Analyzing Sialic Acids Using High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection

Analytical Biochemistry 283, 3–9 (2000) doi:10.1006/abio.2000.4643, available online at http://www.idealibrary.com on

THEMATIC REVIEW Analyzing Sialic Acids Using High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection Jeffrey S. Rohrer Dionex Corporation, 1228 Titan Way, Sunnyvale, California 94088

Sialic acids are a large family of N- and O-substituted neuraminic acids. The amino group of neuraminic acid is linked to an acetyl or glycolyl group, yielding N-acetylneuraminic acid (Neu5Ac) 1 and Nglycolylneuraminic acid (Neu5Gc), respectively. Replacement of the amino group of neuraminic acid with a hydroxyl group yields 3-deoxy-D-glycero-D-galacto-2nonulosonic acid (KDN). O-substitution of one or more of the hydroxyl groups of Neu5Ac, Neu5Gc, and KDN with methyl, acetyl, lactoyl, sulfate, and phosphate groups yields the large family of sialic acids. The terminal positions of many glycoproteins and glycolipids are occupied by sialic acids. The structures, biosynthesis, cellular locations, and functions of sialic acids have been reviewed (1, 2). Since those reviews, additional sialic acids have been identified (3). The oligosaccharides of glycoproteins have many biological functions and some of these functions have been attributed to an oligosaccharide’s sialic acids (4). For example, when a mammalian serum glycoprotein’s oligosaccharides lose Neu5Ac residues, the exposed galactose residues are recognized by the hepatic asialoglycoprotein receptor and the glycoprotein is removed from circulation (5). In some cases the loss of Neu5Ac causes reduced activity (6). The importance of Neu5Ac to a glycoprotein’s serum half-life, and possibly its activity, emphasizes the need to determine the Neu5Ac content of a glycoprotein when assaying its function and/or its efficacy as a pharmaceutical. The separation and detection of carbohydrates using HPAE-PAD was first reported in 1983 (7). In that 1 Abbreviations used: CHO, Chinese hamster ovary; HPAE, High performance anion-exchange chromatography; KDN, 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid; KDO, 3-deoxy-D-manno-2-octulosonic acid; 4MU-Neu5Ac, 2-[4-methylumbelliferone]-␣-ketoside; Neu5Ac, N-acetylneuraminic acid; Neu5Gc, N-glycolylneuraminic acid; PAD, Pulsed amperometric detection; Tg, thyroglobulin.

0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

Jeffery S. Rohrer

paper the authors showed that carbohydrates could be separated by anion-exchange chromatography using a pellicular anion-exchange resin and a sodium hydroxide eluent. This strong alkaline eluent was ideally suited for detection of the separated carbohydrates by pulsed amperometry on a gold working electrode. Earlier, Johnson et al. (8) had shown that pulsed amperometry, a technique where changing potentials are applied to an electrode for compound detection, electrode cleaning, and electrode activation, could be used detect carbohydrates in alkaline solution on a platinum working electrode. The major advantage of PAD is that it is a direct detection technique, and therefore; carbohydrates require no derivatization for sensitive detection. Since 1983, HPAE-PAD has been used to determine free carbohydrates in a variety of matrices (e.g., fermentation broths and urine) and bound carbohydrates after release from a glycoconjugate (e.g., glycoprotein) or matrix (e.g., bacterial cell wall). The basic principles of HPAE-PAD and many of its applications for carbohydrate analysis have been reviewed (9 –11). Manzi et al. (12) first reported an HPAE-PAD determination of sialic acids. While Neu5Ac and Neu5Gc are stable in alkaline eluents, sialic acids containing Oacyl substituents are unstable at alkaline pH. Because they were interested in identifying and purifying sialic acids containing O-substitutions, their separation used a CarboPac PA1 column with an acetic acid/sodium acetate eluent and required the addition of 0.3 M sodium hydroxide to the column effluent to detect the sialic acids by PAD. Shortly thereafter, Lee (9) reported the separation of Neu5Ac and Neu5Gc and Blithe et al. (13) reported the determination of Neu5Ac in human choriogonadotropin ␤-subunit using alkaline eluents (0.1 M NaOH, 0.05 M sodium acetate and 0.15 M NaOH, 0.1 M NaOAc, respectively). Nearly all reported sialic acid separations have used alkaline eluents. 3

