Glycobiology, 2001, Vol. 11, No. 12 1025-1033
© 2001 Oxford University Press
Novel methods for the preparation and characterization of hyaluronan oligosaccharides of defined length
2MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom; 3Oxford Centre for Molecular Sciences, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom; and 4Department of Biomedical Engineering/ND20, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195
Received on May 31, 2001; revised on August 31, 2001; accepted on August 31, 2001.
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Abstract |
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Hyaluronan is a ubiquitous glycosaminoglycan of high molecular weight that acts as a structural component of extracellular matrices and mediates cell adhesion. There have been numerous recent reports that fragments of hyaluronan have different properties compared to the intact molecule. Though many of these results may be genuine, it is possible that some activities are due to minor components in the preparations used. Therefore, it is important that well-characterized and highly purified oligosaccharides are used in cell biological and structural studies so that erroneous results are avoided. We present methods for the purification of hyaluronan oligomers of defined size using size exclusion and anion-exchange chromatography following digestion of hyaluronan with testicular hyaluronidase. These preparations were characterized by a combination of electrospray ionization mass spectrometry, matrix-assisted laser desorption/ionization mass spectrometry with time-of-flight analysis, and fluorophore-assisted carbohydrate electrophoresis. Hyaluronan oligomers ranging from tetrasaccharides to 34-mers were separated. The 4- to 16-mers were shown to be homogeneous with regard to length but did contain varying amounts of chondroitin sulfate. This contaminant could have been minimized if digestion had been performed with medical-grade hyaluronan rather than the relatively impure starting material used here. The 18- to 34-mer preparations were mixtures of oligosaccharides of different lengths (e.g., the latter contained 87% 34-mer, 10% 32-mer, and 3% 30-mer) but were free of detectable chondroitin sulfate. In addition to oligomers with even numbers of sugar rings, novel 5- and 7-mers with terminal glucuronic acid residues were identified.
Key words: electrospray ionization mass spectrometry/fluorophore-assisted carbohydrate electrophoresis/hyaluronan/hyaluronidase/matrix-assisted laser desorption/ionization mass spectrometry.
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Introduction |
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Hyaluronan (HA) is a large, nonsulfated glycosaminoglycan that has important structural and biological roles within the extracellular matrices surrounding animal cells (Camenisch and McDonald, 2000
; Toole, 2000). It is composed of a repeating disaccharide subunit of D-glucuronic acid (ß1
3) and N-acetyl-D-glucosamine (ß14) (Weissman and Meyer, 1954). There are an increasing number of HA-binding proteins that exhibit significant differences in their specificity, affinity and regulation (Day, 1999, 2001). HA oligosaccharides of defined length have been used extensively to characterize these protein–HA interactions (Hardingham and Muir, 1973; Hascall and Heinegård, 1974; Bertrand and Delpech, 1985; Tammi et al., 1998; Kahmann et al., 2000; Lesley et al., 2000; Mahoney et al., 2001). In addition, low-molecular-weight fragments of HA (including short oligomers) have been reported to have biological activities not associated with the parent molecule. For example, such HA preparations have been shown to promote angiogenesis (West et al., 1985; Rahmanian et al., 1997), induce expression of inflammatory mediators in alveolar macrophages (McKee et al., 1996; Noble et al., 1996; Horton et al., 1999) and inhibit tumour growth in vivo (Zeng et al., 1998) as well as having many other effects (Oertli et al., 1998; Fitzgerald et al., 2000; Ohkawara et al., 2000).
Though the above activities may all be due to HA, it is clearly essential that well-characterized and highly purified oligosaccharide preparations of defined size are used in both cell biological and structural studies so that erroneous results are avoided (Camenisch and McDonald, 2000). For instance, the pro-inflammatory effects of "low-molecular-weight HA" on interleukin-12 and tumor necrosis factor upregulation in monocytes, in fact, results from contaminating DNA (Filion and Phillips, 2001). Furthermore, production of HA oligomers free from chondroitin sulfate has been shown recently to be of importance in establishing the role of HA in the accumulation of malaria parasite–infected erythrocytes in placenta (Beeson et al., 2000; Chai et al., 2001).
