Inc. in my form reserved.
CHROMATOGRAPHIC
ANALYSIS
OF
METHYL
GLYCOSIDES
dried in a vacuum concentrator (Speed Vat, Savant Instruments) or under a nitrogen flow in 13 X 50-mm glass tubes with a conical bottom. Methanolic hydrogen chloride solution (0.625 M) was made by adding acetyl chloride (1.16 ml) dropwise to dry methanol (25 ml), and 320 ~1 of this reagent was pipetted into each tube. After addition of 80 ~1 of methyl acetate and flushing with nitrogen, the tubes were closed with Teflon-lined screw caps and kept at 82-84°C for 16 h. The tubes were cooled, and 80 ~1 of t-butanol was added to each sample prior to evaporation. Dry methanol (50 pl), pyridine (5 pl), and acetic anhydride (5 gl) were added successively with intermediate mixing for re-N-acetylation of the amino sugars (twice these volumes would be used if more than 50 nmol of amino sugars were present (19)). After 30 min at room temperature the solutions were evaporated to dryness. Dry samples were dissolved in 50-200 gl of water, and samples other than pure standards were passed through a 0.22~pm filter (Millipore). The fatty acid methyl esters of glycolipid samples were extracted into chloroform (50-200 ~1) from the aqueous solution (8). Chromatography The Dionex HPLC system (Series 45OOi) consisted of a BioLC Gradient Pump Module (GPM-II), a Liquid Chromatography Module (LCM-2), a Pulsed Amperometric Detector Module (PAD-II), and an Eluant Degas Module for sparging the eluants with helium. The column was a Waters Dextro-Pak plastic cartridge (8 X 100 mm) compressed in an RCM 100 radial compression module. The Dextro-Pak column was stored in acetonitrile:water (20:80) and flushed with water before runs. A Cl8 cartridge (Guard Pak Resolve C18, Waters) was used as a precolumn. The column system was eluted with water at a flow rate of 1 ml/min. In some experiments, a gradient of O-3% acetonitrile was applied. Detection was by PAD using a gold working electrode. NaOH (100 mM) was added as a postcolumn reagent at a flow rate of 1.0 ml/min. The following pulse potentials and durations were used: E, = 0.05 V (tl = 360 ms), E, = 0.80 V (t2 = 120 ms), E3 = -0.60 V (t3 = 420 ms). The response time was set to 3 s. RESULTS
Figure 1 shows the separation of eight methyl glycosides derived from standard monosaccharides. Impurities and unreacted monosaccharides, if present, eluted rapidly from the column before the first peak of methyl galactoside. Retention times and relative responses are given in Table 1. Most of the parent monosaccharides gave more than one peak. However, when the peaks overlapped, quantification was still possible by using subsidiary peaks or simultaneous equations (10). On the other hand, the multiplicity of peaks also affected the
WITH
B
AMPEROMETRIC
133
DETECTION
* I
I
0
I
1
I
I
10
20 TIME
I
1
I
30
(MINI
FIG. 1. Elution profiles of standard methyl glycosides. Dextro-Pak (8 X 100 mm) cartridge eluted with water, postcolumn NaOH addition (100 mM) for PAD (300 nA full scale). The peaks were from: (A) 1, galactose; 4, mannose; 7, fucose; 8, N-acetylneuraminic acid; and (B) 2, glucose; 3, glucuronic acid; 5, N-acetylgalactosamine; 6, N-acetylglucosamine. The amount of each sugar was 2.5 nmol.