4

JEFFREY S. ROHRER

FIG. 1. Sialic Acids determined by HPAE-PAD. (A) The separation of a standard containing 200 pmol each Neu5Ac, KDN, and Neu5Gc. (B) The separation of a 0.1 N HCl digestion of human serum transferrin (3.3 ␮g injected). (C) The separation of an Arthrobacter ureafaciens neuraminidase digestion of human serum transferrin (3.3 ␮g injected). Peaks 1, 2, and 3 are Neu5Ac, KDN, and Neu5Gc, respectively. KDN was added as an internal standard to both the acid and neuraminidase digests of human transferrin. The conditions for acid and neuraminidase digestion have been previously described (38). The following separation conditions were the same for all separations. Sialic acids were separated on a CarboPac PA10 column set (guard and separator columns) with 100 mM sodium hydroxide and a sodium acetate gradient from 70 to 300 mM over the first 10 min of the separation. The eluent was held at the final gradient conditions for 1 min and then returned to the starting conditions over the next minute. The flow rate was 1.0 mL/min and an injection was made every 27 min. The pulsed amperometry waveform is depicted in Fig. 3. The column and amperometry cell were housed in a chromatography oven set at 30°C and the injection volume was 20 ␮L.

SEPARATION CONDITIONS

Neu5Ac and Neu5Gc are easily separated with any of three different CarboPac columns using a variety of eluent conditions. Most reported separations use a CarboPac 2 PA1 column eluted either isocratically with a NaOH/sodium acetate eluent (e.g., 0.1 M NaOH, 0.15 M sodium acetate) or with a constant [NaOH] and a gradient of sodium acetate (13–33). The CarboPac PA100 has been used with the same eluents (34 –37). Figure 1A shows that the CarboPac PA10 column separates Neu5Ac, KDN, and Neu5Gc (38). This separa2

CarboPac is a registered trademark of the Dionex Corporation.

tion is similar to that achieved on the PA1 column, but with the advantage that Neu5Gc is less retained on the PA10 column, which permits a faster separation (39). The gradient separations shown in Fig. 1 require only 27 min per injection, and if needed, the run time can be reduced. There are advantages to using a sodium acetate gradient rather than an isocratic eluent for separating Neu5Ac and Neu5Gc. The gradient separation allows for greater Neu5Ac retention compared to isocratic elution, while still eluting Neu5Gc in a reasonable time. The greater retention of Neu5Ac reduces the possibility of interference from unretained or poorly retained electroactive compounds (e.g., reduced Triton X-100) that may be present in the sample. Isocratic

REVIEW: CHROMATOGRAPHIC ANALYSIS OF SIALIC ACIDS

conditions that increase Neu5Ac retention cause long Neu5Gc retention times (⬎20 min) (39). Using isocratic elution, peak width increases with retention time. With a sodium acetate gradient, the more strongly retained Neu5Gc has a small peak width, which increases sensitivity. Therefore, gradient conditions are recommended when it is important to detect Neu5Gc when it is only a small percentage of the total sialic acid content of a sample. This is the often true when analyzing therapeutic glycoproteins expressed in Chinese hamster ovary (CHO) cells that, unlike noncancerous human cells, synthesize Neu5Gc. Because of possible Neu5Gc immunogenicity, it may be important to quantify low amounts of Neu5Gc in therapeutic glycoprotein preparations (40). An additional advantage of the PA10 column is that it is, unlike the PA1, compatible with some organic solvents, which can be used for cleaning a column contaminated with organic compounds. Neu5Ac, KDN, and Neu5Gc can also be separated on a PA1 column using a sodium hydroxide/sodium nitrate eluent (41). The sodium nitrate concentrations used for these separations are much lower than the sodium acetate concentrations used for similar separations. For example, using a 100 mM NaOH/100 mM sodium acetate eluent causes Neu5Ac, KDN, and Neu5Gc to elute at 4.68, 6.91, and 19.98 min, respectively, while the retention times with a 100 mM NaOH/5 mM sodium nitrate eluent are 5.65, 6.77, and 22.98 min, respectively. As noted above, the separation of base-labile sialic acids is possible at neutral pH with PAD accomplished by adding sodium hydroxide to the column effluent (12). There have been two additional reports of using this method for base-labile sialic acids (19, 42). Baselabile sialic acids are more commonly analyzed by labeling with a fluorophore such as 1, 2-diamino-4, 5-methylenedioxybenzene or o-phenylenediamine 䡠 2HCl followed by a reversed-phase chromatography separation (43, 44). The 1,2-diamino-4,5-methylenedioxybenzene method was combined with mass spectrometry to identify new base-labile sialic acids (3). These methods can also be used for Neu5Ac and Neu5Gc determinations, but require the analyst to prepare the derivatizing reagent, derivatize the sample (usually after acid hydrolysis) and standards, and filter or centrifuge the sample. In addition to the extra labor, these methods require the analyst to assume that the sample’s sialic acids are completely labeled and that both the standards and samples have been labeled to the same extent. Without the assistance of a method that directly detects sialic acids (e.g., HPAE-PAD) it is difficult to optimize both sample hydrolysis and the efficiency of labeling in the sample matrix. Sialic acid polymers have also been analyzed using HPAE-PAD. Many organisms synthesize these molecules and studies have shown that they have potential

