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Ion-spray mass spectrometry for identification of the nonreducing terminal sugar of glycosaminoglycan
Introduction
Results
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
Ion-spray mass spectrometry for identification of the nonreducing terminal sugar of glycosaminoglycan
Various oligosaccharides from hyaluronic acid, which have glucuronic acid or N-acetylglucosamine at the nonreducing terminal, were prepared by digestion with a combination of testicular hyaluronidase and [beta]-glucuronidase. These oligo saccharides were analyzed by negative-mode ion-spray mass spectrometry (MS) with an atmospheric pressure ion source. Introduction of collisionally activated dissociation tandem mass spectrometry (CAD-MS/MS) produced ions derived from cleavage of the glycosidic bonds, allowing the structure to be analyzed. The CAD-MS/MS spectrum showed an intense and characteristic fragment ion at m/z 193 for oligosaccharides having glucuronic acid at the nonreducing terminal. On the other hand, this ion was not observed in the spectra of oligosaccharides having N-acetylglucosamine at the nonreducing terminal. Therefore, the fragmentation pattern revealed by CAD-MS/MS provides useful information for distinguishing glucuronic acid and N-acetylglucosamine at the nonreducing terminal of oligosaccharides derived from hyaluronic acid and other glycosaminoglycans. This ion-spray CAD-MS/MS technique was also applied successfully to the characterization of glycosaminoglycans reconstructed by glycotechnology.
Key words: glycosaminoglycan/glycotechnology/hyaluronic acid/ion-spray mass spectrometry/nonreducing terminal sugar
Introduction
It has been shown that a glycosaminoglycan (GAG) chain has many kinds of domain structures performing specific physiological functions such as anticoagulant or antithrombotic activities (Lane et al., 1984; Maimone, 1990). However, the relationship between the biological function and structure of GAGs is not yet fully understood. Therefore, it is important to develop a method for reconstructing GAG oligosaccharides to investigate their biological function and to obtain their active domains. Recently, various attempts have been made to chemically synthesize GAGs (Goto and Ogawa, 1993). On the other hand, enzymatic reconstruction of oligosaccharides has been achieved using the transglycosylation reaction of glycosidase (Abe et al., 1988; Usui et al., 1988; Bardales and Bhavanandan, 1989).
Testicular hyaluronidase is an endo-[beta]-N-acetylhexosaminidase that hydrolyzes the internal [beta]-N-acetylhexosaminidic linkage binding the d-glucuronic acid residue in hyaluronic acid and chondroitin sulfate (Meyer et al., 1960). Besides this hydrolytic reaction, it is also known that hyaluronidase catalyzes transglycosylation as a reverse reaction (Weissman, 1955; Hoffman et al., 1956; Schechter and Berger, 1966; Highsmith et al., 1975; Rodén et al., 1989). We have also succeeded in reconstructing GAG using the transglycosylation reaction of testicular hyaluronidase (Takagaki et al., 1994; Saitoh et al., 1995). However, it was not easy to analyze the reconstructed GAG structure, particularly the nonreducing terminal sugars.
In recent years, soft ionization, which allows mass analysis of natural-state oligosaccharides, which are thermolabile and poorly volatile compounds, has been developed (Busch and Cooks, 1982). Using this technology, Carr and Reinhold (1984), Reinhold et al. (1987), and Takagaki et al. (1991, 1992) have successfully analyzed oligosaccharides derived from GAG sugar chains. We therefore applied ion-spray MS to analysis of the nonreducing terminal sugars of GAG, and the results indicated that ion-spray MS is useful for this purpose.
Results
Ion-spray MS and CAD-MS/MS spectra of oligosaccharides
When the oligosaccharides derived from hyaluronic acid were measured by ion-spray MS, they showed characteristic multiply charged ions produced by proton abstraction. Each oligosaccharide had more than one characteristic ion species, and the major observed multiply charged ions and their relative intensities are shown in Table I. Next, the most intense peak was selected as the precursor ion, and then the nonreducing terminal sugar of each oligosaccharide was analyzed from its fragmentation pattern on MS/MS.Disaccharides. The CAD-MS/MS spectra of the [M-H]- ion (m/z 396) of GlcA-GlcNAc and GlcNAc-GlcA are shown in Figure
Table I.
