Glycobiology, 2001, Vol. 11, No. 9 759-767
© 2001 Oxford University Press
An ultrasensitive chemical method for polysialic acid analysis
2Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan, ROC, and 3Biology Department, Johns Hopkins University, Baltimore, Maryland 21218, USA
Received on April 17, 2001; revised on June 13, 2001; accepted on June 21, 2001.
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An ultrasensitive method for analysis of polysialic acid (polySia) chains, using fluorescence-assisted high-performance liquid chromotography was developed. The new method is a substantial improvement of our earlier method in which the reducing terminal Sia residues of a homologous series of oligo/polySia hydrolytically released during derivatization reaction were simultaneously labeled with a fluorogenic reagent, 1,2-diamino-4,5-methylenedioxybenzene (DMB) in situ. We first studied extensively the stability of oligo/polySia in the acid (0.02 M trifluoracetic acid) used for 1,2-diamino-4,5-methylenedioxybenzene derivatization under various conditions of reaction time and temperature, analyzing the hydrolytic products by high-performance anion exchange chromatography with pulsed electrochemical detection (HPAEC-PED). Then we optimized the reaction conditions to minimize degradation of the parent polySia while maintaining high derivatization rate. Using a DNAPac PA-100 column rather than a MonoQ column, baseline resolution of polySia peaks up to DP 90 with a detection threshold of 1.4 femtomol per resolved peak was achieved. The new method was used to analyze the degree of polymerization of a polySia-containing glycopeptide fraction derived from embryonic chicken brain, and the results were compared with those obtained by HPAEC-PED.
Key words: acid stability of polysialic acid/1,2-diamino- 4,5-methylenedioxybenzene/neural cell adhesion molecule/polysialic acid analysis
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Introduction |
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In higher vertebrates, the
2,8-linked homopolymer of Neu5Ac (polysialic acid; referred to as polySia in this article) is expressed in neural cell adhesion molecules (NCAMs) most abundantly in neuronal tissues during embryonic development and pertinently expressed in postnatal and adult animal on the surface of some cells where reorganization of the tissue is taking place. PolySia is also reexpressed on a number of human tumors; thus it is an oncodevelopmental antigen (Troy, 1995
). Antibodies, often in combination with endo-N-acylneuraminidase (Endo-N), are used as diagnostic probes for polySia. In addition to a large number of publications on cell- and tissue-specific distribution and developmentally regulated expression of polySia, the studies on enzymes involved in its biosynthesis have been reported recently (Eckhardt et al., 1995; Nakayama et al., 1995; Scheidegger et al., 1995; Oka et al., 1995; Kojima et al., 1996; Wood et al., 1997; Ong et al., 1998; Angata et al., 1998, 2000, 2001; Kudo et al., 1998; Sevigny et al., 1998; Close and Colley, 1998; Close et al., 2000). However, the reactivity to these biological reagents does not yield precise information on the degree of polymerization (DP) of polySia, which is fundamentally important in understanding the molecular mechanism of biosynthesis of polySia and fine tuning of cell–cell and cell-adhesive interactions by polySia. PolySia expressed on embryonic NCAM is believed to be longer than 100 (Rutishauser and Landmesser, 1996) presumably based on the published value (DP > 55) determined for glycopeptides derived from human neuroblastoma cells (Livingston et al., 1988). Indeed, this was the only report that showed the presence of extended polySia chains in neural cells until our recent determination of the DP of polySia chains in polySia-bearing glycopeptides derived from chicken brains of various developmental stages (Inoue et al., 2000). However, some technical difficulties remain in determination of DP of polySia, and the DP values of extended polySia chains on the NCAM can be controversial.
In view of the proposed biological significance of polySia chains preferentially expressed on NCAM during embryonic stages and much less in adult, it is important to determine exact DP values of the polySia chains and their developmental stage–dependent change. Apart from DP determination, development of a convenient and sensitive method that can be used for monitoring polySia during isolation and purification is urgent for chemical studies of polySia-bearing glycan chains. Recently we analyzed developmental pattern of polysialylation expressed in chicken brain by chromatographic separation of the polySia-bearing glycopeptides, compositional analysis, and using two different high-performance chromatographic methods for polySia analysis (Inoue et al., 2000). One method, high-performance anion-exchange chromatography with pulsed electrochemical detection (HPAEC-PED) showed excellent resolution of high DP polySia but relatively large amounts (more than 10 µg) were required due to the sensitivity limit of the detector. In the HPLC with fluorescence detection (FD) method, oligo/polySia chains were derivatized with the 1,2-diamino-4,5-methylenedioxybenzene (DMB) reagent and the products were separated on a MonoQ HR 5/5 column. HPLC-FD gave more sensitive and specific detection of oligo/polySia than HPAEC-PED, but the peak resolution on this column was only up to about 25. Some other problems remained to be answered in polySia analysis: (1) the stability of oligo/polySia under conditions used for pretreatment and during derivatization; (2) the yields of DMB labeling; and (c) DP dependency of the detector response.
