Glycobiology Advance Access originally published online on September 15, 2005
Glycobiology 2006 16(1):65-72; doi:10.1093/glycob/cwj037
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Quantification of glycosaminoglycans by reversed-phase HPLC separation of fluorescent isoindole derivatives
Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, MO 63110
1 To whom correspondence should be addressed; e-mail: ljzhang{at}wustl.edu
Received on July 22, 2005; accepted on September 3, 2005
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Abstract |
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Glycosaminoglycans (GAGs) are linear polysaccharides made by all animal cells. GAGs bind to hundreds of proteins, such as growth factors, cytokines, chemokines, extracellular matrix components, protease inhibitors, proteases, and lipoprotein lipase, through carbohydrate and protein interactions. These interactions control many multicellular processes. The increased use of GAGs isolated from cells and small tissue samples in bioassays and binding experiments demands a sensitive and robust quantification method. We have developed such a method, which is based on a popular assay for amino acid analysis. We have refined it to enhance GAG quantification. It allows the quantification of glucosamine- and galactosamine-containing GAGs after the reversed-phase separation of their fluorescent isoindole derivatives. The derivatives are created by the reaction of o-phthaldialdehyde and 3-mercaptopropionic acid (3MPA) with the amino group of hexosaminitol monosaccharides generated from GAG acid hydrolysis and sodium borohydride reduction. The advantages of our method include automatic derivitization, a simple chromatograph with clean separation of glucosaminitol and galactosaminitol derivatives from contaminating amino acids, excellent sensitivity with 0.04 pmol detection, and linearity from 2.5 to 1280 pmol. A major advantage is that it can be readily implemented in any laboratory with typical reversed-phase high performance liquid chromatography (HPLC) equipment.
Key words: assay / glycosaminoglycan / hydrolysis / isolation / OPA
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Introduction |
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TopAbstractIntroductionResults and discussionMaterials and methodsAcknowledgmentsReferences |
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Glycosaminoglycans (GAGs) are linear polysaccharides comprised of different repeating uronic acid-hexosamine disaccharides. Most animal cells express them in abundance. GAGs decorate the cell membrane and fill the extracellular space. Genetic defects in heparan and chondroitin sulfate GAG biosynthesis lead to multiple signaling pathway dysfunctions with serious developmental consequences (Perrimon and Bernfield, 2000
; Selleck, 2000; Sugahara et al., 2003). The GAG chains of proteoglycans are assembled in the Golgi by enzymes, including specific sulfotransferases, encoded by over 40 genes. Because of the vast expression repertoire of the GAG assembly enzymes, GAGs may have a sulfation pattern, chain length, and fine structure unique to each cell (Esko and Lindahl, 2001). Cell surface GAGs turn over within 1/8–1/3 of a cell cycle (Yanagishita and Hascall, 1992). This means their quantity and fine structures are able to rapidly change in response to a variety of environmental factors. We aim to discover the biological activity of GAG sequences and understand their structure and the nature of their action. Our research is guided by the presumption that highly specific GAG sequences regulate specific biological functions. Whether GAGs are isolated, digested, fractionated, or purchased, their molar concentration must be readily determined to make sense of their activity in bioassays and binding experiments.
We have endeavored to develop a solid quantification method for routine GAG analysis that can be implemented with instruments available in a general laboratory setting. Due to the small quantity of GAGs from a flask of cultured cells and the undersulfated GAGs that may be present in some biological samples, we felt that the carbazole (Platzer et al., 1999) and the Alcian blue (Bjornsson, 1998) assays are not sensitive or reliable enough for routine GAG quantification. We decided on a method that would also focus on GAG quantification, rather than a general method for carbohydrates, but could be readily automated and would be sensitive enough for small samples. Our method allows the separation and quantification of glucosamine- and galactosamine-containing GAGs. Fluorescent isoindole derivatives are created by the reaction of o-phthaldialdehyde (OPA) and 3-mercaptopropionic acid (3MPA) with the amino group of hexosaminitol monosaccharides generated from GAG acid hydrolysis and sodium borohydride reduction. The derivatives are then separated by reversed-phase high performance liquid chromatography (HPLC). Figure 1 shows the derivitization reaction and the structures of the glucosaminitol and galactosaminitol amino sugars that are derivitized. Methods used for more general carbohydrate analysis include derivitization methods specific for the carbonyl moiety of reducing sugars (Hase, 1996) or high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) (Lee, 1996). These methods have high utility for GAG quantification, but for just this requirement they are unnecessarily time consuming, or expensive (specialized equipment for HPAEC/PAD).
