- Email: [email protected]
A method of purification of partially methylated alditol acetates in the methylation analysis of glycoproteins and glycopeptides
ANALYTICAL
BIOCHEMISTRY
136,
187- 19 1 ( 1984)
A Method of Purification of Partially Methylated Alditol Acetates Methylation Analysis of Glycoproteins and Glycopeptides
in the
MARK E. LOWE* AND Bo NILSSON~,' *Pediatric Metabolism Branch, NIADDK; TLaboratory of Pathology, NCI, National Institutes of Health, Bethesda, Maryland 20205 Received May 16, 1983 A method for methylation analysis of intact glycoproteins is described. Starting with intact glycoprotein, the oligosaccharides are methylated, hydrolyzed, reduced, and acetylated. The partially methylated alditol acetates are then separated from noncarbohydrate contaminants on a silica gel G column. Partially methylated hexitol acetates are eluted from the column with petroleum ether:ethyl acetate (l:l, v/v) and partially methylated N-acetylhexosaminitol acetates are subsequently eluted with methanol. Analysis by gas-liquid chromatography/mass spectrometry of the partially methylated alditol acetates shows no interfering contaminants. This method circumvents the need to make pronase glycopeptides and avoids the pitfalls of other methylation procedures. KEY WORDS: glycoprotein; methylation; silica; glycopeptide; N-linked; ohgosaccharide.
An important step in the structural analysis of oligosaccharides is the methylation analysis. This procedure identifies the locus of substitution of the various sugars. Normally, oligosaccharides or glycopeptides are methylated, not intact glycoproteins ( 1,2). During studies to determine structures of the oligosaccharides of human c+macroglobulin, we encountered problems with the methylation analysis of glycopeptides obtained after pronase digestion (unpublished work, Lowe, M.). Recovery of the partially methylated alditol acetates was variable, ranging from quantitative to negligible. Moreover, the gas-liquid chromatography patterns of the methylated samples were complicated by numerous noncarbohydrate peaks which severely limited the quantitation of the sugar derivatives. Such problems are often encountered when the pronase digestion is incomplete and the resulting glycopeptides retain a large pep’ Person to whom correspondence should be sent. Present address: University of Lund, Chemical Center, Dept. of Carbohydrate Chemistry, P.O. Box 740, S-220-07 Lund, Sweden.
tide moiety. The presence of large peptides alters the solubility properties of the methylated glycopeptides, making extraction from an aqueous phase inefficient, and results in variable recoveries of the methylated glycopeptides. Large peptides also increase the amount of noncarbohydrate material in the samples. The removal of the contaminating noncarbohydrate material by chromatography on silica gel columns is described in this paper. The procedure reliably separates the partially methylated alditol acetates from most contaminants, thereby, allowing the methylation analysis of N-linked oligosaccharides on intact glycoproteins. The method can also be used to purify partially methylated alditol acetates obtained after methylation of glycopeptides. MATERIALS
AND
METHODS
Silica gel G was obtained from Merck. Fetuin was purchased from Sigma. Human haptoglobin l-1 was a gift of Dr. Jerzy Osada (Bio-Pharmaceutical Institute, Wroclaw, Poland). Pronase-digested human thrombin (3) 187
0003-2697184 $3.00 Copyright 0 1984 by Academic Press. Inc. All rights of reproduction in any form reserved.
188
LOWE AND NILSSON
was kindly provided by Dr. McDonald Horne (NIH, Bethesda). a*-Macroglobulin was prepared from human plasma by published procedures (4,5). Glycopeptides obtained by pronase digestion of laminin and the keratan sulfate linkage region from monkey cornea (6) were provided by Dr. John Hassell and Dr. Steve Ledbetter (NIH, Bethesda). Partially desialylated transfenin was a gift from Dr. David Zopf (NIH, Bethesda). Pronase glycopeptides of rabbit hepatic binding protein were prepared as previously described (7,8). The standard partially methylated derivatives of mannose were prepared by the Hakomori procedure (1) employing only one-half the amount of dimsyl sodium required for complete methylation. Methylation of glycopeptides was accomplished by published procedures (1). The methylation procedure for the intact glycoproteins was as follows. Five to fifteen milligrams of glycoprotein in a vial was dissolved in dry dimethyl sulfoxide (DMSO).* The volume of DMSO required to solubilize each glycoprotein varied considerably, so DMSO was added in aliquots while sonicating the sample until the glycoprotein was in solution. Several grains of triphenylmethane were added as an indicator (9), and the vial was sealed and flushed with nitrogen. Methylation was accomplished using dimsyl sodium and methyl iodide (1). After the methylation, excess methyl iodide was removed by evaporation, water was added and the samples were dialyzed exhaustively against distilled water. O-linked oligosaccharides are liberated by P-elimination, degraded by “peeling” during treatment with dimsyl sodium, and lost on dialysis, After concentration the samples to dryness, they were acid-hydrolyzed, reduced, and acetylated as previously described ( 10). The dried, partially methylated alditol acetates and contaminants were dissolved in a small amount of petroleum ether:ethyl acetate * Abbreviations used: GLC, gas-liquid chromatography; GLC/MS, gas-liquid chromatography/mass spectrometry; DMSO, dimethyl sulfoxide.