sensitivity of the method. The limit of detection (signal to noise ratio, 2:l) using isocratic elution with water was about 200 pmol for methyl fucoside (four peaks), whereas as little as 25 pmol of methyl glucoside, methyl mannoside, or methyl galactoside could be quantified (one major peak). Consequently, the best results are obtained when at least 0.5 nmol of each sugar can be injected per analysis. The sensitivity for fucose and other late-eluting components could be increased by gradient elution (see below). We applied the method to the analysis of several glycoproteins and glycopeptides. Figure 2 shows chromatograms of methanolysis products from glycophorin A, fetuin, and purified glycopeptides from fetuin. In the glycophorin A sample (Fig. 2A), all peaks except the first peaks derived from mannose and N-acetylgalactosamine were resolved. By injecting twice as much sample, even fucose could be quantified at 300 nA full scale. The carbohydrate composition of fetuin was also easily analyzed, although the commercial preparation contained a considerable amount of glucose. The elution profile of the glycoprotein methanolysate was otherwise very similar to that of the glycopeptide sample (Figs. 2B and 2C). Methanolysates of ganglio- and globoseries glycosphingolipids were also analyzed, and examples of the elution profiles are given in Fig. 3. An additional peak eluting after the third peak of methyl galactoside was
134
LAMP10 TABLE
DISCUSSION
1
Retention Times and Relative Sugar Derivatives Obtained
PAD Responses by Methanolysis Time
Parent sugar
Peak”
Galactose Glucose Glucuronic
1 2 3
acid
Mannose
4
N-Acetylgalactosamine
5
N-Acetylglucosamine
6
Fucose
7
N-Acetylneuraminic
acid
8
AND FINNE
Percentage* 74.1 5.7 20.2 100 38.0 17.0 44.6 7.3 92.7 8.6 5.3 63.0 12.1 11.0 15.2 84.8 5.7 10.2 54.4 29.7
100
(min) 4.6 5.4 6.0 5.4 6.0 23.5 27.1 6.9 8.0 6.9 11.5 13.4 14.7 15.9 8.6 17.4 9.0 11.3 16.3 18.4 26.0
of PAD response’ 1.00
1.06 0.74
0.88 0.99
Although anion-exchange chromatography with PAD (1,2,4) offers at present one of the most. sensitive and convenient methods of sugar analysis of glycoproteins, the method has some drawbacks. The most serious is that not all sugars can be analyzed from the same hydrolyzate, because sialic acid is lost. Consequently, different sugars require different optimum conditions of hydrolysis (5). In the present study, we have applied methanolysis to circumvent these drawbacks. We have used the methanolysis conditions worked out by Chaplin (8) for an improved release and yield of methyl glycosides from glycoconjugates. The advantage of the methanolysis procedure is that not only sialic acid but even uranic acid can be analyzed in addition to the neutral
0.99 0.46
0.47
sugars
and hexosamines.
Furthermore,
the current
results extend the use of PAD to the analysis of the monosaccharide composition of glycolipids also. Unlike methods employing acid hydrolysis, methods based on methanolysis give several derivatives for each monosaccharide (a- and P-pyranosides and furanosides and small amounts of free sugars). The production of essentially single main components after methanolysis has been reported (12). However, as discussed before
’ Refers to Fig. 1. * Percentage of total peak areas of each sugar. c Ratio of total peak areas of each sugar to that of galactose.
present in each sample. It was derived from the chloroform extraction step used for the removal of fatty acid
methyl esters, and as judged from its retention time and control experiments, was identified as ethanol, present. in chloroform as a stabilizer. The peak could be removed by evaporating the sample and redissolving it in water. Alternatively, the fatty acid methyl esters can be removed by hexane extraction after methanolysis (6). Table 2 shows the results of the sugar composition analysis of two reference glycoconjugates. The values were calculated on the basis of the response factors obtained for the free monosaccharides. In general the results were very similar to those obtained by other methods. The glucose observed for the commercial preparation of fetuin was due to contamination, as glucose was not present in the purified glycopeptides from fetuin (Fig. 2). The somewhat higher amounts of N-acetylneuraminic acid in GM1 ganglioside reflect the better yield of the sugar derivative obtained from the bound form compared to that obtained from the free form. Figure 4 shows that gradient elution with acetonitrile could be applied for improving the detection of the later eluting compounds. For example, O-3% acetonitrile in the eluant made it possible to detect low amounts of glucuronic acid, which were present in marine sponge glycopeptides.
1..1,.;,,.;:. A.c ;
&!LLAn A1 1 45 4 6 .-.