Various methods for the production of HA oligomers have been described. Most of these involve the digestion of polymeric HA with the endohydrolase, testicular hyaluronidase, followed by purification by size exclusion chromatrography (Hardingham and Muir, 1973; Hascall and Heinegård, 1974; Tammi et al., 1998; Lesley et al., 2000) and/or ion exchange chromatography (Holmbeck and Lerner, 1993; Toffanin et al., 1993; Almond et al., 1998; Tammi et al., 1998; Chai et al., 2001), or by reverse-phase ion-pair high-performance liquid chromatography (HPLC) (Cramer and Bailey, 1991). These methods result in even-numbered oligosaccharides for which the minimal size is HA4 (i.e., 2 disaccharide units in length). The use of hyaluronate lyase (Price et al., 1997; Chai et al., 2001) and sonication (Kubo et al., 1993) have also been described; digestion with the former generates a modified unsaturated uronic acid moiety on the nonreducing termini and, therefore, is best avoided.
A range of techniques has been employed to assess the sizes of the oligomers produced. These were originally determined from the ratio of total glucuronic acid (GlcUA) concentration to that of the reducing end N-acetyl glucosamime (GlcNAc) group (Hardingham and Muir, 1973; Cramer and Bailey, 1991) or the nonreducing terminal GlcUA (Hascall and Heinegård, 1974). However, other methods, including capillary electrophoresis (Grimshaw, 1997; Hong et al., 1998), high pH anion-exchange chromatography (Lauder et al., 2000), mass spectrometry (Price et al., 1997; Schiller et al., 1999; Yeung and Marecak, 1999; Chai et al., 2001) and nuclear magnetic resonance spectroscopy (Chai et al., 2001), have more recently been employed. Many of the methods used have provided little information on the purity of the oligomer preparations and have limited capabilities to determine the molecular masses of longer oligosaccharides (i.e., greater than 
HA16). In addition, apart from notable exceptions (Holmbeck and Lerner, 1993; Lesley et al., 2000), there have been few attempts to purify oligomers of greater than about HA20.
Here we describe the purification of HA oligosaccharides ranging from HA4 to HA34, including novel 5-mer and 7-mer species with terminal GlcUA groups, using size exclusion chromatography and ion-exchange HPLC. Oligomers were characterized using a combination of electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and fluorophore-assisted carbohydrate electrophoresis (FACE).
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Purification of low-molecular-weight HA oligomers
Polymeric HA was incubated with testicular hyaluronidase for a range of times, and the resulting digests were analyzed by anion-exchange chromatography. As can be seen from Figure 1, a series of well-separated and symmetrical peaks were eluted from the column. These were analyzed by MS (as described in detail later) and found to correspond to HA oligosaccharides of increasing length and thus of increasing negative charge. A digest of 5 min (Figure 1A) resulted in HA oligomers ranging from hexasaccharides (HA6) to greater than 30-mers. As the time of digest was increased, the average size of the oligosaccharides decreases, with a larger proportion of low molecular weight species formed. A digestion time of 1 h (Figure 1C) was selected, on the basis of relative peak areas, as optimal for the large-scale preparation of HA4 to HA16 with a significant proportion of these being HA6, HA8 and HA10.