| Molecule-related ions (m/z) | ||||
| [M-H]- | [M-2H]2- | [M-3H]3- | [M-4H]4- | |
| Oligosaccharides | ||||
| A-N | 396 (100) | |||
| A-N-A | 572 (100) | |||
| A-N-A-N | 775 (100) | 387 (38) | ||
| A-N-A-N-A | 951 (14) | 475 (100) | ||
| A-N-A-N-A-N | 1157 (70) | 577 (100) | ||
| A-N-A-N-A-N-A-N | 767 (100) | 510 (25) | ||
| N-A | 396 (100) | |||
| N-A-N | 599 (100) | |||
| N-A-N-A | 775 (100) | 387 (20) | ||
| N-A-N-A-N | 977 (75) | 488 (100) | ||
| N-A-N-A-N-A-N | 1355 (25) | 677 (100) | ||
| PA-oligosaccharides | ||||
| A-N-A-N-A-N-A-N-PA | 805 (15) | 536 (100) | 402 (75) | |
| N-A-N-A-N-A-N-A-N-PA | 907 (16) | 604 (69) | 452 (100) | |
Figure 1. CAD-MS/MS spectra of the [M-H]- ion (m/z 396) obtained from the disaccharides, GlcA-GlcNAc (A) and GlcNAc-GlcA (B). The spectra were acquired after injection of the samples dissolved in a mobile phase of 0.5 mM ammonium acetate/acetonitrile (50:50 by volume). Figure 3. CAD-MS/MS spectra of the [M-H]- ion (m/z 775 and 467) obtained from the tetrasaccharides, GlcA-GlcNAc-GlcA-GlcNAc (A), GlcNAc-GlcA-GlcNAc-GlcA (B) and GlcA-GulNAc(6-O-sulfate)-GlcA-GalNAc(6-O-sulfate) (C). Table II. 
Figure 2. CAD-MS/MS spectra of the [M-H]- ion (m/z 572 and 599) obtained from the trisaccharides, GlcA-GlcNAc-GlcA (A) and GlcNAc-GlcA-GlcNAc (B).
Oligosaccharides
Fragment ions (m/z)
A-N
193
A-N-A
175
193
396
A-N-A-N
175
193
396
572
A-N-A-N-A
175
193
396
572
775
A-N-A-N-A-N
175
193
396
572
775
951
A-N-A-N-A-N-A-N
175
193
396
572
775
951
1154
N-A
175
N-A-N
175
396
N-A-N-A
175
396
599
N-A-N-A-N
175
396
599
775
N-A-N-A-N-A-N
175
396
599
775
977
Application of CAD-MS/MS to identification of the nonreducing terminal sugar of a newly reconstructed oligosaccharide
CAD-MS/MS was applied for identifying the nonreducing terminal sugars of newly reconstructed oligosaccharides, which were synthesized using the transglycosylation reaction of testicular hyaluronidase. PA-hexasaccharide, whose reducing terminal had been labeled with a fluorescent reagent, PA, as an acceptor, and PA-unlabeled hexasaccharide having glucuronic acid at the nonreducing terminal as a donor, were incubated with hyaluronidase under conditions optimal for the transglycosylation reaction at 37°C for 1 h. The reaction products were analyzed by tracing the fluorescence of the PA by HPLC. Two peaks of newly reconstructed products were observed, which were PA-oligosaccharides elongated by the addition of disaccharide units to the acceptor, PA-hexasaccharide (Figure
Figure 4. HPLC chromatograms of the transglycosylation reaction products. (A) PA-hexasaccharide. (B) PA- hexasaccharide and PA-unlabeled hexasaccharide, which were used as an acceptor and a donor, respectively, were incubated with hyaluronidase. (C) PA-hexasaccharide and PA-unlabeled heptasaccharide were also incubated with hyaluronidase. The reaction mixtures were then subjected to HPLC (PALPAK Type S). The conditions for HPLC are described in Materials and methods. Arrows indicate the elution positions of PA-oligosaccharide standards: 1, hexasaccharide; 2, octasaccharide; 3, decasaccharide. Peaks I, II, and III were pooled, respectively. Next, PA-unlabeled heptasaccharide having N-acetylglucosamine at the nonreducing terminal and PA-hexasaccharide, as a donor and an acceptor, respectively, were incubated with hyaluronidase under optimal conditions and analyzed by HPLC. One peak was found to appear more prominently at the midpoint between the standard PA-octasaccharide and the PA-decasaccharide (Figure Figure 6. CAD-MS/MS spectrum of the [M-4H]4- ion (m/z 452) obtained from the transglycosylation products. PA-hexasaccharide as an acceptor and PA-unlabeled heptasaccharide as a donor were incubated with testicular hyaluronidase. A reaction product considered to be PA-nonasaccharide (fraction II in Figure 4C) was recovered, and then subjected to ion-spray CAD-MS/MS.