5

roles in processes such as cell growth, differentiation, and fertilization (45). ␣2,8-linked polymers of Neu5Ac, Neu5Gc, and KDN were separated on a PA100 column with a constant concentration of sodium hydroxide and a gradient of sodium nitrate (41). Degrees of polymerization up to 80 could be resolved. The separations using the PA100 were faster than those achieved with the PA1. The maximum degree of polymerization that could be discerned using a sodium acetate gradient rather than a sodium nitrate gradient was 60. Recently, these separations were confirmed and extended to separations of a ␣2-9 Neu5Ac polymer, a Neu5Gc␣2-5-O- glycolyl polymer, and a Neu5Gc9SO 4capped Neu5Gc␣2-5-O- glycolyl polymer (46). RELEASE OF SIALIC ACIDS FROM GLYCOCONJUGATES

To quantify the sialic acids attached to a glycoconjugate they must first be released. An acid or enzyme treatment is used for this release. Historically, glycoproteins were treated with 0.1 N sulfuric acid at 80°C for 1 h (47). These or similar conditions have been used with HPAE-PAD (16, 19, 27, 29, 30, 42). In some cases the mild sulfuric acid solutions were neutralized with sodium hydroxide prior to HPAE-PAD analysis (16, 19, 29), although the purpose of this treatment is unclear. Sulfate ions could affect the chromatography, but neutralization does not remove sulfate. It is unlikely that direct injection of a typical sample volume (⬍50 ␮L) would affect the chromatography, because 5 ␮L of 1.5 N sulfuric acid can be injected onto a PA1 column, which was using a weaker eluent, with no adverse effect on the chromatography (48). In one publication sulfate was removed by treatment with barium carbonate (30). To avoid potential problems with sulfate most scientists have used volatile acids to release sialic acids for HPAE-PAD analysis. Dilute hydrochloric acid (e.g. 0.1N at 80°C for 1 h) has been the most common choice (17, 18, 20 –23, 26 –28, 36, 38, 41). Figure 1B shows a sialic acid analysis of human serum transferrin in which HCl was the hydrolysis acid. Dilute trifluoroacetic acid has also been used (13, 27, 30, 32, 38, 42). Acetic acid hydrolysis (e.g., 2 M at 80°C for 3 h) improves the recovery of O-substitutions found on some sialic acids (49) and has been used to release sialic acids prior to HPAE-PAD analysis (30, 38, 41, 42, 50). HPAE-PAD was used to determine that among formic, acetic, and propionic acids, propionic provided the highest preservation rate of O-substitution during acid hydrolysis (42). That study and one describing the optimization of acid hydrolysis for 3-deoxy-D-manno-2octulosonic acid (KDO, a carboxylic acid found in some glycoconjugates) determinations are good examples of how to optimize acid hydrolysis for releasing sialic acids from glycoconjugates (50). In these studies the analyst chooses an acid, acid concentration, and tem-