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Size exclusion chromatography on Bio-Gel P-6 was used as the initial step in the large-scale purification of HA4 to HA10. Figure 2 shows a typical chromatogram for 100 mg of HA digest on a column with a total elution volume of 177 ml, where the fractions obtained were analyzed using anion-exchange HPLC. For example, fraction 8 was shown to contain significant amounts of oligomers HA14 to HA18, whereas fraction 27 is predominantly HA8. To select for oligomers of low molecular weight (i.e., HA6 to HA10) fractions 21 to 31 were pooled (pool A) and further purified by anion-exchange chromatography (see Figure 3). The peaks were analyzed by ESI-MS as described below. In addition to even-numbered oligosaccharides (i.e., HA4, HA6, HA8, and HA10), odd-numbered oligomers, HA5 and HA7, were obtained (see below). Milligram quantities of even-numbered oligosaccharides were purified (e.g., 25 mg of HA8 resulted from the digestion of 300 mg HA, corresponding to 8% of the starting material), whereas the yield for odd numbered oligosaccharides was considerably lower (
0.2% for HA5 and HA7). Oligomers of HA12 to HA24 were produced from a 1 h digest and then purified in a single step by anion-exchange chromatography with the P1 gradient (not shown).
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Analysis of HA oligomers by ESI-MS
ESI-MS was used in the negative ion mode to analyze oligomers purified by anion-exchange chromatography. As shown in Figure 4, for each oligomer a series of negatively charged species of different m/z (mass/charge) ratio are seen. The theoretical ion series arising from HA4 to HA14, as well as the average molecular masses, are shown in Table I. As described previously, a particular compound does not exhibit all the possible charge states; the smaller HA oligomers exist mainly as the mono- or di-anion, whereas the 12- to 14-mers exist predominantly as the hexa- and penta-anions (Price et al., 1997
; Chai et al., 2001). This is illustrated by the ESI-MS spectrum of HA12 as shown in Figure 4. At a cone voltage of –10 V four species are seen (A series: A3 to A6), corresponding to the [M-3H]3–, [M-4H]4–, [M-5H]5– and [M-6H]6– anions, respectively. The absence of other species at this low voltage indicates a high degree of sample purity; for instance if HA14 was present this would generate a [M-nH]n– series, giving rise to m/z signals of 444.54 [M-6H]6–, 533.65 [M-5H]5–, and so on, which are not seen. As the cone voltage is increased a proportion of the oligosaccharide is fragmented resulting in an additional series of negatively charged species. For example, HA12 at a cone voltage of –20 V gives ions (B series in Figure 4) corresponding to HA11 (with a theorectical mass of 2089.73 Da; see Table I) which arises from the loss of a GlcNAc residue from the reducing end of the 12-mer. Similarly, at a cone voltage of –30 V three ion series, corresponding to HA12 (A series), HA11 (B series), and HA10 (C series) are seen.
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Fragmentation clearly complicates the assignment but does provide "sequence" information. Such information has allowed us to confirm the identity of the odd numbered oligosaccharides, HA5 and HA7, purified on ion exchange chromatography (Figure 3). These species have molecular masses (952.8 ± 1.0 and 1332.5 ± 0.8 Da, respectively) that are consistent with saturated 5- and 7-mers having nonreducing and reducing terminal GlcUA residues (see Table I). HA5 and HA7 fragment to produce ions corresponding to HA4 and HA6, respectively, at high cone voltages (data not shown). On ion exchange purification the HA5 and HA7 species eluted after HA6 and HA8, respectively, which is not surprising given their greater charge to mass ratios.
Purification of high-molecular-weight HA oligomers
Polymeric HA was digested for 5 min to generate longer oligosaccharides (see Figure 1). These were fractionated by gel filtration on a Waters Protein PAK-125 column (data not shown), and the eluent from 12 to 20 min was collected and pooled (pool B). Oligosaccharides in pool B were subsequently purified by anion-exchange HPLC using gradient P2 (see Figure 5); this gradient was adapted from that used previously (i.e., P1) to give greater resolution of high-molecular-weight oligosaccharides. As can be seen from Figure 5 a wide range of oligomer lengths can be separated (from HA8, or less, to greater than HA34). However, the yield of each individual species is relatively low (1–2% of starting material for HA12 to HA24 and 0.5% of starting material for HA34).