For chemical studies of oligo/polySia-bearing compounds, some procedures inevitably result in cleavage of some sialyl linkages. This complication often hampers the development of methods for isolation and analysis of oligo/polySia. To circumvent such problems, we first carried out careful studies on the lability/stability of oligo/polySia under various conditions we use during isolation and chemical analysis of oligo/polySia and oligo/polySia-bearing molecules. Second, we established the ultrasensitive HPLC-FD method of oligo/polySia analysis by improving the conditions for DMB derivatization and chromatographic system so that peaks up to DP 
90 with a detection threshold of 1.4 femtomol per peak could be resolved. Third, we compared the present method with previously established HPAEC-PED method when the DP of polySia expressed in an embryonic chicken brain sample was determined. Finally, we showed application of the newly established method for polySia analysis in a small amount of brain tissue.
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Results and discussion |
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Stability of oligo/polySia during prolonged incubation at near neutral pH
Prolonged incubation of oligo/polySia-bearing compounds with enzymes such as proteases is often necessary during isolation and structural analysis. In a previous study using thin-layer chromatography (TLC) we reported that polySia was stable and no lower oligoSia was detected by incubation of colominic acid at pH 7.4 for 3 days at 37°C, whereas during treatment at pH 5.6 and 37°C for 1 day or at pH 6.4 and 80°C for 7 h extensive cleavage of the internal
2,8-linkages occurred and produced lower oligoSia in high yields (Kitazume et al., 1992). In our recent studies on the chemical analysis of embryonic chicken brain NCAM, we incubated the delipidated brain tissue with nonspecific bacterial proteinase in 0.1 M Tris–HCl buffer (pH 8.0) at 37°C for 3 days (Inoue et al., 2000). We examined whether any cleavage of sialyl linkages occurred under these conditions using model compounds, a mixture of oligoNeu5Ac, A9–11(a mixture of DP 9, 10, and 11; in about 14%, 67%, and 18%, respectively ), and A19–28 (DP 19–28), and HPAEC-PED, which is much more sensitive and quantitative than TLC previously used. No evidence of cleavage of sialyl linkage in these samples was obtained (see Figure 1a). At pH 7.0, a minute amount of lower oligoSia was detected during incubation of these model oligoSia for 3 days at 37°C, and at pH 6.0, the production of lower oligoSia was extensive in accord with the previous results (data not shown). However, all preparative chromatographic procedures for polySia-bearing compounds used in our study were carried out at pH 8.0.
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Stability of oligo/polyNeu5Ac in relatively strong acid used for DMB-derivatization
Recently, we have developed a new highly sensitive method of oligo/polySia analysis by DMB derivatization (Lin et al., 1999
), in which the derivatization of oligo/polySia was found to occur most efficiently in 0.02 M trifluoracetic acid (TFA), and stability of oligo/polySia under such conditions had been an arguable subject. We examined the stability of model free oligo/polySia chains incubated in 0.02 M TFA at various temperatures, in analyzing oligo/polySia with HPAEC-PED before and after acid treatment. The results showed that, when lower oligoSia (DP 2–5) were used as the parent compounds, they were shown to be remarkably stable in 0.02 M TFA, and the recovery of the parent compounds was > 80% at temperatures below 20°C even after 48 h at10°C, or 4 h at 20°C, but the recovery became significantly low above 37°C (Figure 2a). Higher oligomers, A9–11 and A19–28, also showed 80% recovery after prolonged treatment at 10°C, and after 2 h at 20°C (Figure 2b). However, at higher temperatures, and even at 20°C after 4 h, the survival rate for the higher oligoSia was significantly lower than lower oligoSia. Thus at 50°C, less than 40% of A19–28 remained after 30 min. We then examined how the DP distribution profile for the mixtures of oligo/polySia changes from the original compound after the treatment. Figure 1a–e clearly showed that in spite of formation of a series of (Neu5Ac)n (n = 1–18) during the acid treatment, DP distribution profile for A19–28 remained unchanged during the acid treatment at 10–37°C, and even at 50°C for 30 min. In lower polySia, A9–11, the DP distribution profile almost unchanged after 2 h treatment at 50°C in 0.02 M TFA, and only the peak area of parent compounds decreased (data not shown). In the case of polydisperse mixtures containing a wide range of DP, such as commercially available colominic acid, the acid treatment resulted in a small shift in DP distribution profiles at 10°C and 20°C (Figure 3b and c), and a significant change at 37°C and 50°C (Figure 3d and e). Monomeric Neu5Ac was the major product accumulated during the acid treatment under all conditions (Figures 1 and 3). In view of the reported stability of external 2,8-sialyl linkage (Nadano et al., 1986; Manzi et al., 1994), we first expected the accumulation of diSia after the acid treatment, but it was not the case. As described and discussed later, the difference in apparent first-order rate constants of the hydrolysis of 2,8-sialyl linkages in 0.02 M TFA was small, if any, between diSia and polySia. Apparent faster rates of depolymerization of higher oligoSia and polySia are thus simply accountable by the increasing numbers of reaction sites of hydrolysis.