6

JEFFREY S. ROHRER

perature for each hydrolysis. Samples are hydrolyzed and aliquots are removed at set times and analyzed for released sialic acids to determine the optimum hydrolysis time for each set of conditions. The optimum time is when the ratio of sialic acids released to sialic acids destroyed is maximized. Because no sample derivatization and subsequent purification(s) are required, the multiple samples required to determine optimum hydrolysis conditions can be quickly prepared for and assayed by HPAE-PAD. A substrate blank and an acid blank should be included to test for possible electrochemical interferences (23). When volatile acids are used, the samples are usually dried in a vacuum concentrator, without applied heat, and reconstituted in de-ionized water. A neuraminidase (sialidase) can also be used to release bound sialic acids prior to HPAE-PAD analysis (17, 18, 20, 22–27, 29, 31, 34 –38, 41). There are a number of commercially available neuraminidases with defined linkage specificities (51). Samples treated with Arthrobacter ureafaciens (18, 20, 22, 24, 25, 31, 35–38, 41), Vibrio cholerae (17, 26, 31), Clostridium perfringens (24, 29, 34), and Newcastle disease virus (34) neuraminidases have been directly analyzed for released sialic acids by HPAE-PAD. Figure 1C shows an HPAE-PAD analysis of an A. ureafaciens neuraminidase digest of human serum transferrin. In this example 83 ␮g of transferrin was treated with 1 mU of neuraminidase in a total volume of 200 ␮L 0.1 M sodium acetate pH 5.0 for 18 h at 37°C. The digest was diluted to 500 ␮L with deionized water and 25 ␮L (3.3-␮g transferrin) was injected. Although neuraminidase digests are usually conducted in buffers containing strong anion-exchange eluent ions (e.g., acetate and phosphate,) there have been no reports of retention time shifts caused by sample injection. Conditions for neuraminidase digestion can be optimized in the same manner described for acid hydrolysis (23). As with acid hydrolysis, a substrate blank and an enzyme blank should be included to test for possible electrochemical interferences. When analyzing an unknown sample that is not in short supply, acid and neuraminidase treatments should be used. Acid hydrolysis is always a balance between release and destruction, and therefore; a neuraminidase treatment may provide a more accurate assessment of a sample’s sialic acid content. Conversely, it is possible that a neuraminidase treatment may not release all the sialic acids from a sample and that the acid treatment will provide the more accurate answer and/or aid in the development of an optimized neuraminidase treatment. APPLICATIONS

The most common application of HPAE-PAD sialic acid analysis is the assay of sialic acids released from

glycoconjugates. HPAE-PAD assays of sialic acids released from glycoproteins (16 –18, 21–23, 28, 29, 31, 32, 37, 38), glycolipids (14, 26, 36), tryptic glycopeptides (18, 20), pronase glycopeptides (28), and a synthetic glycoconjugate have been reported (27). The sialic acid content of an oligosaccharide can be determined after it is released from a glycoconjugate (14, 17, 22). The assay of sialic acids from gangliosides (14, 26) required oligosaccharide release from the lipid and subsequent removal of the lipid prior to HPAE-PAD analysis. It is likely that the lipid would foul the CarboPac column. HPAE-PAD was used to make the first identification of a glycosylinositol phospholipid anchor containing a sialic acid (36). In this application the lipid was not removed prior to injection. Sialic acids were released from tryptic glycopeptides, which were first separated by reversed-phase chromatography, to identify and characterize glycosylation attachment sites (18, 20). Sialic acids were determined from 8 ␮g of recombinant erythropoietin that was first separated by gel electrophoresis and then electroblotted to a polyvinylidene fluoride membrane (21). The sialic acid contents of other glycoproteins have been determined in the same manner (22, 32). The total sialic content of cells has been determined by HPAE-PAD (15, 19, 28). Cell lysates of cultured fibroblasts from three sialuria (a rare inborn error in metabolism caused by excessive sialic acid synthesis) patients were analyzed for Neu5Ac and CMP-Neu5Ac (15). This determined the different forms of soluble sialic acid. Standards and samples were in 25 mM sodium dihydrogen phosphate, pH 8.5, which had no noted effect on the chromatography. Another publication reported the determination of the Neu5Ac and Neu5Gc contents of platelets and precursors (19). To liberate sialic acids, the cells were treated with 0.1 N sulfuric acid for 1 h at 100°C. HPAE-PAD sialic acid analysis was also used to show that a mutation (lec32) in CHO cells nearly abrogates CMP-Neu5Ac synthetase activity (28). The amount of bound Neu5Ac in the cells was determined by preparing pronase glycopeptides and then determining the sialic acids releasing by mild acid treatment. Cytoplasmic fractions were analyzed for Neu5Ac and CMP-Neu5Ac using a gradient separation. The elution order of their separation was Neu5Ac, Neu5Gc, CMP-Neu5Ac, and CMP-Neu5Gc. Grollman et al. (34) analyzed cell surface membrane and secreted thyroglobulin (Tg) from a rat thyroid cell line grown in the presence or absence of Tg for ␣2–3and ␣2– 6-linked sialic acids. Purified Tg was treated with Clostridium perfringens and Newcastle disease virus neuraminidases. The Newcastle disease virus neuraminidase treatment established the amount of ␣2–3-sialic acid and the amount of ␣2– 6-sialic acid by difference from the total sialic acids determined by the C. perfringens neuraminidase treatment. Tg from cells