(2:l v/v) and applied to a silica gel column (2.0-ml bed volume) that was equilibrated in the same solvent. The column was washed with 6.0 ml of this solvent. The wash contained no sugars and was discarded. The hexose derivatives were eluted with 6.0 ml of petroleum ether:ethyl acetate (1: 1 v/v). One fraction of 6.0 ml was collected as soon as the elution was begun. The hexosamine derivatives were then eluted with 6.0 ml of methanol. As before, one fraction of 6.0 ml was collected when the elution was started. The two fractions containing the sugars were evaporated to dryness and the residues were dissolved in chloroform prior to analysis by gas-liquid chromatography/mass spectrometry. The partially methylated alditol acetates were quantitated on a Hewlett-Packard 5840 gas chromatograph fitted with a fused silica capillary column (25 m X 0.2 mm) of OV-1 W.C.O.T. (Hewlett-Packard) and programmed from 180 to 240°C at 4’/min. Gasliquid chromatography/mass spectrometry (GLC/MS) (11) was done on a Hewlett-Packard 5992A instrument equipped with a glass column (1 m X 2.0 mm) packed with 3% SP2340 on Supelcoport 100/120 (Supelco, Inc.) at 170-240”. Mass spectra were recorded at 70 eV with an ion source temperature of 140°C. RESULTS AND DISCUSSION
Partially methylated alditol acetates in hydrolysates of derivatized glycoproteins can be purified on silica gel before analysis by GLC and GLC/MS. Elution first with petroleum ether:ethyl acetate 2: 1 (v/v) removed noncarbohydrate contaminants but no carbohydrate derivatives. The partially methylated hexitol acetates are subsequently eluted with petroleum ether:ethyl acetate 1: 1 (v/v). Finally, the partially methylated ZV-acetyl-hexosaminitol acetates are recovered by elution with methanol. Table 1 compares the relative proportions of all partially methylated alditol acetates from mannose before and after silica column chro-
METHYLATION TABLE
OF GLYCOPROTEINS
1
RELATIVE MOLAR PROPORTIONS OF PARTIALLY METHYLATED MANNITOL ACETATES BEFORE AND AFTER SILICA GEL CHROMATOGRAPHY
Position of methyl group 2,3,4,6
2,X4 2,3,6 and 3,4,6
2,4.6 W 24 2.6 3,4
56 4.6 2 3 and 4 6
T value”
Before
After
l.OOh 1.52 1.33 1.42 1.I9 1.93 1.59 1.87 1.68 1.55 2.16 2.36 1.87
l.Oh 1.5 2.6’ 1.3 1.4 2.1 2.7 0.3 1.2 1.1 2.6 1.9’ 3.1
l.ob 1.7 2.4‘ 1.3 1.4 2.2 2.6 0.3 1.3 1.0 2.6 2.0‘ 3.0
’ Retention time relative to 1,5di-O-acetyl-2,3,4,6-tetraO-methyl-glucitol on an OV- 1 column. ’ 2,3,4,6-O-Me-Man is set to 1.O. ‘Determined in combination.