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Selection of anion-exchange column used in the HPLC separation of DMB-oligo/polySia
It should be emphasized that for the above experiments using HPAEC-PED (Figure 3), injection of 10 µg of colominic acid was necessary for each chromatographic run, which is not sufficiently sensitive for studying oligo/polySia chains expressed on glycoproteins such as embryonic NCAM. In preliminary experiments using the published DMB derivatization conditions (0.02 N TFA for 2 h at 50°C), we have found that resolution of DMB-polySia was greatly improved (DP up to
80) by using a CarboPac PA-100 column (used in HPAEC-PED) instead of previously used MonoQ HR 5/5. Furthermore, we found that use of a DNAPac PA-100 and elution with a gradient concentration of NaNO3 resulted in further improvement in resolution of DMB-polySia up to DP 90. Thus, the resolution of the DMB-polySia on a column originally developed for DNA (DNAPac PA-100) was superior to that on a column generally used for carbohydrates (CarboPac PA-100).
Determination of the optimized conditions for DMB derivatization
From the results of the acid treatment described above, we chose some different reaction temperature and reaction time that do not cause extensive cleavage of internal sialyl linkages and compared the yields of the DMB derivatives obtained under each of these conditions. The results for A9–11, and A19–28 are given in Table I. Among the conditions tested, reaction at 10°C for 48 h gave the highest total derivatization yields and satisfactory survival rate of the parent compounds. Results for high molecular weight colominic acid showed a similar trend as for these model compounds. When Neu5Ac monomer was reacted at 10°C for 48 h, the yield of DMB-Neu5Ac was 65% of the maximum value that was attained by 2.5 h reaction at 55°C. Thus we set the reaction at 10°C for 48 h as the optimal derivatization conditions for polySia analysis.
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Comparing the elution profile of A19–28 derivatized with DMB under optimized conditions (Figure 4) with the DP-distribution of the same compounds analyzed by HPAEC-PED (Figure 1), we concluded that DMB/HPLC-FD method can be used not only for a diagnostic tool for the identification of occurrence of polySia but also for its DP analysis. Next, we examined if DMB/HPLC-FD can be applied to DP analysis of high DP polySia compound such as colominic acid. HPAEC-PED elution profiles for two different samples of colominic acid col-N and col-Q are given in Figure 5a and b, respectively. Col-N is a commercial sample (average DP
100) and col-Q is a major fraction separated from another commercial colominic acid sample (average DP 50) purchased from the same supplier (Nacalai Tesque, Kyoto) by preparative chromatography on a MonoQ HR 10/10 column. This fraction was eluted from the MonoQ column a little earlier than polySia-glycopeptides derived from embryonic chicken brain (Inoue et al., 2000). As shown in Figure 5, col-N was highly polydisperse and peaks of DP 1 up to 50 were resolved by HPAEC-PED. Col-Q also contained low DP species, but major components appeared to be a mixture of DP > 30, as was expected from the elution from the preparative MonoQ column. Elution profiles of these colominic acid samples analyzed by DMB/HPLC-FD are given in Figure 6a–c. Due to the acidic condition during derivatization, the proportion of low DP species was higher in DMB/HPLC-FD than HPAEC-PED profiles. However, resolution of high DP region by DMB/HPLC-FD is marked as can be seen in Figure 6a, DP 90 being resolvable. The sensitivity of this method is also remarkable. In Figure 6b, only 20 ng was injected in contrast to 25 µg injected for HPAEC-PED shown in Figure 5a. Moreover, the elution profile for col-Q clearly shows that major species in this sample are of DP 34–70 (Figure 6c).