REVIEW: CHROMATOGRAPHIC ANALYSIS OF SIALIC ACIDS

grown in the presence of Tg had less ␣2– 6-sialic acids and the same amount of ␣2–3-sialic acids. HPAE-PAD has been used to purify sialic acids. Manzi et al. (12) reported that individual sialic acids could be purified when up to 2 ␮M of total sialic acids was loaded on a semipreparative PA1 column. To remove the sodium acetate (NaOH was not used.) column fractions were passed through a Dowex 50 (H⫹ form) column and then dried. In another publication, the column effluent was passed through a cation micromembrane suppressor to replace the sodium ions in the eluent with hydronium ions prior to fraction collection and subsequent drying (19). They reported a 70 – 95% recovery of purified sialic acids. HPAE-PAD sialic acid analysis has been used to characterize sialic acid polymers (24). Homopolymers of Neu5Ac, Neu5Gc, and KDN, all ␣2– 8-linked were sequentially treated with sodium periodate, sodium borohydride, and neuraminidase prior to HPAE-PAD analysis. This procedure determined the polymer type, size, and the number of chains bound to a glycoprotein of known molecular weight. To make these determinations, reduced Neu5Ac, 5-acetamido-3,5-dideoxy-L-arabinohepusonic acid, and Neu5Ac and were separated (in the order listed). Acid hydrolysis and neuraminidase digestion were used to determine the chain lengths of HPAE-separated sialic acid polymers (41). HPAE-PAD sialic acid analysis was one of the tools used to demonstrate the existence of an extracellular sialidase in CHO cells (34). The observation that some glycosidically bound sialic acid in mucin glycoproteins reacts as free sialic acid in the thiobarbituric acid assay was confirmed by HPAE-PAD (30). In addition to sialic acid analysis of glycoproteins, HPAE-PAD was used to analyze the reaction products of individual steps of the thiobarbituric acid assay. The donor specificity of Trypanosoma cruzi trans-sialidase was determined using a set of derivatives of 2-[4-methylumbelliferone]-␣ketoside (4MU-Neu5Ac) as the donor and lactose as the acceptor (25). HPAE-PAD sialic acid analysis was used to determine if the ketosidic linkage between Neu5Ac and 4MU survived the derivatization process. Each of seven reductive amination products of 4MU-Neu5Ac were treated with Arthrobacter ureafaciens neuramindase and analyzed for Neu5Ac. A recent publication showed that HPAE-PAD could be used to follow the conversion of the Neu5Ac on the sialoglycoconjugates of nerve cells to Neu5Gc (33). This application required a separation of Neu5Ac and N-propanoylneuraminic acid, an intermediate in their conversion of Neu5Ac to Neu5Gc. APPLICATIONS FOCUSED ON METHOD RUGGEDNESS

Three publications have reported the suitability of HPAE-PAD for accurate and reproducible determina-