0
5:5
6
10
8
20 TIME
30
(MINI
FIG. 2. Chromatograms of glycoprotein and glycopeptide methanolysates. (A) 2.5 I.cg glycophorin A, 300 nA full scale; (B) 5 pg fetuin; 100 nA full scale; (C) fetuin glycopeptides; 300 nA full scale.
CHROMATOGRAPHIC
ANALYSIS
OF
METHYL
GLYCOSIDES
WITH
AMPEROMETRIC
135
DETECTION
TABLE
2
Monosaccharide Composition of Glycoconjugates Determined after Methanolysis Fetuin Monosaccharide
Ratio”
Galactose Glucose Mannose IV-Acetylgalactosamine N-Acetylglucosamine N-Acetylneuraminic acid
GM1 Ref.
(22) b
Ratio”
1 0.15 f 0.01
1 0
0.75
0.75 0.25 1.25
0.48
1.08
0.69
* 0.08
0.21 k 0.01 1.20 f 0.10 1.09 * 0.10
ganglioside
0.47
n Monosaccharides were analyzed after methanolysis the values were calculated using the response factors values are given as the mean values (n = 6) +- SD. b Expected values based on the references given.
Ref.
(23) b 1
1 k 0.01
0.5 0 0.5 0 0.5
0 f 0.02 0 * 0.03
by HPLC and in Table 1. The
too close to some of the monosaccharides to be analyzed. These included rhamnose, 2-deoxygalactose, 2deoxyglucose, inositol, mannitol, galactitol, glucitol, N-acetylglucosaminitol, N-acetylmannosamine, and glucoheptose. The best suited of the monosaccharides tried was 3-0-methylglucose. As a methyl glycoside it gives three peaks between the main peaks of mannose and N-acetylgalactosamine and could, if required, be used as an internal standard. Interestingly, the PAD
0
10
20 TIME
30
(MINI
FIG. 3.
Chromatograms of glycosphingolipid methanolysates. (A) Ceramide dihexoside, (B) asialo-GM2, (C) GMl. Detection was by PAD at 300 nA full scale. Peak “S” is a solvent peak from the extraction of fatty acid methyl esters (see text).
(20), the less abundant isomers may be lost due to the presence of water, if the samples are dried after methanolysis. We have used the method including methyl acetate as a water scavenger during methanolysis (8), which is required for obtaining optimum yields of the methyl glycosides released. Ideally, methods of monosaccharide analysis should include an internal standard that could be applied to the sample at the first step of the procedure. In the anionexchange chromatography-PAD procedure based on acid hydrolysis, 2-deoxyglucose is used as an internal standard, but can be added only after the hydrolysis step due to its acid lability (6). We have tried several monosaccharides as internal standards added before or after methanolysis, but most of them were not useful as internal standards, due to overlapping with or elution
B
1
r 0
I
6 7
I 10
I TIME
FIG. 4.
I 20
I
I
I
30
(MINI
Gradient elution profile of methanolysis products from marine sponge glycopeptides. (A) Isocratic elution with water; (B) gradient elution: O-l min, water; l-10 min, O-3% acetonitrile; lo-22 min, 3% acetonitrile; 22-23 min, 3-O% acetonitrile; 23 min, water.