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To see if we can estimate the distribution of the amount of material from the elution profile, we next examined DP dependence of the peak area for model (Neu5Ac)n, for which the yield of the DMB-derivatives of parent compounds was > 50% of total. The parent peak area per weight of the starting material (total Neu5Ac) significantly decreased with DP, while the parent peak area per mol of the starting material remained relatively constant (Table II). It is difficult to evaluate DP-dependence of these values for high DP polySia for which yields of DMB-derivatives of parent compounds are small. Nevertheless, we can assume that peak area of DMB-(Neu5Ac)n is proportional to mol of (Neu5Ac)n if the fluorescence intensity of Q, the quinoxalinone unit formed by reaction of the reducing terminal Sia with DMB (Lin et al., 2000
), is not influenced by the attachment of (Neu5Ac)n–1. Fluorescence emission spectrum for each of the different (Neu5Ac)n–1-Q was virtually indistinguishable. We also examined the DP dependence of PED response (peak area) for model (Neu5Ac)n and found that PED response per mol of compound was relatively unchanged irrespective to DP for wide DP range 2–54, whereas PED response per mol of Neu5Ac residue showed large decrease with increasing DP values, suggesting the large contribution of the carboxylate group of terminal Neu5Ac to electrochemical detection for these compounds. Although it was difficult to accurately determine the molar response values for polySia for both PED and FD, the DP distribution profile for a colominic acid sample obtained by DMB/HPLC-FD superimposed on that obtained by HPAEC-PED (without prehydrolysis), suggesting that both profiles show distribution based on mol of oligo/polySia chains rather than mol of Neu5Ac residues (Figure 7a). Apparent molar response of FD for DMB-(Neu5Ac)n estimated from the peak area was approximately 1000-fold of that of PED for (Neu5Ac)n.
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Evaluation of the DMB/HPLC-FD method for the analysis of polySia linked to glycan chains
All model compounds used in the establishment of the new method described above were oligo/polySia chains having free reducing termini. To apply this method to the analysis of polySia linked to core glycan chains, such reducing terminals must be generated. Unfortunately, no enzyme that specifically cleaves
2,3-sialyl linkage between proximal Neu5Ac and external Gal residue of NCAM N-glycans is available. Thus in the previous analysis of chicken brain NCAM polySia by HPAEC-PED, samples were subjected to hydrolysis in 0.1 M acetic acid for 15 min at 60°C before injection. In some experiments we pretreated polySia-containing glycopeptides with concentrated HCl to facilitate lactone formation and thus stabilized high DP polySia during mild acid hydrolysis (see Figure 7 in Inoue et al., 2000). Figure 7 shows peak area distribution of underivatized oligo/polySia in colominic acid before and after treatment with 0.1 M acetic acid for 15 min at 60°C (HPAEC- PED), and compared the results with the peak area distribution of DMB-derivatized oligo/polySia obtained under the optimized conditions (HPLC-FD). As is clearly demonstrated in Figure 7a, the peak area distribution of colominic acid significantly shifted to lower DP after the acid treatment. In contrast, peak area distribution of the DMB-tagged oligo/polySia was similar to that for untreated colominic acid, because the conditions used in DMB derivatization (48 h at 10°C in 0.02 M TFA) were milder than prehydrolysis conditions previously used. Moreover, due to the sensitivity of the fluorescence detector, high DP (DP 70–90) that could not be detected by PED was detected by the new method. Notably, the results given in Figure 7a were obtained by injecting 100 µg (total Neu5Ac) for HPAEC-PED analysis, whereas only 200 ng is needed for DMB-FD. In contrast to PED, FD response was proportional to the concentration of compounds in a wide range and little affected by DP. This wide range of high sensitivity is most important in applying the method to the analysis of samples obtainable in only minute amounts.