7

tions of glycoconjugate sialic acid content (16, 27, 38). Hermentin and Seidat validated an HPAE-PAD assay of Neu5Ac released from ␣ 1-acid glycoprotein by acid hydrolysis (16). They demonstrated that the Neu5Ac content of ␣ 1-acid glycoprotein could be accurately (consistent with literature values) and reproducibly (RSD ⬍ 3%) determined when the amount of Neu5Ac injected was between 0.2 and 1.6 nmol. The method was rugged to variations in the amount of protein hydrolyzed (2.5–150 ␮g), the amount of protein injected (37.5 ␮g was the highest amount tested.), the concentration of phosphate buffered saline in the hydrolyzed sample (the highest concentration was 0.14 M chloride and 0.01 M phosphate), the mode of sample injection (manual or automated), and the system operator. An HPAE-PAD assay was validated for the determination of the Neu5Ac content of a synthetic glycoconjugate in which the sialyl-Tn antigen was conjugated to keyhole limpet hemocyanin (27). Equivalent results were reported for Neu5Ac released by either acid or enzymatic digestion. KDO was used as an internal standard to correct a gradual decrease in the Neu5Ac PAD response of standards that was attributed to system use (loss of response as injections increased) and to sample protein. Because the internal standard was added to samples prior to acid hydrolysis, it also corrected for sample handling and hydrolysis losses. After correcting for changes in the internal standard response, Neu5Ac response was linear between 0.2 and 1.6 nmol injected and the limit of quantification was 0.16 nmol (25-␮L injection). This method was also used to monitor possible Neu5Ac loss and formation of degradation products when the synthetic glycoconjugate was subjected to stressed conditions (e.g., increased temperature). A similar experimental design was used to show the effect of 0.1% trifluoroacetic acid, a common eluent additive for reversed-phase chromatography peptide separations, on the stability of sialic acids on glycopeptides isolated by reversed-phase chromatography (20). The third publication found that the determination of the Neu5Ac and Neu5Gc contents of glycoproteins was accurate and reproducible (38). This publication also showed that Neu5Ac and Neu5Gc responses decreased with recession of the working electrode surface below its original position, which is consistent with the decrease in Neu5Ac peak area response observed with increasing number of injections in the synthetic glycoconjugate assay (27). KDN was used as a post-hydrolysis internal standard to correct for changes caused by working electrode recession. Despite working electrode recession, the Neu5Ac and Neu5Gc contents of three glycoproteins could be accurately (consistent with published values) and reproducibly (RSDs ⬍ 5%) determined by using both external standards and the inter-

8

JEFFREY S. ROHRER

FIG. 2. Triple potential pulsed electrochemical waveform for carbohydrate detection.

FIG. 3. Quadruple potential pulsed electrochemical waveform for carbohydrate detection.

nal standard. The peak area response of the internal standard was suppressed in HCl and TFA hydrolysates. Correcting Neu5Ac and Neu5Gc peak areas for this change gave amounts consistent with those obtained by neuraminidase or acetic acid digestions. This suppression of peak area response was attributed to residual chloride or trifluoroacetate ions. No changes in peak area responses or retention times due to sample injections (⬍5 ␮g protein per injection) were reported. Peak area responses were reported to be linear between 0.01 and 0.5 nmol injected and minimum detection limits (S/N ⬎ 3) were ⬍5 pmol (25 ␮L injection), regardless of the amount of working electrode recession. The authors also noted that the KDN internal standard could not be separated from KDO, the internal standard used in the synthetic glycoconjugate analysis. Others have reported this coelution (30, 50). All the HPAE-PAD publications described here have used a triple potential pulsed amperometry waveform similar or identical to the one depicted in Fig. 2. As

noted earlier, with use the working electrode surface recedes and sialic acid peak area responses decrease. Although this peak area loss does not prevent the accurate and reproducible determination of sialic acids as directly demonstrated in Ref. (38) and indirectly demonstrated by the publications referenced in this review, preservation of peak area response is preferable. Recently a quadruple potential pulsed amperometry waveform was shown to preserve long-term peak area responses of carbohydrates (52). The waveform, shown in Fig. 3, uses reductive rather than oxidative electrode cleaning. This prevents working electrode recession and preserves long-term peak area response. The strengths and weaknesses of these two waveforms have been compared (53). Table 1 shows a comparison of a set of four experiments (data from Ref. 38) that used the triple potential waveform depicted in Fig. 2 to data from a similar set of four experiments using the quadruple potential waveform, shown in Fig. 3. Each set of experiments required 2 weeks of analyses and included analyses of neuraminidase and acid digests of