136
LAMP10
response of this sugar is considerably lower than that of the nonmethylated sugars. Because of the multiple derivatives obtained in methanolysis, and the higher pK, values of the methyl glycosides compared to those of free sugars, the sensitivity of detection is somewhat lower than that with the method involving acid hydrolysis. On the other hand, the constant pattern of peaks obtained for each monosaccharide makes the identification of the sugars more reliable, which is of importance in the analysis of low amounts of samples from biological sources. Although some of the multiple peaks of different sugars overlap, it is possible to quantify the sugars from their other peaks, due to the fairly constant ratios of the derivatives (10). The sensitivity could also be increased by using gradient elution, which is not possible with a refractive index detector (11). Gradient elution has similarly been used to improve the analysis of cyclodextrins using PAD with a membrane reactor (21). Moreover, the lower sensitivity of the methanolysis method compared to the hydrolysis method is balanced by the need of only one sample for the analysis of all sugar components. ACKNOWLEDGMENTS We thank Pia Isomlki, Auli Linnala, Kristiina Miikinen, Johanna Pispa, Anne Puustinen, and Dorothe Spillmann for the preparation of glycoconjugate samples and Irma Isaksson for technical assistance. REFERENCES 1. Townsend, R. R., Hardy, M. R., and Lee, Y. C. (1989) in Methods in Enzymology (Ginsburg, V., Ed.), Vol. 179, pp. 65-75, Academic Press, San Diego, CA. 2. Hardy, M. R. (1989) in Methods in Enzymology (Ginsburg, V., Ed.), Vol. 179, pp. 76-81, Academic Press, San Diego, CA.
AND
FINNE 3. Manzi,
A. E., Diaz,
S., and Varki,
A. (1990)
Anal.
188,
B&hem.
20-32. 4. Lee, Y. C. (1990) 5. Hardy, then.
Anal. Biochem. 189,151-162. M. R., Townsend, R. R., and Lee, Y. C. (1988)
Ad.
Bio-
170,54-62.
6. Sweeley, 7. Bhatti, Biophys.
C. S., and Walker,
B. (1964)
Ad.
Chem.
T., Chambers, R. E., and Clamp, Acta 222,339-347.
8. Chaplin,
M. F. (1982)
9. Cheetham,
36,1461-1466.
J. R. (1970)
Biochim.
123, 336-341.
And.
Biochem.
N. W. H., and
Sirimanne,
P. (1981)
J. Chromatogr.
N. W. H., and Sirimanne,
P. (1983)
Curbohydr.
208,100-103. 10. Cheetham,
Res.
112,1-10. 11. Hjerpe, A., Engfeldt, B., Tsegenidis, (1983) J. ChFOT7ZUtOgF. 259,334-337.
12. Taverna,
M.,
ChFO?TXatOgF.
13. Misevic,
Baillet, 514,
A. E., and
T., and Antonopoulos, Baylocq-Ferrier,
C. A.
D. (1990)
J.
70-79.
G. N., Finne,
J., and Burger,
M. M. (1987)
J. Biol.
Chem.
262,5870-5877. 14. Finne,
J., and Krusius, T. (1982) in Methods in Enzymology (Ginsburg, V., Ed.), Vol. 83, pp. 269-277, Academic Press, San Diego, CA.
15. Hamaguchi, H., and Cleve, H. (1972) Biochem. Commun. 47,459-464. 16. Saito, T., and Hakomori, S. (1971) J. Lipid Res. 17. Svennerholm,
L., and Fredman,
Bzbphys.
Res.
12, 257-259.
P. (1980)
B&him.
Biophys.
Acta
Y. (1976)
Biochim.
Biophys.
Acta
617,97-109. 18. Momoi,
T., Ando,
S., and Magai,
441,488-497. 19. Chaplin, M. F. (1986) in Carbohydrate and Kennedy, J. F., Eds.), pp. l-36,
IRL
Analysis Press,
(Chaplin, Oxford.
M. F.,
N. (1985) Anal. Biochem. 148,424-433. J., Nishimura, Y., Wakai, J., Yasuda, H., Koizumi, K., and Nomura, T. (1989) Anal. Biochem. 179,336-340. 22. Nilsson, B., Norden, N. E., and Svensson, S. (1979) J. Bid. Chem.
20. Jentoft,
21. Haginaka,
254,4545-4553. 23. Svennerholm, L. (1964)
J. Lipid
Res. 5,145-155.