We also examined if the new method is applicable to DP analysis of polySia chains linked to core glycan chains of glycoproteins. In embryonic NCAM it has been reported that polySia chains are linked 2,3 to external Gal residues of N-glycan chains (mainly triantennary) (Kudo et al., 1996). We have published that the first-order rate constant of mild acid hydrolysis of ketosidic bonds in 0.1 M TFA at 80°C was about fourfold larger in Neu5Ac2,3Gal than in Neu5Gc2,8Neu5Gc (Nadano et al., 1986). Here first-order rate constants of hydrolysis of ketoside bonds in Neu5Ac2,3Galß1,4Glc, Neu5Ac2,8Neu5Ac, 2,8-linked (Neu5Ac)10, and (Neu5Ac)21 were determined in 0.02 M TFA at 50°C using HPAEC-PED so that concentrations of all reaction products as well as the reactant at time t could be quantified. The rate constants for 2,8-linked polyNeu5Ac were calculated assuming that every interketosidic linkage is hydrolyzed at the same rate, and formulating the rate equation. The apparent rate constants thus obtained were 2.0 and 2.3 x 10–3 min–1 for 2,8-linked (Neu5Ac)10 and (Neu5Ac)21, respectively (Inoue and Inoue, 2001). When these rate constants are compared with that of the first-order hydrolysis reaction of the ketosidic bond for Neu5Ac2,3Galß1,4Glc, 5.7 x 10–3 min–1, the 2,3-sialyl linkage between the proximal Neu5Ac and the Gal residue is considered to be cleaved significantly faster than internal 2,8-sialyl linkages during the reaction of NCAM-derived polysialylated glycopeptide with DMB under the optimized conditions.
The DP distribution profiles for polySia-containing glycopeptide isolated from embryonic day 12 chicken brain by the HPAEC-PED and DMB/HPLC-FD methods are shown in Figure 7b. For HPAEC-PED, samples were pretreated in 0.1 M acetic acid at 60°C for 15 min, and 8 µg were injected. For DMB/HPLC-FD analysis samples were derivatized with the DMB-reagent under three different conditions, 48 h at 10°C, 30 min and 2 h at 50°C, and 400 ng (total Neu5Ac) were injected. As clearly seen from the figure, DP distribution profiles obtained by the new method are essentially the same as those obtained by HPAEC-PED. For the chicken brain sample similar results were obtained using more vigorous conditions (2 h at 20°C or 30 min at 50°C) of DMB-derivatization than the optimized ones (48 h at 10°C) in contrast to the case for colominic acid. This can be ascribed to the fact that there existed polySia chains with shorter DP values in this sample as compared to colominic acid sample used: As already shown for model compound, the apparent DP distribution profile of lower DP is less sensitive to the conditions of the acid treatment than higher DP. As was the case for colominic acid, peaks of higher DP than those observed by PED were observed by FD due to high sensitivity of FD.
Application of DMB/HPLC-FD to polySia analysis in the homogenate of chicken brain
Apart from DP determination of the purified polySia-containing compounds, DMB/HPLC-FD is applicable for monitoring polySia-containing fraction during isolation and chromatographic separation because of its convenience and high sensitivity. Furthermore, the method can be used as a diagnostic assay for detecting the presence of polySia in cells and tissues. Figure 8 shows an elution profile obtained by injecting DMB-derivatized samples from about 10 mg (wet weight) of embryonic day 12 chicken brain tissue. In this analysis 11 mg of lyophilized brain tissue (corresponding to 80 mg of tissue) was delipidated and the residue was directly derivatized with the DMB reagent as described in Materials and methods. The chromatogram clearly shows the presence of polySia chains with DP as long as 50. It should be emphasized that polySia chains with DP higher than 50 was hardly detectable even by applying this ultrasensitive method directly to the tissue homogenate without purification procedures that had been argued to cause depolymerization of polySia. Detailed studies on developmental profile of NCAM glycoforms with varying DP of polySia chains are reported elsewhere (Inoue and Inoue, 2001).
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Oligo/polySia samples and the determination of total sialic acid
Authentic samples of oligoNeu5Ac (dimer to pentamer) were kindly donated by NGK Insulators (Handa, Japan). Neu5Ac and colominic acid was purchased from Nacalai Tesque (Kyoto, Japan). Col-N is a commercial sample (average DP
100), and col-Q is a major fraction separated from another lot of commercial colominic acid sample (average DP 50) purchased from the same supplier (Nacalai Tesque) by preparative chromatography on a MonoQ HR 10/10 column. (Neu5Ac)10, (Neu5Ac)14–20, and (Neu5Ac)20–28 were prepared by controlled acid hydrolysis of colominic acid followed by chromatographic separation of oligo/polyNeu5Ac on a DEAE-Sephadex column (Nomoto et al., 1982), or by direct HPLC fractionation of colominic acid without prehydrolysis on a MonoQ HR 10/10 (Pharmacia, Uppsala, Sweden) or a DNAPac PA-100 (Dionex, Sunnyvale, CA) column. 3'-sialyllactose and 6'-sialyllactose were purchased from Glyko (Novato, CA) and OGS (Oxford, UK), respectively. Total Neu5Ac in samples were determined by reverse-phase HPLC after maximum hydrolytic liberation of free Neu5Ac in 0.1 M TFA at 80°C followed by DMB derivatization as described previously (Inoue et al., 2000), or by HPAEC-PED (Zhang et al., 1997).