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We have refined a sensitive method for determining the molar concentrations of the disaccharide content of purified GAGs by enhancing the quantitative HPLC separation of glucosamine and galactosamine derivatives obtained from the samples by acid hydrolysis and reduction. This allows us to perform experiments comparing the effect of GAGs from various sources and with different modifications at the same molar concentrations. The peak produced by the glucosamine derivative represents GAGs containing glucosamine, including heparin, heparan sulfate, keratan sulfate, and hyaluronan. The peak produced by galactosamine derivatives represents all of the chondroitin sulfates, including dermatan sulfate. With these two peaks, we readily obtain an estimate of the proportion of glucosamine and galactosamine containing GAGs as well as the total content of GAGs in our samples. These methods are robust and the assay is forgiving, because it is linear over a wide range of concentrations. We have employed these procedures in our laboratory for over a year to process GAG samples from a wide variety of sources.
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Results and discussion |
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TopAbstractIntroductionResults and discussionMaterials and methodsAcknowledgmentsReferences |
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The first obstacle confronted in assay development was the inability to separate glucosamine from galactosamine on our column. Reversed phase chromatography of either standard resulted in two peaks connected by a center smear (Figure 2). These complex signals from glucosamine or galactosamine had similar elution profiles and almost completely overlapped. The source of the problem was the opening and closing of the hemiacetal ring of each amino sugar. In the linear form, the hydroxyl group at C5 can react with two different faces of the C1 aldehyde to generate geometric isomers with up or down orientations of the hydroxyl group on the anomeric C1 carbon, this carbon so named because of this predilection. This phenomenon is called mutarotation because of changes in optical activity after the dissolution of an optically pure solid progresses to the establishment of an equilibrium of mixed anomers.
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A literature search revealed that mutarotation had been addressed by employing high column temperatures, high column pH, or treatment of the column with nonionic detergent (Vrátny et al., 1983; Cheetham and Teng, 1984; Verhaar et al., 1984; Koizumi et al., 1991; Nishikawa et al., 1996). The effect of all of these maneuvers is to accelerate the rate of anomer interconversion sufficiently to cause coelution, but there is always a concomitant reduction of retention time, which decreases resolution.
Because we wanted a clean separation of the signals from glucosamine and galactosamine, we decided to use sodium borohydride reduction to convert these amino sugars into the linear hexosaminitols (Cheetham et al., 1981; Cheng, 1987) shown in Figure 1. This procedure had already been used to advantage by Friedrich Altmann (1992). He achieved 1.4 min separation of glucosaminitol and galactosaminitol using precolumn OPA and ß-mercaptoethanol derivitization, with arginine eluting between the hexosaminitols. In our system, the separation is 2.7 min (Figure 2, bottom); arginine elutes before glucosaminitol; and none of the amino acids released by protein hydrolysis elute between the glucosaminitol and galactosaminitol signals (Figure 3).