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Oligosaccharides up to the length of HA24 were unambiguously identified by ESI-MS, with experimental and theoretical molecular masses in good agreement (data not shown). However, the molecular masses and purities of longer oligosaccharides were difficult to determine by this method due to the increased occurrence of fragmentation even at low cone voltages. In addition, as the length of oligomer increases, the mass differences between species of similar size and charge becomes smaller, thus making assignment difficult. Consequently, MALDI-TOF MS, which does not result in sample fragmentation or the generation of multiple ion species, was investigated.
Analysis of HA oligomers by MALDI-TOF MS
We evaluated several matrices for the analysis of HA oligosaccharides by MALDI-TOF MS. These included 2,5-dihydroxybenzoic acid, which has been used recently for the analysis of HA oligomers (Schiller et al., 1999; Yeung and Marecak, 1999), but this matrix did not generate good signals. However, high-quality spectra were seen with a co-matrix of 2,4,6-trihydroxyacetophenone with triammonium citrate (Jovanovic et al., 2000). In the positive ion mass spectra, the major species were generally a mixture of metal ion adducts, such as [M+Na]+ and [M+K]+, whereas [M-H]– was the predominant species in the negative ion mode. Consequently, negative ion spectra were recorded in most cases, because they were easier to interpret, particularly for complex samples. Figure 6 shows a typical negative ion spectrum, which was obtained for peak 16 purified from pool B by anion-exchange HPLC (see Figure 5). Species consistent with the masses for the monovalent anions of HA30, HA32, and HA34 were observed. Additional peaks were also seen at +62 Da for HA32 and HA34, and these probably correspond to copper ion adducts, which are likely to be artifacts of the target used. Table II summarizes the results obtained for other peaks from anion-exchange chromatography (Figure 5) analyzed by MALDI-TOF MS. As shown in the table, the experimental masses obtained are in reasonable agreement with the theoretical molecular weights; any slight differences are believed to arise from the method of calibration. The results presented here demonstrate that MALDI-TOF MS is suitable for the analysis of a wide range of HA oligosaccharide sizes. In addition, MS analyses indicate that the HA oligomers produced here are of high purity. This was further investigated by FACE analysis.
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Analysis of HA oligomers by FACE
Aliquots of each of the purified HA preparations and a series of standards were fluorotagged prior to analysis by electrophoresis as described in Materials and methods. The position of each band in relation to the derivatized standards can be used to identify the HA oligosaccharide(s) present (Calabro et al., 2000b
). Figure 7 shows a FACE gel of HA oligomers, identified as HA4, HA6, HA8, HA10, HA12, HA14, and HA16 by ESI-MS (see Analysis of HA oligomers by ESI-MS). As can be seen in Figure 7A (29 ms exposure), all preparations contained a single major band corresponding to the expected oligomer length. When gels were overexposed (600 ms), visualisation of minor components is apparent (Figure 7B). The mobilities of these species suggested that they are oligosaccharides of chondroitin sulfate (Calabro et al., 2000a). This was verified by further digestion of portions of each sample with both chondroitinase ABC and Streptococcal hyaluronidase followed by analyses with FACE. In each case characteristic 
Di disaccharides from chondroitin sulfate were observed in the expected amounts relative to the DiHA disaccharides derived from the dominant HA oligomer (data not shown). Integrated optical density (IOD) intensities of fluorescence for the properly exposed gel (Figure 7A) indicate that these contaminants are present at between 1% (for HA6) and 15% (for HA16) of the total oligosaccharide (data not shown). This analysis also shows that these HA oligomer preparations are homogeneous with regard to length with undetectable or very small quantities of longer or shorter HA oligosaccharides present in a particular sample.