TABLE 1

Average Peak Areas of Sialic Acid Standards from Four Experiments Run with a Triple Potential Waveform (Fig. 2) and Four Experiments Run with a Quadruple Potential Waveform (Fig. 3) Triple potential waveform

Quadruple potential waveform

Number of injections a

Neu5Ac area (⫻10 5)

KDN area (⫻10 5)

Neu5Ac area (⫻10 5)

Number of injections

Neu5Ac area (⫻10 5)

KDN area (⫻10 5)

Neu5Ac area (⫻10 5)

107 107 65 65

8.57 7.50 6.95 6.73

6.27 5.46 5.14 5.14

12.1 10.7 9.97 9.72

107 53 110 45

24.3 23.2 24.2 24.4

23.3 22.4 22.9 23.5

41.2 40.2 42.3 41.7

Note. The data from the triple potential waveform were from a deeply recessed (350 ␮M) working electrode (38). The standard contained 200 pmol of each sialic acid and was analyzed using the method in Fig. 1. Each set of four experiments was conducted over a 2-week period and the experiments are listed in chronological order. a The number of injections of standard per experiment. Experiments with lower injection numbers had approximately the same number of injections of neuraminidase and acid digestions of glycoproteins.

REVIEW: CHROMATOGRAPHIC ANALYSIS OF SIALIC ACIDS

glycoproteins. After four experiments with the triple potential waveform, sialic acid peak areas had decreased 18.0 –21.5%. In contrast, four experiments with the quadruple potential waveform showed no significant change in sialic acid peak areas. No effects of prior sample injection on the peak area responses of standards were observed. Rocklin et al. (52) reported that a 14-day continuous analysis of a standard containing Neu5Ac and Neu5Gc showed a 6.6% decrease of Neu5Gc area and a 2.4% increase of Neu5Ac area. The data presented both here and in Ref. (52) suggest that the quadruple potential waveform should benefit those writing system suitability criteria for HPAE-PAD sialic acid assays. REFERENCES 1. Schauer, R. (1991) Glycobiology 1, 449 – 452. 2. Varki, A. (1992) Glycobiology 2, 25– 40. 3. Klein, A., Diaz, S., Ferreira, I., Lamblin, G., Roussel, P., and Manzi, A. E. (1997) Glycobiology 7, 421– 432. 4. Varki, A. (1993) Glycobiology 3, 97–130. 5. Ashwell, G., and Morrell, A. G. (1974) in Advances in Enzymology (Meister, A., Ed.), Vol. 41, pp. 99 –128, Wiley, New York. 6. Takeuchi, M., Inoue, N., Strickland, T. W., Kubota, M., Wada, M., Shimizu, R., Hoshi, S., Kozutsumi, H., Takasaki, S., and Kobata, A. (1989) Proc. Natl. Acad. Sci. USA 86, 7819 –7822. 7. Rocklin, R. D., and Pohl, C. A. (1983) J. Liq. Chromatogr. 6, 1577–1590. 8. Hughes, S., and Johnson, D. C. (1981) Anal. Chim. Acta 132, 11–22. 9. Lee, Y. C. (1990) Anal. Biochem. 189, 151–162. 10. Townsend, R. R. (1994) J. Chromatogr. Library 58, 181–209. 11. Lee, Y. C. (1996) J. Chromatogr. A 720, 137–149. 12. Manzi, A., Diaz, S., and Varki, A. (1990) Anal. Biochem. 188, 20 –32. 13. Blithe, D. L, Wehmann, R. E., and Nisula, B. C. (1989) Endocrinology 125, 2267–2272. 14. Wang, W., Erlansson, K., Lindh, F., Lundgren, T., and Zopf, D. (1990) Anal. Biochem. 190, 182–187. 15. Seppala, R., Teitze, F., Krasnewich, D., Weiss, P., Ashwell, G., Barsh, G., Thomas, G., Packman, S., and Gahl, W. (1991) J. Biol. Chem. 266, 7456 –7461. 16. Hermentin, P., and Seidat, J. (1991) in Protein Glycosylation: Cellular, Biotechnological, and Analytical Aspects (Condradt, H. S., Ed.), Vol. 15, pp.185–188, VCH Publishers, Cambridge, UK. 17. Lloyd, K. O., and Savage, A. (1991) Glycoconjugate J. 8, 493– 498. 18. Rohrer, J. S., and White, H. B., III (1992) Biochem. J. 285, 275–280. 19. Budd, T. J., Dolman, C. D., Lawson, A. M., Chai, W., Saxton, J., and Hemming, F. W. (1992) Glycoconjugate J. 9, 274 –278. 20. Rohrer, J. S., Cooper, G. A., and Townsend, R. R. (1993) Anal. Biochem. 212, 7–16.