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Formation of fluorescent isoindoles from OPA requires a free amino group on the molecule to be derivitized and a –SH group from a thiol such as ß-mercaptoethanol. At alkaline pH, the reaction is rapid and robust. It is the current method of choice for amino acid determination (Kutlán et al., 2002). Our initial experiments with OPA hexosamine derivatives employed ß-mercaptoethanol. With this reducing agent, the hexosaminitols were separated about 1 min on our column, and they eluted late; 70% buffer B was required for elution at 20 min. We improved this performance by employing 3MPA as the reducing agent. The OPA derivatives eluted earlier with much less buffer B, and resolution was markedly improved. This effect must be due to the replacement of the –OH (from mercaptoethanol) with –COOH (from mercaptopropionate) in the fluorescent products. Molár-Perl and colleagues (Kutlán et al., 2002; Kutlán and Molár-Perl, 2003) have investigated the stability of OPA derivatives and have discovered the generation of secondary species can be inhibited by a molar excess of the –SH reducing agent in the reaction buffer. We have followed their suggestion of a 1:50 OPA to 3-MPA molar ratio.
After the adoption of 3MPA in the OPA reaction, the elution position of the hexosaminitol derivatives was easily set between 10 and 15 min by manipulating the amount of methanol in buffer A. The next step of method development was to assay the retention times of amino acid derivatives that might co-purify in GAG preparations. In our initial prospecting, we employed phosphate buffer at pH 6.4 in buffer A. At this pH, histidine was smeared from 10 to 20 min, across the elution positions of the amino sugar alcohols. Lowering the pH to 4.6 with acetate buffer solved this problem but moved the elution position of arginine between glucosaminitol and galactosaminitol. We investigated more alkaline conditions and eventually settled on pH 7.2. As shown in Figure 3, we achieve a clean separation of the hexosaminitols from the amino acids that have similar retention times on our column.
Our final conditions are Buffer A, 0.05 M monobasic and dibasic sodium phosphate, pH 7.2 in 25% methanol; Buffer B, methanol/water/tetrahydrofuran at 70:30:3 volume ratios; Column temperature 35°C; and flow rate 0.8 mL/min. The binary pump is programmed as shown by the % buffer B trace in the top panel of Figure 2. The limits of detection of the assay, defined as peak heights at three times baseline noise levels, was 0.04 pmol for the glucosaminitol and the galactosaminitol derivative. Table I summarizes the accuracy and precision for this assay. Standards were assayed in septuplicate at five of the concentrations used for external standard curves. The calibration curves for the values in Table I, for 10 standards ranging from 2.5 pmol to 1280 pmol hexosaminitols, where x is the area under the curve for the analyte peak and where y is the estimated hexosaminitol concentration, were y = 0.36466x + 1.51835, r2 = 0.9969 for glucosaminitol and y = 0.56985x + 1.61184, r2 = 0.9954 for galactosaminitol. In Table I, relative standard deviations (RSD), (SD/mean) x 100(%), give an estimate of precision and accuracy is estimated by relative error, ([standard-measured value]/standard) x 100(%). While relative error is elevated at the lowest analyte concentration, the assay is linear over a 500-fold concentration range. The wide range greatly enhances the utility of this method of GAG quantification.
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This quantification assay relies on the conversion of complex polysaccharides to simple monosaccharides before analytical separation. The acetyl amino sugars must also be de-acetylated before the OPA/3MPA derivitization reaction is possible. We use vapor-phase acid hydrolysis to accomplish all of this. It cleaves sugars and removes acetyl and sulfate groups, although not necessarily in that order, to yield glucosamine and galactosamine (Figure 4a). GAGs cannot survive the protracted hydrolysis used to liberate amino acids from proteins. To optimize our hydrolysis procedure, we tested six different hydrolysis times ranging from 0.5 to 5.5 h. We employed a sample containing equivalent amounts of chondroitin sulfate A and heparan sulfate as well as norleucine as an internal recovery standard. Aliquots of this sample were dried and hydrolyzed in quadruplicate at each test interval. A surprising feature of the results was a peak eluting before the expected monosaccharide derivatives. It is most prominent after the shortest hydrolysis period, then it declines as the magnitude of the glucosaminitol and galactosaminitol isoindole derivatives concomitantly increase (Figure 4a). After 3 h of hydrolysis, it is essentially gone, and it is not present after 5.5 h of hydrolysis. Because of this behavior, we think it is likely to be partly digested GAG polysaccharides with free amine groups. This makes it useful for trouble-shooting GAG hydrolysis, to find the interval when the digestion is virtually complete. Figure 4b shows that this interval, 3 h, corresponds to the peak recovery of glucosaminitol from heparan sulfate and galactosaminitol from chondroitin sulfate. Figure 4b also suggests that digestion for up to an additional hour does not result in appreciable signal loss.
