created: 16th October 1997, last updated: 16th January 1998 ,© 1998 ABRF
Ping Du and Kalyan R. Anumula
SmithKline Beecham, R&D, King of Prussia, PA 19406, USA
ABSTRACT
In the current method, glycoprotein derived monosaccharides were separated in a short time (25 min) using a Waters Symmetry C-18 (3.9 x 150 mm, 5 um) column which is comparable to the time needed for analysis by HPAEC-PAD. The monosaccharides in glycoprotein acid hydrolysates (20% TFA at 100 degrees C for 6-7 h) are derivatized with fluorescent anthranilic acid and separated using solvents as described earlier with a modified gradient. The monosaccharide composition determined by this method was in excellent agreement with the expected, and is very reproducible. Recovery of the monosaccharides from the glycoprotein hydrolysates was >80%. Relative standard deviation for the compositions was determined and the precision was less than 3% (at <15 pmol level). Because of the very high sensitivity and specificity, proteins blotted onto PVDF membrane following SDS-PAGE can be easily identified as glycoproteins. As little as 0.1 ug of bovine fetuin (contains ~15% w/w carbohydrate) loaded onto the gel can be easily analyzed by this method.
INTRODUCTION
Carbohydrate composition analysis of glycoproteins is a challenging problem in glycobiology. An accurate determination of the monosaccharide composition of a glycoprotein is a first step in learning about its glycosylation. The present communication describes a rapid HPLC method for quantitative determination of monosaccharides in glycoproteins. The monosaccharides found in glycoproteins were separated on a Waters Symmetry C-18 column in 15 or 25 min using either a 7.5 cm or a 15 cm column, respectively, following hydrolysis and derivatization with fluorescent anthranilic acid (1). The time required for HPLC separation was reduced by 50-70% in this method and it compares favorably to the analysis by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). This method has been validated with respect to accuracy, precision, ruggedness and reproducibility using well characterized proteins. The other methods available for the analysis of monosaccharides have been reviewed recently (2).
EXPERIMENTAL PROCEDURES
A. Hydrolysis of Glycoproteins
Glycoproteins 30-50 ug were hydrolyzed in 0.5 mL of 20% trifluoroacetic acid in 1.6 mL polypropylene screw-cap freeze vials at 100 degrees C for 6-7 h (1). To prevent any accidental evaporation of the sample during hydrolysis, the caps were sealed with 4-5 layers of Teflon tape. Samples were dried overnight using a vacuum centrifuge evaporator (Speed-Vac, Savant) without heat.
Glycoproteins (<5 ug) were hydrolyzed in 100 uL of 20% TFA as described above in a crimp-top-sealed 0.3 mL glass microvials and were dried in a vacuum centrifuge evaporator after removing the aluminum crimp-top seals (1).
B. Derivatization of Monosaccharides with Anthranilic Acid
Preparation of anthranilic acid reagent: A solution of 4% sodium acetate·3H2O and 2% boric acid in methanol was prepared first. Anthranilic acid (30 mg) and sodium cyanoborohydride (20 mg) were dissolved in 1.0 mL of the acetate-boric acid-methanol solution (1).
Glycoprotein hydrolysates were dissolved in 100 uL of 1% sodium acetate.3H2O and an aliquot of 50 uL was transferred to a new screw-cap freeze vial. Samples were mixed with 100 uL of the anthranilic acid and capped tightly. Vials were heated at 80 degrees C in a heating block (Pierce Reacti-Therm) for 45-60 min. After cooling to ambient temperature, the samples were made up to 1.0 mL with HPLC solvent A and mixed vigorously on a Vortex mixer in order to expel the hydrogen evolved during the reaction. Duplicate injections of 50 uL were made from each vial for analysis.
For analysis using microvials the dried hydrolysates were dissolved in 10 uL of 1% fresh sodium acetate.3H2O and mixed with 50 uL of anthranilic acid reagent in the same hydrolysis vial. The vials were recapped and heated at 80 degrees C for 45 min in a heating block. After cooling to ambient temperature the samples were made up to 250 uL with HPLC solvent A. The vials were mixed thoroughly on a Vortex mixer and 50 uL were injected from each vial for analysis.
C. HPLC Analysis of Anthranilic Acid-Monosaccharide Derivatives
Monosaccharide derivatives were separated on a Waters Symmetry C-18 reversed-phase HPLC column (3.9 x 150 mm, 5 um and 4.6 x 75 mm, 3.5 um) using 1-butylamine-phosphoric acid-tetrahydrofuran mobile phase system. The separations were carried out at 17 degrees C using a flow rate of 1 mL/min. Solvent A consisted of 0.2% 1-butylamine, 0.5% phosphoric acid, and 1% tetrahydrofuran (inhibited) in water and solvent B consisted of equal parts of solvent A and acetonitrile. Table 1 describes the gradient used for separation of the monosaccharides.