BIOCHEMISTRY
197,132-136
(19%)
Sugar Analysis of Glycoproteins and Glycolipids after Methanolysis by High-Performance Liquid Chromatography with Pulsed Amperometric Detection’ Anja Lampio and Jukka Finne* Department of Biochemistry, University of Helsinki, Unioninkatu 35, SF-001 70, Helsinki, Finland; and *Department of Medical Biochemistry, University of Turku, Kiinamyllynkatu 10, SF-20520 Turky Finland
Received
January
30,199l
A procedure for the analysis of the monosaccharide composition of glycoproteins and glycolipids by methanolysis and high-performance liquid chromatography with pulsed amperometric detection is described. The advantage over previous methods is the analysis of underivatized methyl glycosides of all glycoconjugate monosaccharides including sialic acid and uranic acid in a single chromatographic step at the subnanomolar level.
0 1991
Academic
Press,
Inc.
Anion-exchange chromatography with PAD’ is a novel and sensitive method for analyzing underivatized monosaccharides and oligosaccharides (l-3). In most applications, carbohydrates are separated on the anionexchange column at alkaline pH, which is also a prerequisite for the electrochemical detection of sugars. When an eluant of low alkalinity is used, or in applications using lower pH with other types of columns (4), postcolumn alkalinization becomes necessary. As reported by Hardy et al. (5) the monosaccharide composition of glycoconjugates can be conveniently determined at high sensitivity by high-performance anion-exchange chromatography and PAD after acid hydrolysis. However, sialic acid, an important component of glycoproteins and glycolipids, was destroyed under the conditions used for the analysis and was therefore not quantified. In gas-liquid chromatography, methanolysis has been widely used for the monosaccharide analysis of glycoconjugates (6-8). The advantages are the high yields of sugars including sialic acid and a more
reliable peak identification due to the multiple peaks obtained for each monosaccharide. Analysis of underivatized methyl glycosides of monosaccharides by high-performance liquid chromatography on reversed-phase columns has been reported (912), but these studies do not include sialic acid. Moreover, the detection methods used do not seem to have the same sensitivity as PAD. In the present paper we report on the analysis at the subnanomolar level of all constituent sugars of glycoproteins and glycolipids, including sialic acid and uranic acid, after methanolysis using high-performance liquid chromatography and PAD. MATERIALS
AND
METHODS
Reagents and Materials Thirty percent (w/w) NaOH solution was purchased from Merck (Darmstadt,. Germany). Glucoheptose, 2deoxygalactose, and 3-0-methylglucose were obtained from Pfanstiehl Laboratories (Waukegan, IL), and other monosaccharides and fetuin were from Sigma (St. Louis, MO). Glycopeptides were prepared from fetuin and from the marine sponge Microciona prolifera (13) after pronase digestion (14). Human glycophorin A was isolated by the method of Hamaguchi and Cleve (15): Neutral glycolipids were purified from mammalian redcell membranes by the method of Saito and Hakomori (16). Gangliosides were isolated from pig brain (17) and purified by anion-exchange chromatography followed by chromatography on latrobeads 6RS-8060 silica gel (18). Ultrapure water (Mill;-&, Millipore) was used in the preparation and analysis of the samples. Methanolysis
1 This study was supported by grants from Foundation and the Finnish Academy. ’ Abbreviation used: PAD, pulsed amperometric
the
Sigrid detection.
Jus6lius
Methanolysis of carbohydrates was performed by the method of Chaplin (8). All samples (0.5-200 nmol) were
132 All
Copyright 0 1991 rights of reproduction
0003-2697/91$3.00 by Academic Press, Inc. in my form reserved.