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Because the purpose of this assay is to quantify GAGs, we were concerned about amino acids only insofar as they might interfere. After using this assay to analyze dozens of samples from diverse sources such as fly embryos, mouse growth plates, Chinese hamster ovary (CHO) cell lines, and mouse joints, we have been gratified that amines co-purifying in our experimental GAG preparations have not proved to be an obstruction to our analysis. Figure 5 compares a commercial heparan sulfate isolated from bovine kidney (top, HS bov) to GAGs we isolated from a skeletal cell line (CFK2), a CHO cell line (CHO K1) and paws from BALB/c mice (bottom, paws). The peak heights of the internal norleucine standard in each sample are a rough indication of the relative GAG concentrations. These are typical results chosen to contrast samples rich in heparan sulfate (bovine kidney) or chondroitin sulfate (mouse paws) with cell lines expressing lower but near equivalent amounts of these GAGs. The GAGs from the CFK2 and CHO K1 cell lines were isolated, each preparation, from one flask of cells. The chromatographic signals for these cell lines shown in Figure 5 represent 0.5% of each preparation. Quantification of the peaks yielded 63.0 pmol of GlcN–OH and 22.2 pmol of GalN–OH for the CFK2 cells, and 8.7 pmol of GlcN–OH and 14.8 pmol of GalN–OH for the CHO K1 cells. In our tissue preparation example (Figure 5, bottom), the aliquot assayed represents 0.4% of the GAGs isolated from the paws of three mice. Quantification of the peaks yielded 95.2 pmol of GlcN–OH and 1310 pmol of GalN–OH. The purity of the paw GAGs with respect to co-purifying amines compares favorably to the commercial heparan sulfate preparation.
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These samples were selected as examples to illustrate the utility of our procedures for GAG research. CFK2 cells produce cartilage matrix components, including cartilage-specific proteoglycans and type II collagen, after extended cell culture. They also exhibit mineralization of focal cell nodules (Bernier et al., 1990; Bernier and Goltzman, 1993). CHO K1 is one of the first subclones of the original Chinese Hamster Ovary cell line isolated by Puck et al. (1958). It produces relatively simple heparan and chondroitin sulfates, because it expresses a limited set of sulfotransferases. This feature has made CHO cells useful for the production of GAG-deficient mutants and for assessing the effect of GAG modifying enzymes by transduction of their genes into these cells (Esko et al., 1985; Bame and Esko, 1989; Bai and Esko, 1996; Bai et al., 1999). For example, to assess the effect of 3-O-sulfotransferase, an enzyme not present in CHO cells (Zhang et al., 2001a,b), accurate quantification of GAGs isolated from control and transduced cells are essential before bioassay. Although CHO cells do not produce copious amounts of GAGs, the recovery shown in Figure 5 is sufficient for multiple mass spectrometry experiments, which often employ stable metabolic labeling with 34SO4 and frequently involve digestion with heparanases and chondroitinases to produce defined fragments. In a similar vein, we are currently exploring the role of GAGs in the joint specificity of rheumatoid arthritis in a mouse model. Assessment of GAGs isolated from paws versus knees or hips in our gel-shift assays requires the use of equivalent concentrations of GAGs from these tissues. The isolation and quantification of GAGs from these joints was the first use of the procedures described in this article.