matography. All derivatives were recovered in about the same proportions. This method is useful for purification of partially methylated alditol acetates obtained from methylation of glycopeptides (Table 2). Glycopeptides from laminin, thrombin, rabbit hepatic binding protein, and cornea1 keratan sulfate linkage region were prepared by pronase digestion. The glycopeptides were methylated, hydrolyzed, reduced, acetylated, and analyzed by GLC/MS. The partially methylated alditol acetates were then fractionated on a silica gel column (see Materials and Methods) and analyzed again by GLC/MS. As can be seen from Table 2 all of the partially methyIated hexitol acetates for each sample were recovered in about the same proportions after silica gel chromatography. The hexosamine derivatives are separated from the partially methylated hexitol acetates on the silica gel column (eluted with methanol). Since the partially methylated N-acetylhexosaminitol acetates cannot be accurately quantitated relative to the hexose derivatives there is no ad-
189
vantage for eluting them together. All of the expected hexosamine derivatives were recovered and easily identified by GLC/MS. Methylation analysis of intact glycoproteins is difficult to interpret due to interference by noncarbohydrate material in the GLC analyses. If the carbohydrate content of the glycoprotein is low, quantitation of the partially methylated alditol acetates is virtually impossible by GLC. By removing contaminants on a silica gel column before analysis by GLC, an accurate quantitation of the partially methylated alditol acetates can be achieved. Table 3 shows the recoveries of partially methylated alditol acetates after methylation of intact glycoproteins and subsequent silica gel chromatography. These values are in agreement with published results and what is obtained from methylation of glycopeptides. Very few noncarbohydrate components are seen in the GLC pattern (Fig. 1) of the partially methylated hexitol acetates from a2-macroglobulin purified on the silica column. The major contaminants elute well ahead of any partially methylated sugars and do not interfere with the quantitation of these sugars. The mass spectra of the sugar derivatives gave no indication for comigrating contaminants. The advantages of this procedure are many. The problem of poor solubility of the methylated glycopeptides in chloroform is completely avoided. The procedure is faster than analyzing glycopeptides since there is no need to prepare glycopeptides. Relative recoveries of the various derivatives agree with the values obtained by methylation analysis of glycopeptides, and the samples are free of major contaminants. The method allows the determination of sugar linkages present in the starting material without the complication of losing components because the glycopeptides are fractionated during the chloroform extractions. This method provides a good starting point for structural studies on the oligosaccharides of glycoproteins. The major disadvantage with this method is that the methylation analysis pattern can be complicated if several different N-linked carbohydrate
190
LOWE AND NILSSON TABLE 2
PARTIALLY METHYLATEDALDITOLACETATESOBTAINEDFROM METHYLATIONOFGLYCOPEPTIDES: RELATIVE MOLAR PROPORTIONSBEFOREAND AI-TER SILICA GEL COLUMN CHROMATOGRAPHY Thrombin
Laminin Sugar derivatives 2,3,4-O-Me-Fuc 2,3,4-6-O-Me-Man 2,3,4,6-O-Me-Gal 3,4,6-O-Me-Man 2,4,6-O-Me-Gal 2,3,4-O-Me-Gal 3,6-O-Me-Man 2,4-O-Me-Man 3,4-O-Me-Man 3,4,6-O-Me-GlcN(Me)Ac 3,6-0-Me-GlcN(Me)Ac 6-0-Me-GlcN(Me)Ac 3-0-Me-GlcN(Me)Ac
Rabbit hepatic binding protein
Keratan sulfate linkage region
T-value”
Before
After
Before
After
Before
After
Before
After
0.68 1.00 1.04 1.33 1.38 1.58 1.68 1.93 1.87 2.35 2.89 3.81 4.06
0.4 0.6 1.5 2.8 4.8 0 I.06 2.6 1.1 +d + + +
0.4 0.7 1.6 2.9 4.6 0 l.oh 2.5 1.2 + + + +
0.3 0 0 2.1 0 1.9 0 1.O’ 0 0 + 0 +
0.2 0 0 1.9 0 2.1 0 1.0’ 0 0 + 0 +
0 0 0 1.0 1.2 2.1 l.Oh 1.1 0 0 + 0 0
0 0 0 1.1 1.2 1.9 1.0* 1.0 0 0 + 0 0
0.7 0 0 1.9 2.1 0 0 1.0’ 0 + + 0 +
0.5 0 0 2.0 2.1 0 0 1.0’ 0 + + 0 +
’ Retention time relative to l,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-glucitol. * 3,6-O-Me-Man is set to 1.0. ’ 2,4-O-Me-Man is set to 1.O. d Present but not quantitated. TABLE 3
a2-
Transferrin
Fetuin Sugar derivatives 2,3,4-O-Me-Fuc 2,3,4,6-O-Me-Man 2,3,4,6-O-Me-Gal 3,4,6-O-Me-Man 2,4,6-O-Me-Gal 2,3,4-O-Me-Gal 3,6-O-Me-Man 2,4-O-Me-Man 3,6-0-Me-GlcN(Me)Ac 6-O-Me-GlcN(Me)Ac 3-0-Me-GlcN(Me)Ac
T-value ’ 0.68 1.oo 1.04 1.33 1.38 1.58 1.68 1.93 2.89 3.81 4.06
h
c
0 0
0 0
0.2 1.1 1.5 1.2 0.8 1.0’ +g 0 0
0 1.1 1.8 1.1 1.2 1.0’ + 0 0
Haptoglobin
b
d
b
0
0
0 1.4 1.9 0.5 0 0.1 1.0’
0 2.2 1.9 0 0 0.2 1.O’
0.2 0 0.2 1.3 0.6 1.1 0.4 1.0’
+
+
0 0
0 0
a Retention time relative to 1,5-di-O-acetyl-2,3,4,6-tetra-O-methybglucitol. b Values obtained after silica gel column chromatography. ’ Values obtained from Ref. (IO). d Values obtained from Ref. (I 3). ’ Values obtained from methylation analysis of pronase glycopeptides. ‘2,4-O-Me-Man is set to 1.O. gPresent but not quantitated.