CHROMATOGRAPHIC
ANALYSIS
OF
METHYL
GLYCOSIDES
dried in a vacuum concentrator (Speed Vat, Savant Instruments) or under a nitrogen flow in 13 X 50-mm glass tubes with a conical bottom. Methanolic hydrogen chloride solution (0.625 M) was made by adding acetyl chloride (1.16 ml) dropwise to dry methanol (25 ml), and 320 ~1 of this reagent was pipetted into each tube. After addition of 80 ~1 of methyl acetate and flushing with nitrogen, the tubes were closed with Teflon-lined screw caps and kept at 82-84°C for 16 h. The tubes were cooled, and 80 ~1 of t-butanol was added to each sample prior to evaporation. Dry methanol (50 pl), pyridine (5 pl), and acetic anhydride (5 gl) were added successively with intermediate mixing for re-N-acetylation of the amino sugars (twice these volumes would be used if more than 50 nmol of amino sugars were present (19)). After 30 min at room temperature the solutions were evaporated to dryness. Dry samples were dissolved in 50-200 gl of water, and samples other than pure standards were passed through a 0.22~pm filter (Millipore). The fatty acid methyl esters of glycolipid samples were extracted into chloroform (50-200 ~1) from the aqueous solution (8). Chromatography The Dionex HPLC system (Series 45OOi) consisted of a BioLC Gradient Pump Module (GPM-II), a Liquid Chromatography Module (LCM-2), a Pulsed Amperometric Detector Module (PAD-II), and an Eluant Degas Module for sparging the eluants with helium. The column was a Waters Dextro-Pak plastic cartridge (8 X 100 mm) compressed in an RCM 100 radial compression module. The Dextro-Pak column was stored in acetonitrile:water (20:80) and flushed with water before runs. A Cl8 cartridge (Guard Pak Resolve C18, Waters) was used as a precolumn. The column system was eluted with water at a flow rate of 1 ml/min. In some experiments, a gradient of O-3% acetonitrile was applied. Detection was by PAD using a gold working electrode. NaOH (100 mM) was added as a postcolumn reagent at a flow rate of 1.0 ml/min. The following pulse potentials and durations were used: E, = 0.05 V (tl = 360 ms), E, = 0.80 V (t2 = 120 ms), E3 = -0.60 V (t3 = 420 ms). The response time was set to 3 s. RESULTS
Figure 1 shows the separation of eight methyl glycosides derived from standard monosaccharides. Impurities and unreacted monosaccharides, if present, eluted rapidly from the column before the first peak of methyl galactoside. Retention times and relative responses are given in Table 1. Most of the parent monosaccharides gave more than one peak. However, when the peaks overlapped, quantification was still possible by using subsidiary peaks or simultaneous equations (10). On the other hand, the multiplicity of peaks also affected the
WITH
B
AMPEROMETRIC
133
DETECTION
* I
I
0
I
1
I
I
10
20 TIME
I
1
I
30
(MINI
FIG. 1. Elution profiles of standard methyl glycosides. Dextro-Pak (8 X 100 mm) cartridge eluted with water, postcolumn NaOH addition (100 mM) for PAD (300 nA full scale). The peaks were from: (A) 1, galactose; 4, mannose; 7, fucose; 8, N-acetylneuraminic acid; and (B) 2, glucose; 3, glucuronic acid; 5, N-acetylgalactosamine; 6, N-acetylglucosamine. The amount of each sugar was 2.5 nmol.
sensitivity of the method. The limit of detection (signal to noise ratio, 2:l) using isocratic elution with water was about 200 pmol for methyl fucoside (four peaks), whereas as little as 25 pmol of methyl glucoside, methyl mannoside, or methyl galactoside could be quantified (one major peak). Consequently, the best results are obtained when at least 0.5 nmol of each sugar can be injected per analysis. The sensitivity for fucose and other late-eluting components could be increased by gradient elution (see below). We applied the method to the analysis of several glycoproteins and glycopeptides. Figure 2 shows chromatograms of methanolysis products from glycophorin A, fetuin, and purified glycopeptides from fetuin. In the glycophorin A sample (Fig. 2A), all peaks except the first peaks derived from mannose and N-acetylgalactosamine were resolved. By injecting twice as much sample, even fucose could be quantified at 300 nA full scale. The carbohydrate composition of fetuin was also easily analyzed, although the commercial preparation contained a considerable amount of glucose. The elution profile of the glycoprotein methanolysate was otherwise very similar to that of the glycopeptide sample (Figs. 2B and 2C). Methanolysates of ganglio- and globoseries glycosphingolipids were also analyzed, and examples of the elution profiles are given in Fig. 3. An additional peak eluting after the third peak of methyl galactoside was