Whenever an investigator decides to isolate GAGs from a new source, we recommend the use of tracer amounts of radiolabeled GAGs during the isolation procedure to troubleshoot GAG recovery. Routine recoveries after diethylaminoethyl (DEAE)-Sephacel and final ethanol precipitation should be greater than 90%. The binding capacity of the DEAE-Sephacel beads is affected by pH. In practice, small sample isolation is robust between pH 5 and 8. For more concentrated samples that approach the bead capacity, it is important to make sure the pH is adjusted between pH 5 and 6. The quality of the isolated GAGs is also important. We recently have discovered that our isolation procedure produces GAGs pure enough to allow us to detect nonsulfated and monosulfated disaccharides by LC mass spectrometry (data not shown), a result not achieved by past methods of GAG isolation (Kuberan et al., 2004; Lawrence et al., 2004).
Toyoda et al. (1997, 1999) has described a derivitization method where fluorescent compounds are formed from the conjugation of 2-cyanoacetamide with reducing sugars. This is effected by use of a 10 m postcolumn reaction coil heated to 110°C. For GAG analysis, the method involves the separation of heparan from chondroitin sulfates by chondroitinase ABC digestion followed by ethanol precipitation to recover the heparan sulfate. Either chondroitin or heparan sulfate analysis is then pursued by complete enzymatic digestion to unsaturated disaccharides. In the case of heparan sulfate, a mixture of heparin lyases I, II, and III is employed. Quantification depends on the use of 6–8 standard curves for each disaccharide. The advantage of this method is, of course, the information about disaccharide composition. But it has drawbacks for simple GAG quantification: extensive and expensive enzyme treatment, to which some oligosaccharide structures are resistant (Toyoda et al., 1997), separate column runs for chondroitin and heparan sulfates, good separation of disaccharides, but with overlap at the baseline. This overlap, coupled with the necessity for several standard curves, increases the experimental error of an estimate of total chondroitin or heparan sulfate.
Other methods with advantages for more general carbohydrate analysis, which may also be used for GAG quantification, include derivitization methods specific for the carbonyl moiety of reducing sugars (Hase, 1996) and HPAEC with PAD (Lee, 1996). Both may be used to assess the carbohydrate composition of any class of glycoprotein. They also have utility for the analysis of disaccharides and oligosaccharides after enzymatic digestion (Midura et al., 1994). Compared to the OPA method, derivitization of sugar carbonyls is complicated or time consuming and generally cannot be accomplished on-instrument by autosampler injector programming. It defeats the problem of mutarotation but requires the postreaction removal of the labeling reagent by extraction and/or adequate column separation of reactants from the labeled sugars for analysis. However, like OPA derivitization, sugar carbonyl derivitization adds bulky hydrophobic groups that facilitate reversed-phase separation. Addition of bulky groups is not needed for HPAEC separation, which defeats the smearing of mutarotation, perhaps, because it is conducted at relatively high pH. PAD detection is as sensitive as the fluorescent detection of OPA derivatives; therefore it does not require sugar derivitization. It is also more susceptible to interference by lipids, peptides, and amino acids present in real samples (Lee, 1996; Weitzhandler et al., 1996). A commendable recent application of HPAEC-PAD to monosaccharide analysis (Campo et al., 2001) demonstrates high precision and reproducibility, albeit over a narrower concentration range than our assay (160-fold versus 500-fold range). However, the analysis of GAGs in plasma and serum samples entailed significant sample preparation including two different ion exchange steps requiring a total of four columns before HPAEC-PAD analysis. Glucosamine was separated from galactosamine by 1.4 min in these samples but without the clean baseline resolution of galactosamine.
In summary, the advantages of our method include an estimate of the molar concentration of GAGs that is obtained from a simple chromatograph featuring a clean separation of glucosaminitol and galactosaminitol from contaminating amino acids. We achieve excellent sensitivity and linearity over a wide concentration range.