Figure 5. CAD-MS/MS spectrum of the [M-3H]3- ion (m/z 536) obtained from the transglycosylation reaction products. PA-hexasaccharide as an acceptor and PA-unlabeled hexasaccharide as a donor were incubated with testicular hyaluronidase under conditions (pH 7.0) optimal for the transglycosylation reaction for 1 h. A reaction product considered to be PA-octasaccharide (fraction I in Figure 4B) was recovered and then subjected to ion-spray CAD-MS/MS.
Discussion
Although there are some existing experimental methods for identification of the reducing terminal sugars of GAGs (Eisenberg, 1974; Ögren and Lindahl, 1975; Matsue and Endo, 1987), it is difficult to identify and distinguish them. From the present results, an important aspect of the fragmentation pattern on CAD-MS/MS was clarified. In the case of oligosaccharides having glucuronic acid at the nonreducing terminal, the CAD-MS/MS spectrum showed an intense peak at m/z 193 corresponding to glucuronic acid at the nonreducing terminal, which was derived by cleavage of the nonreducing site of the O-glycosidic bond (Figure
Figure 7. Fragmentation of the glucuronic acid at the nonreducing terminal. Recently, more attention has been directed toward remodeling of carbohydrate chains using glycosidases using glycotechnology (Abe et al., 1988; Usui et al., 1988; Bardales and Bhavanandan, 1989), and we have also succeeded in reconstructing glycosaminoglycan (GAG) using the transglycosylation reaction of testicular hyaluronidase (Takagaki et al., 1994; Saitoh et al., 1995). Testicular hyaluronidase is an endo-[beta]-N-acetylhexosaminidase that hydrolyzes the internal [beta]-N-acetylhexosaminidic linkage binding the d-glucuronic acid residue in hyaluronic acid and chondroitin sulfate (Meyer et al., 1960). Besides this hydrolytic reaction, it is also known that hyaluronidase catalyzes transglycosylation as a reverse reaction (Weissman, 1955; Hoffman et al., 1956; Schechter and Berger, 1966; Highsmith et al., 1975; Rodén et al., 1989; Takagaki et al., 1994). The mechanism of action of this enzyme in GAG reconstruction has been revealed to be as follows: di- or trisaccharides, GlcA[beta]1->3GlcNAc or GlcNAc[beta]1->4GlcA[beta]1->3GlcNAc, are released from the nonreducing terminal, and then the oligosaccharides are rapidly transferred to the glucuronic acid residue at the nonreducing terminal of another oligosaccharide chain as an acceptor. The newly reconstructed oligosaccharides as a reaction product have a glucuronic acid or N-acetylglucosamine residue at the nonreducing terminal. It is important to identify the nonreducing terminal sugar of the reconstructed GAG. The method described in this article can be used for purified samples. Therefore, after purification of the reconstructed oligosaccharide by HPLC, the nonreducing terminal sugar can be identified. As currently there is no simple method for identification of the nonreducing terminal sugar of GAGs, the present method is expected to be useful for detailed structural analysis of GAGs that have been reconstructed by glycotechnology.
Materials and methods
Materials
Hyaluronic acid was prepared from umbilical cord and further purified by Dowex 1-X2 chromatography as described previously (Nakamura et al., 1990). Chondroitin 6-sulfate (from shark cartilage) was purchased from Seikagaku Kogyo Co. (Tokyo, Japan). Bovine testicular hyaluronidase (type-S) and [beta]-glucuronidase (from Escherichia coli) were purchased from Sigma Chemical Co. (St. Louis, MO), and bovine testicular hyaluronidase was purified according to the method of Borders and Raftery, 1968. Bio-Gel P-4 was obtained from Bio-Rad (Richmond, CA). 2-Aminopyridine (PA) was purchased from Wako Pure Chemical Co. (Osaka, Japan) and recrystallized from hexane. Other reagents and chemicals were obtained from commercial sources.