Table 1. Gradient program used for a 15 cm Symmetry column
The HPLC system consisted of an HP1100 automated binary system with a pump, vacuum degasser, temperature controlled column compartment and an autosampler. The monosaccharide derivatives were detected with a Jasco FP-920 fluorescence detector or a Waters 474 scanning fluorescence detector. The following settings were used on the fluorescence detector: excitation wavelength 360 nm; emission wavelength 425; photo multiplier gain 100 and slow response. Data was collected on Waters Expert Ease chromatographic data management system version 3.2 for Vax network for the first 15 or 25 min. Total HPLC cycle time was 30-40 min depending on the column length. Also, a Gilson 121 fluorescent detector fitted with 305-395 nm excitation and 430-470 nm emission filters was used for quantitation of the monosaccharides.
RESULTS
A. Monosaccharide Composition Analysis of a rIgG
Monosaccharide standard derivatives consisting of glucosamine, galactosamine, galactose, mannose, glucose and fucose were completely separated from excess reagent and from each other on a 15 cm column in 25 minutes using this HPLC method (Fig. 1). IgGs with low amount of carbohydrate (1.5-3% w/w) are difficult analyze for their monosaccharide composition by HPAEC-PAD method due to excessive interference by amino acids and peptides (1). Figure 1 shows a typical chromatogram of the monosaccharides obtained from a rIgG. Due to specific detection of monosaccharides in this method, the monosaccharide composition of the IgGs can be easily analyzed. The quantitative data showed that the monosaccharide composition determined by this method was in excellent agreement with the expected as determined from its oligosaccharide map. Recovery of the monosaccharides from glycoprotein hydrolysates was > 80%.
Figure 1. Typical Chromatograms Obtained with a 15 cm Waters Symmetry Column
B. Validation of the Method
B. Validation of the Method
Accuracy of the method was determined by analyzing a well characterized rIgG with and without spiked monosaccharide standards. The standards were added after the hydrolysis step. Table 2 shows the data from this study. Moles of monosaccharide per mol of rIgG were calculated from each experiment and the recovery of the spiked standards was assumed to be 100%. The data (after subtraction of the spiked amount) showed less than 2% difference between two samples (with and without spikes). Therefore, the results suggest that the method has good accuracy.
Table 2. Monosaccharide Composition of rIgG with and without Monosaccharide Spikes
Precision of the method was determined by preparing three replicates of working standards and three replicates of samples with duplicate injections from each replicate. Table 2 shows relative standard deviation (RSD) was less than 3% for a set of six analyses. Reproducibility of this method was determined by analyzing the same sample on three different occasions and the results are shown in Table 3. The method is very reproducible with an RSD of less than 2% for assay to assay and less than 3% for within assay.
Table 3. Monosaccharide Composition of rIgG from Reproducibility Study
1 %RSDs for a set of six analyses (precision)
2 Day to day variation (reproducibility)
3 Theoretical composition calculated from its known carbohydrate structures
C. Monosaccharide Analysis with 75 mm Column
Composition analysis of glycoproteins was also performed with a Waters Symmetry C-18, 4.6 x 75 mm, 3.5 um column. The following conditions were used for this column. Solvent system A and B were the same as in the case of 15 mm column. The gradient consisted of 7% B for 5 min followed by a linear increase to 20% B from 5 to 15 min. The column was washed with 100% B for 6 min and equilibrated with 7% B for 7 min. Data were collected for 16 min. Figure 2 shows a typical chromatogram of the monosaccharides obtained from a 75 mm column. An increase in peak intensity was observed due to faster elution of the peaks, which further enhanced the sensitivity of the method. Therefore the injection volume can be reduced without increase in the initial sample used for hydrolysis. In addition, the reduction in injection volume helps in reducing the peak width of the excess reagent in the chromatogram.
Figure 2. Typical Chromatograms Obtained with 7.5 cm Waters Symmetry Column and with 15 cm Column with Internal Standard Maltoheptaose
CONCLUSIONS
1. Monosaccharide derivatives were separated on a Waters Symmetry C-18 column (3.9 mm x 150 mm, 5 um) in 25 min.
2. The method was validated using a 150 mm column and found to be precise and rugged.
3. The monosaccharides could be separated in 15 min using a Waters Symmetry C-18, 4.6 x 75 mm, 3.5 um column.
4. The high sensitivity of this method (< 25 fmol per injection) allows identification of protein bands as glycoprotein on PVDF membrane. Amounts as low as 0.1 ug of fetuin could be analyzed easily.
REFERENCES
1. Anumula, K. R. (1994) Anal. Biochem. 220, 275-283.
2. Townsend, R. R. (1993) Am. Chem. Soc. Symp. Ser., 529, 86-101
CORRESPONDING EDITOR Gerald M. Carlson
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