Derivatization of oligo/polySia with DMB
The reagent for the DMB derivatization of sialic acid (Hara et al., 1987) was slightly modified: the reaction mixture contained finally 2.7 M DMB (Dojinbo, Kumamoto, Japan), 9 mM sodium hydrosulfite, and 0.5 M ß-mercaptoethanol (Inoue et al., 1996). For routine analysis, a twice as much concentrated stock reagent containing 40 mM TFA was added 1:1 (v/v) to the sample solution to make a final volume of 40–200 µl. The concentration of TFA (5–20 mM), temperature (0–50°C), and reaction time (30 min–70 h) were varied for optimization. The optimized reaction conditions were determined as 10°C and 48 h in the presence of 20 mM TFA. The reaction was stopped by adding one-fifths volume of 0.2 M NaOH (for oligoSia) or 1 M NaOH (for polySia). The addition of NaOH was necessary to hydrolyze lactones formed during the reaction (Cheng et al., 1998).
HPLC systems
For HPAEC-PED analysis, DX-500 ion chromatography system (Dionex, Sunnyvale, CA, USA) was used with an ED-40 electrochemical detector and a CarboPac PA-100 column. Elution of oligo/polySia was performed at 1 ml/min with a concentration gradient of NaNO3 as described previously, while the concentration of NaOH was always kept at 0.1 M (Zhang et al., 1997; Lin et al., 1999). For DMB/HPLC-FD, a Hewlett-Packard HPLC system series 1100 was used with a fluorescence detector and a DNAPac PA-100 column. Fluorescence detector was set at 372 nm for excitation and 456 nm for emission (Lin et al., 2000). Elution was performed at 1 ml/min with segments of linear gradient of NaNO3 made by introducing 1 M NaNO3, 2%, 2%, 3%, 10%, 20%, 25%, 35% in water at 0, 3, 6, 14, 28, 43, and 95 min, respectively.
Chicken brain samples
PolySia-containing glycopeptides were isolated and purified from day 12 embryonic chicken brain (E12) as described previously (Inoue et al., 2000). The sample used in this article was eluted under a slightly included peak earlier than colominic acid (col-Q) from a Sephacryl S-200 column, and under a peak eluted with higher concentrations of NaCl than for col-Q from a MonoQ HR 10/10 column. In polySia analysis of crude brain samples, lyophilized homogenate (11 mg, from 80 mg wet weight of tissue) of embryonic day 12 chick brain was delipidated with a chloroform:methanol:0.01 M Tris–HCl (pH 8.0) mixture (4:8:3, v/v/v). The residue was washed with cold 80% ethanol and directly reacted with 200 µl of the DMB reagent for 48 h with shaking at 10°C. After reaction, 80 µl of clear supernatant separated by centrifugation was removed and alkalinized by adding 20 µl of 1 M NaOH. A 30-µl portion (corresponding to 10 mg of tissue) was injected on a DNAPac PA-100 column.
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This investigation was supported by the National Science Council Grant NSC 90-2311-B-001-021 (to S.I.) and National Health Research Institutes Grant NHRI-EX90-8805BPs (to Y.I.).
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Abbreviations |
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TopAbstractIntroductionResults and discussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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DMB, 1,2-diamino-4,5-methylenedioxybenzene; DP, degree of polymerization; HPAEC, high-performance anion-exchange chromatography; HPAEC-PED, HPAEC with pulsed electrochemical detection; HPLC, high-performance liquid chromatography; HPLC-FD, HPLC with fluorescence detection; NCAM, neural cell adhesion molecule; Neu5Ac, N-acetylneuraminic acid; polyNeu5Ac,
2,8-poly(N-acetylneuraminic acid); oligo/polySia-Q, oligo/polySia tagged by fluorogenic reagent DMB at their reducing termini (Q represents quinoxalinone derivative formed on reaction of Sia with DMB; see Lin et al., 2000); polySia, polysialic acid; Sia, sialic acid; Neu5Acn, 2,8-linked oligo/polyNeu5Ac with DP = n; TFA, trifluoroacetic acid; TLC, thin-layer chromatography.
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1 To whom correspondence should be addressed
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References |
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