134
LAMP10 TABLE
DISCUSSION
1
Retention Times and Relative Sugar Derivatives Obtained
PAD Responses by Methanolysis Time
Parent sugar
Peak”
Galactose Glucose Glucuronic
1 2 3
acid
Mannose
4
N-Acetylgalactosamine
5
N-Acetylglucosamine
6
Fucose
7
N-Acetylneuraminic
acid
8
AND FINNE
Percentage* 74.1 5.7 20.2 100 38.0 17.0 44.6 7.3 92.7 8.6 5.3 63.0 12.1 11.0 15.2 84.8 5.7 10.2 54.4 29.7
100
(min) 4.6 5.4 6.0 5.4 6.0 23.5 27.1 6.9 8.0 6.9 11.5 13.4 14.7 15.9 8.6 17.4 9.0 11.3 16.3 18.4 26.0
of PAD response’ 1.00
1.06 0.74
0.88 0.99
Although anion-exchange chromatography with PAD (1,2,4) offers at present one of the most. sensitive and convenient methods of sugar analysis of glycoproteins, the method has some drawbacks. The most serious is that not all sugars can be analyzed from the same hydrolyzate, because sialic acid is lost. Consequently, different sugars require different optimum conditions of hydrolysis (5). In the present study, we have applied methanolysis to circumvent these drawbacks. We have used the methanolysis conditions worked out by Chaplin (8) for an improved release and yield of methyl glycosides from glycoconjugates. The advantage of the methanolysis procedure is that not only sialic acid but even uranic acid can be analyzed in addition to the neutral
0.99 0.46
0.47
sugars
and hexosamines.
Furthermore,
the current
results extend the use of PAD to the analysis of the monosaccharide composition of glycolipids also. Unlike methods employing acid hydrolysis, methods based on methanolysis give several derivatives for each monosaccharide (a- and P-pyranosides and furanosides and small amounts of free sugars). The production of essentially single main components after methanolysis has been reported (12). However, as discussed before
’ Refers to Fig. 1. * Percentage of total peak areas of each sugar. c Ratio of total peak areas of each sugar to that of galactose.
present in each sample. It was derived from the chloroform extraction step used for the removal of fatty acid
methyl esters, and as judged from its retention time and control experiments, was identified as ethanol, present. in chloroform as a stabilizer. The peak could be removed by evaporating the sample and redissolving it in water. Alternatively, the fatty acid methyl esters can be removed by hexane extraction after methanolysis (6). Table 2 shows the results of the sugar composition analysis of two reference glycoconjugates. The values were calculated on the basis of the response factors obtained for the free monosaccharides. In general the results were very similar to those obtained by other methods. The glucose observed for the commercial preparation of fetuin was due to contamination, as glucose was not present in the purified glycopeptides from fetuin (Fig. 2). The somewhat higher amounts of N-acetylneuraminic acid in GM1 ganglioside reflect the better yield of the sugar derivative obtained from the bound form compared to that obtained from the free form. Figure 4 shows that gradient elution with acetonitrile could be applied for improving the detection of the later eluting compounds. For example, O-3% acetonitrile in the eluant made it possible to detect low amounts of glucuronic acid, which were present in marine sponge glycopeptides.
1..1,.;,,.;:. A.c ;
&!LLAn A1 1 45 4 6 .-.
0
5:5
6
10
8
20 TIME
30
(MINI
FIG. 2. Chromatograms of glycoprotein and glycopeptide methanolysates. (A) 2.5 I.cg glycophorin A, 300 nA full scale; (B) 5 pg fetuin; 100 nA full scale; (C) fetuin glycopeptides; 300 nA full scale.
CHROMATOGRAPHIC
ANALYSIS
OF
METHYL
GLYCOSIDES
WITH
AMPEROMETRIC
135
DETECTION
TABLE
2
Monosaccharide Composition of Glycoconjugates Determined after Methanolysis Fetuin Monosaccharide
Ratio”
Galactose Glucose Mannose IV-Acetylgalactosamine N-Acetylglucosamine N-Acetylneuraminic acid
GM1 Ref.