+ + +
c
Macroglobulin b
0.2 0 0.1 1.3 0.5 1.5 0.5
0.2 0.3 0.3 2.2 0.9 1.1 0.1
I .oJ + + +
I .oJ + 0 +
c
0.3 0.2 0.3 1.8 0.6 0.9 0 1.OJ + 0 +
METHYLATION
macroglobulin (Table 3) nor was it detected in the original studies of transferrin oligosacchar-ides (12). The presence of a 3,6-O-MeMan derivative suggests that a glycoprotein may contain triantennary or tetraantennary oligosaccharides. That knowledge dictates the best procedures for the further separation of the glycopeptides or oligosaccharides for subsequent analysis.
4
,yll 5
23
1
34567 TIME (mid
191
OF GLYCOPROTEINS
L-
FIG. 1. The GLC pattern of the partially methylated hexitol acetates from cur-macroglobulin. The sugars on human cYr-macroglobulin were methylated, hydrolyzed, and acetylated as described. (1) 2,3,4-O-Me-Fuc; (2) 2,3,4,6-O-Me-Man; (3) 2,3,4,6-O-Me-Gal; (4) 3,4,6-O-MeMan; (5) 2,4,6-O-Me-Gal; (6) 2,3,4-O-Me-Gal; (7) 3,6-0Me-Man: (8) 2.4-O-Me-Man.
REFERENCES
1. Hakomori, S. (1964) .I. Biochem. (Tokyo) 55, 205208. 2. Finne, J., Krusius, T., and Rauvala, H. (1980) Carbohydr.
Res. 80, 336-339.
3. Nilsson, B., Home, M. K., and Gralnick, H. R. (1983) Arch. Biochem. Biophys. 224, 127-133. 4. Kureck, T., Kress, L. F., and Laskowski, M.. Sr. (1979) Anal. Biochem. 99, 4 15-420. 5. Vivca, G. R., Travis, J., Hall, P. K., and Roberts, R. C. (1978) Anal. Biochem. 89, 274-278. 6. Nilsson, B., Nakazawa, K., Hassell, J. R., Newsome, D. A., and Hascall, V. C. (1983) J. Biol. Chem. 258,6056-6063.
7. Kawasaki, T., and Ashwell, G. (1976) J. Biol. Chem. 251, 5292-5299.
structures are present. However, examination of the number di-O-substituted Man residues gives an indication of which types of N-linked oligosaccharides are present, which can guide the further structural analysis. The results for the cy2-macroglobulin and asialotransferrin emphasize this point. The samples analyzed after the methylation of the intact glycoprotein revealed a 3,6-O-Me-Man derivative (Table 3). This derivative was not seen in the analysis of glycopeptides from CQ-
Lowe, M., and Nilsson, B. (1983) J. Biol. Chem. 258, 1885-1887. 9. Rauvala, J. (1979) Carbohydr. Res. 72, 257-260. 10. Nilsson, B., Norden, N. E., and Svensson, S. (1979) 8.
J. Biol.
Chem.
254, 4545-4553.
I I. Bjomdal, H., Hellerquist, C. G., Lindberg, B., and Svensson, S. (1970) Angew. Chem. Int. Ed. Engl. 9,610-619.
12. Jamieson, G. A., Jett, M., and DeBemardo, S. L. (1971) J. Biol. Chem. 246, 3686-3693. 13. Hutton, M. W. C., Marx, L., Berry, L. R., Debanne, M. T., and Reyoezi, E. (1979) Biochem. J. 181, 633-638.