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Materials and methods |
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GAG isolation from cultured cells
For each 75-cm2 flask of cultured cells, remove medium and incubate with 5 mL of 0.02% EDTA solution at 37°C for 15 min to lift the cells. Spin down the cells and wash in 10 mL phosphate-buffered saline (PBS). Pellet the cells again and resuspend in 200 µL of ß-elimination solution, 1.0 M NaBH4, 0.5 M NaOH, to lyse the cells and cleave GAG chains from proteins. Incubate overnight at 4°C. The next morning, destroy the borohydride by adding 20 µL of glacial acetic acid to each sample. Add 1 mL of water to each sample and 5 µL of 1 mg/mL phenol red to monitor pH. The sample will be acidic (yellow). Neutralize to pH 7 with 1.0 M NaOH, until the color of the sample is slightly pinkish. Centrifuge the 15-mL conical tube for 10 min at 2000 x g. Transfer the supernatant to a 1.5-mL siliconized microcentrifuge tube and centrifuge at 16,000 x g for 10 min to remove any remaining cell debris. Transfer the supernatant to fresh 1.5-mL tubes. Add 5 µL of 20 mg/mL glycogen and 100 µL of DEAE Sephacel beads suspended 1:1 in water. (The DEAE Sephacel beads [Sigma I-6505], which are in ethanol as purchased, were prepared before use by washing three times in 5 volumes of 1.0 M NaCl, 0.01% Triton X-100, 20 mM sodium acetate pH 6.0 and five times in 5 volumes of water.) Agitate by inversion for at least 5 min on a rocker platform. Centrifuge at 16,000 x g for 30 s to pellet the beads and wash by resuspension in 1 mL 0.25 M NaCl, 20 mM sodium acetate pH 6.0. Repeat this wash step three times. Pellet the beads in preparation for GAG elution. Pool the supernatants of four successive 100 µL elution steps: collect GAGs eluted in 2.0 M NaCl, 0.01% Triton X-100, 20 mM sodium acetate pH 6.0, once; and in 1.0 M NaCl, 0.01% Triton X-100, 20 mM sodium acetate pH 6.0, thrice. Centrifuge the pooled eluates for 5 min at 16,000 x g to remove any beads and transfer the supernatant to a new 1.5-mL tube. Add 1.2 mL of 100% ethanol to precipitate the GAGs. Incubate for at least 2 h before centrifugation at 16,000 x g for 15 min. Discard the supernatant. Wash the pellet with 0.5 mL 75% ethanol. Resuspend the precipitate in 50 µL of water, and the GAG preparation is ready for use.
GAG isolation from tissue
To isolate GAGs from tissue, record the wet weight of each sample after washing it in water and blotting it dry. Scissor-mince the sample, transfer it to a 50 mL conical tube, and add 1 mL/g wet weight (but a minimum 5 mL) of ß-elimination buffer diluted 1:1 in water. Incubate the tubes at least 10 min at room temperature. Thoroughly homogenize each sample with a Polytron. Incubate homogenized samples overnight at 4°C. The next morning add 10 mL of water to each tube before transferring samples to 30-mL Oakridge centrifuge tubes. Centrifuge the tubes for 10 min at 12,000 x g. Transfer the supernatants to new 50 mL conical tubes. Add 50 µL of glacial acetic acid per g wet weight tissue, no more than 50 µL at a time to mitigate sample foaming. Add phenol red, 1 µL 1 mg/mL per g wet weight tissue, to each tube. Neutralize to pH 7 with 1.0 M NaOH, until the color of the sample is slightly pinkish. Add 15 mL of phenol/chloroform/isoamyl alcohol (25:24:1) and perform an extraction by inverting tubes for 5 min, loosening caps occasionally to release pressure. Spin samples for 30 min at 860 x g in a swinging bucket centrifuge to separate layers. Transfer the top (aqueous) layer to a new 50 mL conical tube. If it contains residual organic phase, add 10 mL of water, centrifuge again, and transfer the aqueous phase to a new tube. Add 15 mL of 0.25 M NaCl, 0.01% Triton X-100, 20 mM sodium acetate, pH 6.0, to the organic solvent layer saved from the initial extraction and invert several times. Spin 30 min at 860 x g and pool the aqueous layer with the previously collected