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FACE analysis was also done on peaks 11 to 18 (purified by the P2 gradient as described above). In Figure 8 the sizes observed by FACE were indexed on the basis of HA22 that was purified using the P1 gradient and characterized by ESI-MS; for peaks 11 and 16 the sizes are in agreement with the values obtained with MALDI-TOF MS (see Table II). All of these samples contained mixtures of HA oligosaccharides, and their relative amounts can be quantified from the IOD values. For example, peak 11 contains mostly HA24 (61%), but HA22 (13%), HA20 (16%), HA18 (7%), and HA16 (3%) are also present, whereas peak 16 is comprised of HA34 (87%), HA32 (10%), and HA30 (3%). Digestion of peaks 11 to 16 (and HA22) with chondroitinase ABC and Streptococcal hyaluronidase followed by FACE analysis indicated that there were no detectable
Di chondroitin sulfate disaccharides in any of these preparations (data not shown). The level of sensitivity of the analyses was such that a contaminant of chondroitin sulfate at the 1% level would have been readily detected.
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Discussion |
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We have described the purification of HA oligosaccharides of between HA4 and HA34 in length by digestion with testicular hyaluronidase, and their characterization by ESI-MS, MALDI-TOF MS, and FACE analyses. ESI-MS (in the negative ion mode) has been used previously for the analysis of HA oligomers produced with hyaluronate lyase (Price et al., 1997
; Chai et al., 2001) and testicular hyaluronidase (Chai et al., 2001). However, Price et al. (1997) employed a relatively high cone voltage (–25 V) that may explain why the longest oligomer analyzed was a 16-mer; in the Chai et al. (2001) study (–20 V cone voltage) only data for the 12-mers were reported. Here we have shown that lower cone voltages (i.e., –10 V) lead to less fragmentation, thus allowing the unambiguous identification of longer HA oligosaccharides (i.e., up to HA24). However, the fragmentation of longer oligomers (even at low cone voltages), and the similarities of the observed molecular weights for highly charged species, makes this technique unsuitable for oligosaccharides greater than about HA24.
For this reason we investigated MALDI-TOF MS for the analysis of higher-molecular-weight sugars. The use of this technique has been reported recently for HA4 to HA8 (Schiller et al., 1999) and for polydispersed mixtures of high molecular weight HA oligomers (Yeung and Marecak, 1999), both of which were performed with matrices based on 2,5-dihydroxybenzoic acid. In our study this matrix gave poor results. However, good spectra were achieved with a co-matrix of 2,4,6-trihydroxyacetophenone and triammonium citrate for a wide range of oligomer sizes (i.e., HA8 to HA34). This co-matrix has been used previously for acidic oligosaccharides (Jovanovic et al., 2000). It is expected that HA oligomers of considerably higher molecular weight could be analyzed in this way as we see excellent signal to noise for the largest HA species (34-mers) made here (as shown in Figure 6).
FACE has recently been developed for the characterization of HA and other glycosaminoglycans (Calabro et al., 2000a,b). This technique allows the sizing of HA oligomers from tetrasaccharides to greater than 50-mers. For low-molecular-weight HA species (HA4 to HA20) the size can be determined relative to sugar standards (Figure 7). Longer HA molecules can be identified by indexing the gel with a HA oligomer of known size. In our case, HA22, characterized by ESI-MS, was used (see Figure 8). In general, MALDI-TOF MS, which can size longer oligomers than ESI-MS, would be an excellent way to calibrate HA standards for use in FACE analysis. Although, the results from MS and FACE analysis were in good general agreement, the latter method has the advantage of being able to quantitate the levels of minor species present. The HA6 preparation, for example, was found to contain 1% of chondroitin sulfate oligosaccharides (see Figure 7). Peak 16, on the other hand, though it contained no detectable chondroitin sulfate, was shown to be a mixture of HA34 (87%), HA32 (10%), and HA30 (3%). These species were also seen in MALDI-TOF MS, but this technique gave different values for their relative amounts (50%, 42% and 8%, respectively; Table II). It seems likely that this discrepancy results from an over estimation by MALDI-TOF MS of shorter oligomers (i.e., less charged species), due to their preferential desorption from the matrix.