BIOCHEMISTRY
136,
187- 19 1 ( 1984)
A Method of Purification of Partially Methylated Alditol Acetates Methylation Analysis of Glycoproteins and Glycopeptides
in the
MARK E. LOWE* AND Bo NILSSON~,' *Pediatric Metabolism Branch, NIADDK; TLaboratory of Pathology, NCI, National Institutes of Health, Bethesda, Maryland 20205 Received May 16, 1983 A method for methylation analysis of intact glycoproteins is described. Starting with intact glycoprotein, the oligosaccharides are methylated, hydrolyzed, reduced, and acetylated. The partially methylated alditol acetates are then separated from noncarbohydrate contaminants on a silica gel G column. Partially methylated hexitol acetates are eluted from the column with petroleum ether:ethyl acetate (l:l, v/v) and partially methylated N-acetylhexosaminitol acetates are subsequently eluted with methanol. Analysis by gas-liquid chromatography/mass spectrometry of the partially methylated alditol acetates shows no interfering contaminants. This method circumvents the need to make pronase glycopeptides and avoids the pitfalls of other methylation procedures. KEY WORDS: glycoprotein; methylation; silica; glycopeptide; N-linked; ohgosaccharide.
An important step in the structural analysis of oligosaccharides is the methylation analysis. This procedure identifies the locus of substitution of the various sugars. Normally, oligosaccharides or glycopeptides are methylated, not intact glycoproteins ( 1,2). During studies to determine structures of the oligosaccharides of human c+macroglobulin, we encountered problems with the methylation analysis of glycopeptides obtained after pronase digestion (unpublished work, Lowe, M.). Recovery of the partially methylated alditol acetates was variable, ranging from quantitative to negligible. Moreover, the gas-liquid chromatography patterns of the methylated samples were complicated by numerous noncarbohydrate peaks which severely limited the quantitation of the sugar derivatives. Such problems are often encountered when the pronase digestion is incomplete and the resulting glycopeptides retain a large pep’ Person to whom correspondence should be sent. Present address: University of Lund, Chemical Center, Dept. of Carbohydrate Chemistry, P.O. Box 740, S-220-07 Lund, Sweden.
tide moiety. The presence of large peptides alters the solubility properties of the methylated glycopeptides, making extraction from an aqueous phase inefficient, and results in variable recoveries of the methylated glycopeptides. Large peptides also increase the amount of noncarbohydrate material in the samples. The removal of the contaminating noncarbohydrate material by chromatography on silica gel columns is described in this paper. The procedure reliably separates the partially methylated alditol acetates from most contaminants, thereby, allowing the methylation analysis of N-linked oligosaccharides on intact glycoproteins. The method can also be used to purify partially methylated alditol acetates obtained after methylation of glycopeptides. MATERIALS
AND
METHODS
Silica gel G was obtained from Merck. Fetuin was purchased from Sigma. Human haptoglobin l-1 was a gift of Dr. Jerzy Osada (Bio-Pharmaceutical Institute, Wroclaw, Poland). Pronase-digested human thrombin (3) 187
0003-2697184 $3.00 Copyright 0 1984 by Academic Press. Inc. All rights of reproduction in any form reserved.
188
LOWE AND NILSSON
was kindly provided by Dr. McDonald Horne (NIH, Bethesda). a*-Macroglobulin was prepared from human plasma by published procedures (4,5). Glycopeptides obtained by pronase digestion of laminin and the keratan sulfate linkage region from monkey cornea (6) were provided by Dr. John Hassell and Dr. Steve Ledbetter (NIH, Bethesda). Partially desialylated transfenin was a gift from Dr. David Zopf (NIH, Bethesda). Pronase glycopeptides of rabbit hepatic binding protein were prepared as previously described (7,8). The standard partially methylated derivatives of mannose were prepared by the Hakomori procedure (1) employing only one-half the amount of dimsyl sodium required for complete methylation. Methylation of glycopeptides was accomplished by published procedures (1). The methylation procedure for the intact glycoproteins was as follows. Five to fifteen milligrams of glycoprotein in a vial was dissolved in dry dimethyl sulfoxide (DMSO).* The volume of DMSO required to solubilize each glycoprotein varied considerably, so DMSO was added in aliquots while sonicating the sample until the glycoprotein was in solution. Several grains of triphenylmethane were added as an indicator (9), and the vial was sealed and flushed with nitrogen. Methylation was accomplished using dimsyl sodium and methyl iodide (1). After the methylation, excess methyl iodide was removed by evaporation, water was added and the samples were dialyzed exhaustively against distilled water. O-linked oligosaccharides are liberated by P-elimination, degraded by “peeling” during treatment with dimsyl sodium, and lost on dialysis, After concentration the samples to dryness, they were acid-hydrolyzed, reduced, and acetylated as previously described ( 10). The dried, partially methylated alditol acetates and contaminants were dissolved in a small amount of petroleum ether:ethyl acetate * Abbreviations used: GLC, gas-liquid chromatography; GLC/MS, gas-liquid chromatography/mass spectrometry; DMSO, dimethyl sulfoxide.