aqueous phase. If the pooled aqueous material looks turbid, spin again for 30 min to get rid of particles and transfer to a new 50-mL conical tube. Add 250 µL of DEAE Sephacel beads (1:1 in water) to each sample. At this point, trace amounts of 35S labeled GAGs, may be added to monitor GAG binding and recovery. Rock 20 min or overnight at 4°C. Centrifuge at 860 x g for 10 min to spin down beads. Wash the beads 4 times with 10 mL of 0.25 M NaCl, 0.01% Triton X-100, 20 mM sodium acetate, pH 6.0. Spin down the beads and aspirate the supernatant. Pool the supernatants of four successive 500 µL elution steps: collect GAGs eluted in 2.0 M NaCl, 0.01% Triton X-100, 20 mM sodium acetate pH 6.0, twice; and in 1.0 M NaCl, 0.01% Triton X-100, 20 mM sodium acetate pH 6.0, twice. Centrifuge at 860 x g for 10 min to get rid of any beads transferred in the pooled eluates. Transfer the supernatant to a new 15-mL conical tube, and add 10 mL of cold 100% ethanol. Invert the tube several times to mix and incubate at 4°C for 2 h or overnight. Spin down the precipitated GAGs at 860 x g for 30 min. Aspirate the ethanol and wash the pellet with 5 mL of 75% ethanol. Spin down again, aspirate the ethanol, and allow the pellet to dry briefly at room pressure or under vacuum. Dissolve pellet in a minimal volume of water and transfer to a microcentrifuge tube.
GAG quantification
The steps are acid hydrolysis, sodium borohydride reduction, precolumn derivitization with OPA/3MPA reagent and reversed-phase HPLC separation with fluorescent detection.
Acid hydrolysis
Aliquots of each GAG sample (5–10 µL) are pipetted into glass inserts for autosampler vials (Agilent part 5181–8872), 3 µL of norleucine (360 pmol) is added as an internal standard. The inserts were previously baked overnight at 500°C to pyrolize any amine contamination. The samples are then dried by centrifugal evacuation at room temperature. Up to 14 sample vials are then placed into a reaction tube (WAT007363, Eldex Laboratories, Napa, CA) for a Waters Pico-Tag workstation. The reaction tubes have a screw cap fitted with a Teflon-stem valve to allow evacuation and nitrogen backfill. Sequencing grade 6 N HCl (200 µL, Pierce 24308) is added to the bottom of the reaction tube before it is capped. The reaction tube is seated into the vacuum/gas exchange port of the Pico-Tag workstation with cap valve opened. The tube is evacuated until the HCl begins to bubble, then the tube is backfilled with dry nitrogen gas (grade 4.8). After four such cycles, the valve on the nitrogen flushed reaction tube is closed. The reaction tube is then placed into the oven of the Pico-Tag workstation and the tube is baked at 100°C for 3 h to allow gas phase hydrolysis of the dried samples. Removing the reaction tube from the oven, releasing the vapor pressure, and removing residual HCl from the samples by centrifugal evacuation terminates hydrolysis.
Sodium borohydride reduction
The samples are rehydrated in 20 µL of water, and 25 µL of 1% NaBH4 is added to begin the reaction. This converts glucosamine and galactosamine liberated by acid hydrolysis into glucosaminitol and galactosaminitol, respectively. The glass inserts are placed in autosampler vials, which are sealed during the reaction. After a minimum of 3 h or overnight at room temperature, the reaction is terminated by adding 5 µL of 2 N acetic acid to each vial, bringing the final sample volume to 50 µL.
Standard solution
The standard solution was made from 50 mM solutions of d-(+)-glucosamine, d-(+)-galactosamine and L-norleucine weighed and volume determined with analytical precision. A mixture containing16 mM glucosamine, 16 mM galactosamine, and 8 mM norleucine (an internal standard) was subjected to sodium borohydride reduction to generate the hexosaminitols. This stock was diluted to produce 10 standard solutions. Aliquots of 5 µL contained 2.5, 5, 10, 20, 40, 80, 160, 320, 640, or 1280 pmol of each hexosaminitol.