The purification methods employed here allowed the production of a wide range of HA oligosaccharide sizes (HA4 to HA34). The low-molecular-weight oligomers (HA4 to HA16 purified with the P1 gradient) were homogenous with regard to length. These included saturated 5- and 7-mers, with terminal GlcUA residues, that to our knowledge have not been reported previously; the HA5 and HA7 species produced by digestion of HA with hyaluronate lyase (Price et al., 1997) also had terminal GlcUA residues, but these species were 18 Da smaller than those we have isolated, indicating that they contained an unsaturated sugar at their nonreducing end. It seems likely that the 5-mer and 7-mer we have identified result from a contaminating hydrolase enzyme in the ovine testicular hyaluronidase preparation used here.
There have been few previous attempts to purify and characterize HA oligosaccharides larger than 20-mers (Holmbeck and Lerner, 1993; Lesley et al., 2000). Here we have produced oligomers up to HA34 and the methodology used, we believe, could be extended (by adjusting digestion times and enzyme to substrate ratios) to prepare still longer molecules. As described above, peaks 7 to 16 were mixtures of HA of different lengths (see Figure 5) where the number of species present in a given sample were similar to those reported previously by Lesley et al. (2000); higher purity might be achieved by re-running the samples on the ion exchange column. The high-molecular-weight oligomers made here are proving useful in the investigation of multimeric protein-HA interactions (Mahoney and Day, unpublished data).
The high-molecular-weight HA preparations (HA22 to HA34) showed no detectable chondroitin sulfate (by FACE analysis). The low-molecular-weight HA oligosaccharides, however, contained significant amounts of this glycosaminoglycan. For instance, there was between 1% and 5% chondroitin sulfate in HA4 to HA10, whereas HA12, HA14, and HA16 contained 7%, 12%, and 15%, respectively. Oligomers of chondroitin sulfate were not identified in any of these samples by MS. This is perhaps not surprising given their low levels, as determined by FACE, with less than 10% in HA4-HA12, and the fact that such oligosaccharides were present as two species of similar intensity in HA14 and HA16 (see Figure 7). In addition, ESI-MS on 6- and 8-mers of chondroitin-4-sulfate (provided by T. E. Hardingham), performed in the negative ion mode under identical conditions to that reported here for HA, indicates that there is significant but partial desulfation of these oligomers even at low cone voltages (data not shown). Therefore, individual chondroitin sulfate species would be difficult to detect by ESI-MS at the levels seen here.
With hindsight chondroitin sulfate contaminants could have been avoided by the use of medical-grade or bacterial HA, which contain very low levels of chondroitin sulfate, rather than the relatively impure preparation from human umbilical cord. In this regard, suitable starting materials could be identified by FACE analysis of chondroitinase digests. In addition, HA preparations should be treated with deoxyribonuclease 1 to remove DNA contaminants (Filion and Phillips, 2001).
Clearly, it is of great importance to detect and quantify such contaminants because they could lead to erroneous results in biological assays (Camenisch and McDonald, 2000). The methodologies described here for the characterization of HA oligosaccharides by MS and FACE can be used to ensure the purity of these important reagents.
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Materials and methods |
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Human umbilical cord HA, reagents for FACE analysis, and all other laboratory reagents unless specified otherwise, were obtained from Aldrich-Sigma. Ammonium hydrogen carbonate was purchased from FISONS, and ammonium acetate was from BDH. Ovine testicular hyaluronidase was supplied by Calbiochem. Dialysis membrane (500 Da cut-off) was obtained from Perbio Science. Bio-Gel P-6 resin (fine) was supplied by Bio-Rad. 2-Aminoacridone-HCl (AMAC) was purchased from Molecular Probes. Phenol red (0.5% w/v) was from Gibco. MONO composition gels (60100) and MONO gel running buffer (70100) were purchased from Glyko. Chondroitinase ABC lyase, Streptococcal hyaluronidase, unsaturated HA, and chondroitin sulfate disaccharide standards were obtained from Seikagaku America.