(2:l v/v) and applied to a silica gel column (2.0-ml bed volume) that was equilibrated in the same solvent. The column was washed with 6.0 ml of this solvent. The wash contained no sugars and was discarded. The hexose derivatives were eluted with 6.0 ml of petroleum ether:ethyl acetate (1: 1 v/v). One fraction of 6.0 ml was collected as soon as the elution was begun. The hexosamine derivatives were then eluted with 6.0 ml of methanol. As before, one fraction of 6.0 ml was collected when the elution was started. The two fractions containing the sugars were evaporated to dryness and the residues were dissolved in chloroform prior to analysis by gas-liquid chromatography/mass spectrometry. The partially methylated alditol acetates were quantitated on a Hewlett-Packard 5840 gas chromatograph fitted with a fused silica capillary column (25 m X 0.2 mm) of OV-1 W.C.O.T. (Hewlett-Packard) and programmed from 180 to 240°C at 4’/min. Gasliquid chromatography/mass spectrometry (GLC/MS) (11) was done on a Hewlett-Packard 5992A instrument equipped with a glass column (1 m X 2.0 mm) packed with 3% SP2340 on Supelcoport 100/120 (Supelco, Inc.) at 170-240”. Mass spectra were recorded at 70 eV with an ion source temperature of 140°C. RESULTS AND DISCUSSION
Partially methylated alditol acetates in hydrolysates of derivatized glycoproteins can be purified on silica gel before analysis by GLC and GLC/MS. Elution first with petroleum ether:ethyl acetate 2: 1 (v/v) removed noncarbohydrate contaminants but no carbohydrate derivatives. The partially methylated hexitol acetates are subsequently eluted with petroleum ether:ethyl acetate 1: 1 (v/v). Finally, the partially methylated ZV-acetyl-hexosaminitol acetates are recovered by elution with methanol. Table 1 compares the relative proportions of all partially methylated alditol acetates from mannose before and after silica column chro-
METHYLATION TABLE
OF GLYCOPROTEINS
1
RELATIVE MOLAR PROPORTIONS OF PARTIALLY METHYLATED MANNITOL ACETATES BEFORE AND AFTER SILICA GEL CHROMATOGRAPHY
Position of methyl group 2,3,4,6
2,X4 2,3,6 and 3,4,6
2,4.6 W 24 2.6 3,4
56 4.6 2 3 and 4 6
T value”
Before
After
l.OOh 1.52 1.33 1.42 1.I9 1.93 1.59 1.87 1.68 1.55 2.16 2.36 1.87
l.Oh 1.5 2.6’ 1.3 1.4 2.1 2.7 0.3 1.2 1.1 2.6 1.9’ 3.1
l.ob 1.7 2.4‘ 1.3 1.4 2.2 2.6 0.3 1.3 1.0 2.6 2.0‘ 3.0
’ Retention time relative to 1,5di-O-acetyl-2,3,4,6-tetraO-methyl-glucitol on an OV- 1 column. ’ 2,3,4,6-O-Me-Man is set to 1.O. ‘Determined in combination.