Derivitization with OPA/3MPA
A 5-µL aliquot of each hydrolyzed and reduced sample is transferred to a fresh autosampler insert vial for precolumn derivitization with a buffered solution of OPA and 3MPA at a 1:50 molar ratio. An automated injection sequence program controls the autosampler to mix 35 µL of the OPA/3MPA solution with the sample by ejecting and withdrawing a 35-µL volume 4 times into the vial insert at 100 µL/min before a final ejection of 35 µL and withdrawal of 20 µL, which is injected for analysis. The OPA/3MPA solution is made by mixing 250 µL of OPA (Acros 13108, CAS 643–79–8), 10 mg/mL in methanol, with 2.0685 mL of 0.4 N Borate, pH 10.2 (Agilent Technologies 5061–3339), 81.5 µL of 3-MPA (Sigma M6750, CAS 10-96-0), and 100 µL of 10 N NaOH for a final volume of 2.5 mL at pH 9.3. The solution is stored at 4°C and is made fresh every 9 days.
Reversed-phase HPLC
The HPLC apparatus is Agilent 1100 series equipment, consisting of a degasser (G1379A), a binary pump (G1312A), an autosampler (G1313A), a temperature regulated column compartment (G1316A), a UV-VIS variable wavelength detector (G1314A), and a fluorescence flow cell (G1321A). Operation of the HPLC equipment and data collection was controlled by Agilent Chemstation software. The OPA derivatives are injected onto a 4.6 x 250 mm C-12 column, a Synergi 4µ MAX-RP 80 Å (Phenomenex 00G-4337-E0), heated to 35°C. At injection, the column was equilibrated with Buffer A, 0.05 M (monobasic and dibasic) sodium phosphate, pH 7.2 in 25% methanol at a flow rate of 0.8 mL/min. Buffer B consisted of methanol/water/tetrahydrofuran at 70:30:3 volume ratios. Buffer B is increased to 8% by a linear gradient between 0 and 3 min, is maintained at 8% between 3 and 18 min, at 55% between 18 and 30.5 min, at 100% between 30.5 and 32.5 min, and at 0% between 32.5 and 35 min (Figure 2, top panel). A 5 min postrun interval at 0% B precedes the initiation of the next precolumn derivitization injection sequence. The fluorescent derivatives of glucosaminitol, galactosaminitol, and the amino acids contained in the GAG preparations were excited at 337 nm and detected at 454 nm in the flow cell.
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Acknowledgments |
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TopAbstractIntroductionResults and discussionMaterials and methodsAcknowledgmentsReferences |
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This work is supported in part by National Institutes of Health Grants: R01GM069968.
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Abbreviations |
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3MPA, 3-mercaptopropionic acid; CHO, Chinese hamster ovary; DEAE, diethylaminoethyl; EDTA, ethylenedinitrilo-tetraacetic acid; GAG, glycosaminoglycan; HPAEC, high-performance anion-exchange chromatography; HPLC, high performance liquid chromatography; OPA, o-phthaldialdehyde; PAD, pulsed amperometric detection
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References |
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Altmann, F. (1992) Determination of amino sugars and amino acids in glycoconjugates using precolumn derivitization with o-phthalaldehyde. Anal. Biochem., 204, 215–219.[CrossRef][Web of Science][Medline]
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Bai X., Wei, G., Sinha, A., and Esko, J.D. (1999) Chinese hamster ovary cell mutants defective in glycosaminoglycan assembly and glucuronosyltransferase I. J. Biol. Chem., 274, 13017–13024.
Bame, K.J. and Esko, J.D. (1989) Undersulfated heparan sulfate in a Chinese hamster ovary cell mutant defective in heparan sulfate N-sulfotransferase. J. Biol. Chem., 264, 8059–8065.
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