Preparation of oligosaccharides
Hyaluronic acid and chondroitin 6-sulfate were partially hydrolyzed by testicular hyaluronidase using the procedure described in a previous report (Takagaki et al., 1992), and then oligosaccharides (GlcA-GlcNAc, GlcA-GlcNAc-GlcA-GlcNAc, GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc, GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc and GlcA-GalNAc(6-O-sulfate)- GlcA-GalNAc(6-O-sulfate)) having glucuronic acid at the nonreducing terminal were purified through a Bio-Gel P-4 column (400 mesh). Oligosaccharides (GlcA-GlcNAc-GlcA and GlcA- GlcNAc-GlcA-GlcNAc-GlcA) having glucuronic acid at both the reducing and nonreducing terminals were further purified with a Bio-Gel P-4 column after [beta]-elimination of tetra- and hexasaccharides in a saturated solution with Ca(OH)2 at 25°C for 4 h. Oligosaccharides (GlcNAc-GlcA and GlcNAc-GlcA- GlcNAc-GlcA) having N-acetylglucosamine at the nonreducing terminal and glucuronic acid at the reducing terminal were further purified with a Bio-Gel P-4 column after [beta]-glucuronidase digestion (Himeno et al., 1974) and [beta]-elimination of tetra- and hexasaccharides.
Mass spectrum measurements
MS and MS/MS spectra were obtained on a Sciex API-III triple-quadrupole mass spectrometer (Thornhill, Ontario, Canada) equipped with an atmospheric pressure ionization source. The mass spectrometer was operated in the negative mode; the ion-spray voltage was set at -4,000 V, the interface plate voltage was -600 V, and the orifice voltage was -100 V. The samples were introduced in 0.5 mM ammonium acetate/acetonitrile (50:50 by volume). A JASCO Familic 100N micro HPLC syringe pump (Harvard Apparatus Inc., MA) was used to deliver the samples at a flow rate of 2 µl/min. Scanning was done from m/z 300 to 1200 during the 1-min scan (six cycles). The collisionally activated dissociation (CAD) spectrum was measured with argon as the collision gas, and the collision energy was 40 eV.
Fluorescence-labeled hexasaccharide
The reducing terminal of the purified hexasaccharide (GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc) was labeled with a fluorescent reagent, using the procedure described in a previous report (Takagaki et al., 1990, 1994; Kon et al., 1991). Briefly, hexasaccharide (1 mg) was dissolved in 100 µl of PA solution (prepared by mixing 1.0 g of PA, 0.76 ml of concentrated HCl, and 2.2 ml of water, giving a final pH of 6.2) and reacted at 100°C for 13 min. Next, the hexasaccharide was reacted with 6 µl of a reducing reagent, which was prepared by dissolving 10 mg of sodium cyanoborohydride in 15 µl of PA solution and 20 µl of water immediately before use, at 90°C for 15 h for reductive amination. Pyridylaminated hexasaccharide (PA-hexasaccharide) was separated by removal of excess reagents with a Sephadex G-15 column, and then used as an acceptor in the transglycosylation reaction.
Conditions for the transglycosylation reaction by testicular hyaluronidase
A typical transglycosylation reaction was carried out as follows (Takagaki et al., 1994). Twenty nanomoles of hyaluronic acid oligosaccharides as donors, 2 nmol of PA-hexasaccharide as an acceptor, and 1.0 NFU of testicular hyaluronidase dissolved in 50 µl of 0.15 M Tris-HCl buffer, pH 7.0, were incubated at 37°C for 1 h. The reaction was terminated by immersion in a boiling water bath at 100°C for 3 min.
HPLC analysis
HPLC analysis was performed using a Hitachi L-6200 equipped with a fluorescence detector (F-1050, Hitachi Co., Tokyo, Japan). The reaction products were eluted through a PALPAK Type S column (4.6 mm × 250 mm) under the following conditions: solution A containing 3% acetic acid adjusted to pH 7.3 with triethylamine and acetonitrile at a ratio of 35:65, and solution B containing the same agents at a ratio of 50:50 were prepared; the column was equilibrated with solution A, and the ratio of solution B to solution A was increased linearly to 100% over 50 min after sample injection; the flow rate was fixed at 1.0 ml/min; the column temperature was 40°C; PA was detected at excitation and emission wavelengths of 320 nm and 400 nm, respectively.
Acknowledgments
We are grateful to Mr. Kaoru Kojima for technical assistance. This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan (Nos. 03304050, 04454153, 05680518, 08457032, 09240202, and 09358013).
Abbreviations
MS, mass spectrometry; CAD, collisionally activated dissociation; MS/MS, tandem mass spectrometry; GAG, glycosaminoglycan; HA, hyaluronic acid; PA, 2-aminopyridine; GlcA, glucuronic acid; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine.
References
2To whom correspondence should be addressed
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