matography. All derivatives were recovered in about the same proportions. This method is useful for purification of partially methylated alditol acetates obtained from methylation of glycopeptides (Table 2). Glycopeptides from laminin, thrombin, rabbit hepatic binding protein, and cornea1 keratan sulfate linkage region were prepared by pronase digestion. The glycopeptides were methylated, hydrolyzed, reduced, acetylated, and analyzed by GLC/MS. The partially methylated alditol acetates were then fractionated on a silica gel column (see Materials and Methods) and analyzed again by GLC/MS. As can be seen from Table 2 all of the partially methyIated hexitol acetates for each sample were recovered in about the same proportions after silica gel chromatography. The hexosamine derivatives are separated from the partially methylated hexitol acetates on the silica gel column (eluted with methanol). Since the partially methylated N-acetylhexosaminitol acetates cannot be accurately quantitated relative to the hexose derivatives there is no ad-
189
vantage for eluting them together. All of the expected hexosamine derivatives were recovered and easily identified by GLC/MS. Methylation analysis of intact glycoproteins is difficult to interpret due to interference by noncarbohydrate material in the GLC analyses. If the carbohydrate content of the glycoprotein is low, quantitation of the partially methylated alditol acetates is virtually impossible by GLC. By removing contaminants on a silica gel column before analysis by GLC, an accurate quantitation of the partially methylated alditol acetates can be achieved. Table 3 shows the recoveries of partially methylated alditol acetates after methylation of intact glycoproteins and subsequent silica gel chromatography. These values are in agreement with published results and what is obtained from methylation of glycopeptides. Very few noncarbohydrate components are seen in the GLC pattern (Fig. 1) of the partially methylated hexitol acetates from a2-macroglobulin purified on the silica column. The major contaminants elute well ahead of any partially methylated sugars and do not interfere with the quantitation of these sugars. The mass spectra of the sugar derivatives gave no indication for comigrating contaminants. The advantages of this procedure are many. The problem of poor solubility of the methylated glycopeptides in chloroform is completely avoided. The procedure is faster than analyzing glycopeptides since there is no need to prepare glycopeptides. Relative recoveries of the various derivatives agree with the values obtained by methylation analysis of glycopeptides, and the samples are free of major contaminants. The method allows the determination of sugar linkages present in the starting material without the complication of losing components because the glycopeptides are fractionated during the chloroform extractions. This method provides a good starting point for structural studies on the oligosaccharides of glycoproteins. The major disadvantage with this method is that the methylation analysis pattern can be complicated if several different N-linked carbohydrate
190
LOWE AND NILSSON TABLE 2
PARTIALLY METHYLATEDALDITOLACETATESOBTAINEDFROM METHYLATIONOFGLYCOPEPTIDES: RELATIVE MOLAR PROPORTIONSBEFOREAND AI-TER SILICA GEL COLUMN CHROMATOGRAPHY Thrombin
Laminin Sugar derivatives 2,3,4-O-Me-Fuc 2,3,4-6-O-Me-Man 2,3,4,6-O-Me-Gal 3,4,6-O-Me-Man 2,4,6-O-Me-Gal 2,3,4-O-Me-Gal 3,6-O-Me-Man 2,4-O-Me-Man 3,4-O-Me-Man 3,4,6-O-Me-GlcN(Me)Ac 3,6-0-Me-GlcN(Me)Ac 6-0-Me-GlcN(Me)Ac 3-0-Me-GlcN(Me)Ac
Rabbit hepatic binding protein
Keratan sulfate linkage region
T-value”
Before
After
Before
After
Before
After
Before
After
0.68 1.00 1.04 1.33 1.38 1.58 1.68 1.93 1.87 2.35 2.89 3.81 4.06
0.4 0.6 1.5 2.8 4.8 0 I.06 2.6 1.1 +d + + +
0.4 0.7 1.6 2.9 4.6 0 l.oh 2.5 1.2 + + + +
0.3 0 0 2.1 0 1.9 0 1.O’ 0 0 + 0 +
0.2 0 0 1.9 0 2.1 0 1.0’ 0 0 + 0 +
0 0 0 1.0 1.2 2.1 l.Oh 1.1 0 0 + 0 0
0 0 0 1.1 1.2 1.9 1.0* 1.0 0 0 + 0 0
0.7 0 0 1.9 2.1 0 0 1.0’ 0 + + 0 +
0.5 0 0 2.0 2.1 0 0 1.0’ 0 + + 0 +
’ Retention time relative to l,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-glucitol. * 3,6-O-Me-Man is set to 1.0. ’ 2,4-O-Me-Man is set to 1.O. d Present but not quantitated. TABLE 3
a2-
Transferrin
Fetuin Sugar derivatives 2,3,4-O-Me-Fuc 2,3,4,6-O-Me-Man 2,3,4,6-O-Me-Gal 3,4,6-O-Me-Man 2,4,6-O-Me-Gal 2,3,4-O-Me-Gal 3,6-O-Me-Man 2,4-O-Me-Man 3,6-0-Me-GlcN(Me)Ac 6-O-Me-GlcN(Me)Ac 3-0-Me-GlcN(Me)Ac
T-value ’ 0.68 1.oo 1.04 1.33 1.38 1.58 1.68 1.93 2.89 3.81 4.06
h
c
0 0
0 0
0.2 1.1 1.5 1.2 0.8 1.0’ +g 0 0
0 1.1 1.8 1.1 1.2 1.0’ + 0 0
Haptoglobin
b
d
b
0
0
0 1.4 1.9 0.5 0 0.1 1.0’
0 2.2 1.9 0 0 0.2 1.O’
0.2 0 0.2 1.3 0.6 1.1 0.4 1.0’
+
+
0 0
0 0
a Retention time relative to 1,5-di-O-acetyl-2,3,4,6-tetra-O-methybglucitol. b Values obtained after silica gel column chromatography. ’ Values obtained from Ref. (IO). d Values obtained from Ref. (I 3). ’ Values obtained from methylation analysis of pronase glycopeptides. ‘2,4-O-Me-Man is set to 1.O. gPresent but not quantitated.