Digestion of HA
HA (100 mg) was dissolved at a concentration of 3.33 mg/ml (30 ml total) in digest buffer (0.15 M NaCl, 0.1 M Na-acetate, adjusted to pH 5.2 with glacial acetic acid) and incubated at 37°C for 30 min. To this, ovine testicular hyaluronidase (0.22 mg; 11,000 U in 10 ml digest buffer) was added, and the solution incubated at 37°C. The digest was assessed at various time points by running 100 µl of the reaction mixture (diluted to 3.5 ml with H2O) on a 75 x 7.5 mm Waters PAK DEAE-5PW anion-exchange column, equilibrated in 5.0 mM ammonium hydrogen carbonate (buffer A) at a flow rate of 1 ml/min. The initial conditions were maintained for 10 min, and HA oligosaccharides were eluted with gradients of 0–50% and 50–95% of buffer B (500 mM ammonium hydrogen carbonate) in buffer A over 15 and 5 min, respectively. Final conditions were maintained for 5 min prior to re-equilibration into buffer A. The eluent was monitored at 210 nm. When the reaction reached the desired point it was stopped by boiling for 5 min, dialyzed against 3 x 10 L H2O for 6 h each at 4°C, lyophilized, and stored at –20°C.
Size exclusion chromatography
The lyophilized reaction mixture from a 1 h digest was resuspended at 100 mg/ml in 0.2 M ammonium acetate and centrifuged (12,000 x g, 2 min) to remove any particulate material. Oligomers were then fractionated by size exclusion chromatography on a 36 x 2.5 cm Bio-Gel P-6 column equilibrated in 0.2 M ammonium acetate at a flow rate of 0.2 ml/min. The eluent was constantly monitored at 214 nm, and 1-ml fractions were collected from 25 to 80 min.
The 5-min digests were resuspended in 20 mM Na-acetate and 100 mM NaCl, purified on a 30 x 0.8 cm Protein PAK-125 column (Waters) equilibrated in the resuspension buffer, and run at 1.0 ml/min. The eluent (monitored at 210 nm) from 12 to 20 min was collected.
Size exclusion fractions were analyzed by running 100 µl on the anion-exchange column, as described in Digestion of HA except that oligomers were eluted with gradients of 0–50% and 50–95% buffer B in A over 40 and 5 min, respectively (denoted here as gradient P1). Fractions containing the desired HA oligomers (as determined by MS) were then pooled, dialyzed extensively against H2O, lyophilized, and resuspended (at 2 mg/ml) in 5 mM ammonium hydrogen carbonate.
Preparative anion-exchange HPLC
Pools resulting from the Bio-Gel P-6 and Protein PAK-125 columns were then further purified by anion-exchange chromatography. For material resulting from the former column the P1 gradient was used, whereas in the latter case, a different elution gradient (P2: 0–35%, 35–80%, and 80–95% buffer B in A over 5, 45, and 5 min, respectively) was employed. Peaks were collected manually, pooled according to their elution position, and lyophilized. These samples were then resuspended in water and relyophilized twice to remove the buffer. The uronic acid content for each oligosaccharide was then determined by the metahydroxybiphenyl reaction (Blumenkrantz and Asboe-Hansen, 1973) as described previously (Kahmann et al., 2000).
ESI-MS
HA oligosaccharides were analyzed by ESI-MS on a BIO-QII-ZS spectrometer (Micromass UK) run in the negative ion mode. The instrument was calibrated with bovine ubiquitin (12 pmol/µl; average molecular mass 8564.85 Da) and scanned over the mass range 200 to 900 Da. A typical sample had a concentration of about 5 pmol/µl and was run in 50% (v/v) acetonitrile and 0.5 mM ammonium acetate. A cone voltage of between –10 V and –35 V was used (depending on the length of the oligosaccharide), with a capillary voltage of –3.25 kV, a source block temperature of 40°C and a desolvation temperature of 120°C. Samples were admitted at a flow rate of 5 µl/min via a syringe pump (Harvard Model 11).