(22) b
Ratio”
1 0.15 f 0.01
1 0
0.75
0.75 0.25 1.25
0.48
1.08
0.69
* 0.08
0.21 k 0.01 1.20 f 0.10 1.09 * 0.10
ganglioside
0.47
n Monosaccharides were analyzed after methanolysis the values were calculated using the response factors values are given as the mean values (n = 6) +- SD. b Expected values based on the references given.
Ref.
(23) b 1
1 k 0.01
0.5 0 0.5 0 0.5
0 f 0.02 0 * 0.03
by HPLC and in Table 1. The
too close to some of the monosaccharides to be analyzed. These included rhamnose, 2-deoxygalactose, 2deoxyglucose, inositol, mannitol, galactitol, glucitol, N-acetylglucosaminitol, N-acetylmannosamine, and glucoheptose. The best suited of the monosaccharides tried was 3-0-methylglucose. As a methyl glycoside it gives three peaks between the main peaks of mannose and N-acetylgalactosamine and could, if required, be used as an internal standard. Interestingly, the PAD
0
10
20 TIME
30
(MINI
FIG. 3.
Chromatograms of glycosphingolipid methanolysates. (A) Ceramide dihexoside, (B) asialo-GM2, (C) GMl. Detection was by PAD at 300 nA full scale. Peak “S” is a solvent peak from the extraction of fatty acid methyl esters (see text).
(20), the less abundant isomers may be lost due to the presence of water, if the samples are dried after methanolysis. We have used the method including methyl acetate as a water scavenger during methanolysis (8), which is required for obtaining optimum yields of the methyl glycosides released. Ideally, methods of monosaccharide analysis should include an internal standard that could be applied to the sample at the first step of the procedure. In the anionexchange chromatography-PAD procedure based on acid hydrolysis, 2-deoxyglucose is used as an internal standard, but can be added only after the hydrolysis step due to its acid lability (6). We have tried several monosaccharides as internal standards added before or after methanolysis, but most of them were not useful as internal standards, due to overlapping with or elution
B
1
r 0
I
6 7
I 10
I TIME
FIG. 4.
I 20
I
I
I
30
(MINI
Gradient elution profile of methanolysis products from marine sponge glycopeptides. (A) Isocratic elution with water; (B) gradient elution: O-l min, water; l-10 min, O-3% acetonitrile; lo-22 min, 3% acetonitrile; 22-23 min, 3-O% acetonitrile; 23 min, water.
136
LAMP10
response of this sugar is considerably lower than that of the nonmethylated sugars. Because of the multiple derivatives obtained in methanolysis, and the higher pK, values of the methyl glycosides compared to those of free sugars, the sensitivity of detection is somewhat lower than that with the method involving acid hydrolysis. On the other hand, the constant pattern of peaks obtained for each monosaccharide makes the identification of the sugars more reliable, which is of importance in the analysis of low amounts of samples from biological sources. Although some of the multiple peaks of different sugars overlap, it is possible to quantify the sugars from their other peaks, due to the fairly constant ratios of the derivatives (10). The sensitivity could also be increased by using gradient elution, which is not possible with a refractive index detector (11). Gradient elution has similarly been used to improve the analysis of cyclodextrins using PAD with a membrane reactor (21). Moreover, the lower sensitivity of the methanolysis method compared to the hydrolysis method is balanced by the need of only one sample for the analysis of all sugar components. ACKNOWLEDGMENTS We thank Pia Isomlki, Auli Linnala, Kristiina Miikinen, Johanna Pispa, Anne Puustinen, and Dorothe Spillmann for the preparation of glycoconjugate samples and Irma Isaksson for technical assistance. REFERENCES 1. Townsend, R. R., Hardy, M. R., and Lee, Y. C. (1989) in Methods in Enzymology (Ginsburg, V., Ed.), Vol. 179, pp. 65-75, Academic Press, San Diego, CA. 2. Hardy, M. R. (1989) in Methods in Enzymology (Ginsburg, V., Ed.), Vol. 179, pp. 76-81, Academic Press, San Diego, CA.
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