+ + +
c
Macroglobulin b
0.2 0 0.1 1.3 0.5 1.5 0.5
0.2 0.3 0.3 2.2 0.9 1.1 0.1
I .oJ + + +
I .oJ + 0 +
c
0.3 0.2 0.3 1.8 0.6 0.9 0 1.OJ + 0 +
METHYLATION
macroglobulin (Table 3) nor was it detected in the original studies of transferrin oligosacchar-ides (12). The presence of a 3,6-O-MeMan derivative suggests that a glycoprotein may contain triantennary or tetraantennary oligosaccharides. That knowledge dictates the best procedures for the further separation of the glycopeptides or oligosaccharides for subsequent analysis.
4
,yll 5
23
1
34567 TIME (mid
191
OF GLYCOPROTEINS
L-
FIG. 1. The GLC pattern of the partially methylated hexitol acetates from cur-macroglobulin. The sugars on human cYr-macroglobulin were methylated, hydrolyzed, and acetylated as described. (1) 2,3,4-O-Me-Fuc; (2) 2,3,4,6-O-Me-Man; (3) 2,3,4,6-O-Me-Gal; (4) 3,4,6-O-MeMan; (5) 2,4,6-O-Me-Gal; (6) 2,3,4-O-Me-Gal; (7) 3,6-0Me-Man: (8) 2.4-O-Me-Man.
REFERENCES
1. Hakomori, S. (1964) .I. Biochem. (Tokyo) 55, 205208. 2. Finne, J., Krusius, T., and Rauvala, H. (1980) Carbohydr.
Res. 80, 336-339.
3. Nilsson, B., Home, M. K., and Gralnick, H. R. (1983) Arch. Biochem. Biophys. 224, 127-133. 4. Kureck, T., Kress, L. F., and Laskowski, M.. Sr. (1979) Anal. Biochem. 99, 4 15-420. 5. Vivca, G. R., Travis, J., Hall, P. K., and Roberts, R. C. (1978) Anal. Biochem. 89, 274-278. 6. Nilsson, B., Nakazawa, K., Hassell, J. R., Newsome, D. A., and Hascall, V. C. (1983) J. Biol. Chem. 258,6056-6063.
7. Kawasaki, T., and Ashwell, G. (1976) J. Biol. Chem. 251, 5292-5299.
structures are present. However, examination of the number di-O-substituted Man residues gives an indication of which types of N-linked oligosaccharides are present, which can guide the further structural analysis. The results for the cy2-macroglobulin and asialotransferrin emphasize this point. The samples analyzed after the methylation of the intact glycoprotein revealed a 3,6-O-Me-Man derivative (Table 3). This derivative was not seen in the analysis of glycopeptides from CQ-
Lowe, M., and Nilsson, B. (1983) J. Biol. Chem. 258, 1885-1887. 9. Rauvala, J. (1979) Carbohydr. Res. 72, 257-260. 10. Nilsson, B., Norden, N. E., and Svensson, S. (1979) 8.
J. Biol.
Chem.
254, 4545-4553.
I I. Bjomdal, H., Hellerquist, C. G., Lindberg, B., and Svensson, S. (1970) Angew. Chem. Int. Ed. Engl. 9,610-619.
12. Jamieson, G. A., Jett, M., and DeBemardo, S. L. (1971) J. Biol. Chem. 246, 3686-3693. 13. Hutton, M. W. C., Marx, L., Berry, L. R., Debanne, M. T., and Reyoezi, E. (1979) Biochem. J. 181, 633-638.