MALDI-TOF MS
MALDI-TOF MS analyses of HA oligosaccharides were done on a TofSpec 2E instrument (Micromass UK) in the reflectron mode. The spectrometer was calibrated using a mixture of angiotensin I (MH+ = 1296.7 Da) and adrenocorticocotrophic hormone amino acids 18–39 (MH+ = 2466.2 Da) at 5 pmol/µl in an 
-cyano-4-hydroxycinnamic acid matrix (10 mg/ml in 30% [v/v] methyl cyanide and 0.1% [v/v] trifluoroacetic acid in water) and scanned over a mass range of 0–10,000 Da. The MALDI matrix was prepared using 2,4,6–trihydroxyacetophenone and triammonium citrate (Jovanovic et al., 2000). In brief, 1.2 µl of a mixture of equal volumes of 20 mg/ml 2,4,6-trihydroxyacetophenone in ethanol and aqueous 20 mM triammonium citrate was deposited onto the stainless steel target and air-dried. To this was added 1.2 µl of sample solution (3 pmol/µl in 50% [v/v] acetonitrile) and allowed to dry. Both positive and negative mass spectra were recorded using the minimum laser energy required to give an observable signal (10:1 signal:noise).
FACE
FACE analysis of HA oligosacccharides was done as described previously (Calabro et al., 2000a,b). Briefly, 50 nmol (or less) of test oligosaccharides and standards (Calabro et al., 2000b) were lyophilized and derivatized by addition of 40 µl of 12.5 mM AMAC in 85% (v/v) dimethyl sulfoxide/15% (v/v) acetic acid followed by incubation for 15 min at room temperature. Then 40 µl of 1.25 M sodium cyanoborohydride in ultrapure water was added followed by incubation for 16 h at 37°C. After derivatization, 20 µl of glycerol was added to each sample prior to electrophoresis (of 5-µl aliquots) on MONO composition gels. These were run and analyzed as described elsewhere (Calabro et al., 2000b). Briefly, the gels are illuminated with UV light (365 nm) from an Ultra Lum Transilluminator and imaged with a Quantix cooled CCD camera from Roper Scientific/Photometrics. Digital images for each gel are taken at two exposures. One exposure oversaturates pixel intensity for the major derivatized structures to allow visualization of less abundant species. The second exposure has all pixels within linear 12-bit depth range and is used for quantification. The images are analyzed using the Gel-Pro Analyzer program version 3.0 (Media Cybernetics).
Portions of each oligosaccharide preparation were also analyzed following digestion with Streptococcal hyaluronidase (type VI-S) and chondroitinase ABC lyase to produce disaccharides (see Calabro et al., 2000a). Briefly, samples were digested for 1 h at 37°C with hyaluronidase (1000 U/ml) followed by 3 h at 37°C with the addition of chondroitinase ABC (100 mU/ml). Digests were then immediately frozen on dry ice and lyophilized until dry on a vacuum concentrator prior to derivatization for FACE analysis.
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Acknowledgments |
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We are very grateful to Aniq Darr for technical assistance; to Drs. Wengang Chai, Paul DeAngelis and Glenn Prestwich for helpful advice on oligomer purification and mass spectrometry; and to Dr. Caroline Milner for proofreading the manuscript. This work was supported by the Medical Research Council; D.J.M. was the recipient of a Biotechnology and Biological Sciences Research Council Studentship.
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Abbreviations |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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AMAC, 2-aminoacridone-HCl; ESI-MS, electrospray ionization mass spectrometry; FACE, fluorophore-assisted carbohydrate electrophoresis; GlcUA, D-glucuronic acid; GlcNAc, N-acetyl-D-glucosamine; HA, hyaluronan; HPLC, high-performance liquid chromatography; IOD, integrated optical density; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
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1 To whom correspondence should be addressed
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