9

21. Weitzhandler, M., Kadlecek, D., Avdalovic, N., Forte, J. G., Chow, D., and Townsend, R. R. (1993) J. Biol. Chem. 268, 5121– 5130. 22. Andersen, D. C., Goochee, C. F., Cooper, G., and Weitzhandler, M. (1994) Glycobiology 4, 459 – 467. 23. Hardy, M. R., and Townsend, R. R. (1994) Methods Enzymol. 230, 208 –225. 24. Ashwell, G., Berlin, W. K., and Gabriel, O. (1994) Anal. Biochem. 222, 495–502. 25. Lee, K. B., and Lee, Y. C. (1994) Anal. Biochem. 216, 358 –364. 26. Ruan, S., and Lloyd, K. O. (1994) Glycoconjugate J. 11, 249 –256. 27. Jorge, P., and Abdul-Wajid, A. (1995) Glycobiology 5, 759 –764. 28. Potvin, B., Raju, T. S., and Stanley, P. (1995) J. Biol. Chem. 270, 30415–30421. 29. Gowda, D. C., Jackson, C. M., Kurzban, G., McPhie, P., and Davidson, E. (1996) Biochemistry 35, 5833–5837. 30. Bhavanandan, V. P., Ringler, N., and Gowda, D. C. (1998) Glycobiology 8, 1077–1086. 31. Monica, T. J., Williams, S. B, Goochee, C. F., and Maiorella, B. L. (1995) Glycobiology 5, 175–185. 32. Zbebska, E., and Koscielak, J. (1999) Anal. Biochem. 275, 171–179. 33. Collins, B. E., Fralich, T. J., Itonori, S., Ichikawa, Y., and Schnaar, R. L. (2000) Glycobiology 10, 11–20. 34. Grollman, E. F., Motoyasu, S., Shimura, Y., Lau, J. T., and Ashwell, G. (1993) J. Biol. Chem. 268, 3604 –3609. 35. Gramer, M., Goochee, C., Chock, V., Brousseau, D., and Sliwkowksi, M. B. (1995) Biotechnology 13, 692– 698. 36. Stahl, N., Baldwin, M., Hecker, R., Pan, K. M., Burlingame, A. L., and Prusiner, S. B. (1992) Biochemistry 31, 5043–5053. 37. Weitzhandler, M., Hardy, M., Co, M. S., and Avdalovic, N. (1994) J. Pharm. Sci. 83, 1670 –1675. 38. Rohrer, J. S., Thayer, J., Weitzhandler, M., and Avdalovic, N. (1998) Glycobiology 8, 35– 43. 39. Technical Note 41 (1998) Dionex Corporation. 40. Noguchi, A., Mukuria, C. J., Suzuki, E., and Naiki, M. (1995) J. Biochem. 117, 59 – 62. 41. Zhang, Y., Inoue, Y., Inoue, S., and Lee, Y. C. (1997) Anal. Biochem. 250, 245–251. 42. Mawhinney, T., and Chance, D. (1994) Anal. Biochem. 223, 164 – 167. 43. Hara, S., Yamaguchi, M., Takemori, Y., Furuhata, K., Ogura, H., and Nakamura, M. (1989) Anal. Biochem. 179, 162–166. 44. Anumula, K. R. (1995) Anal. Biochem. 230, 24 –30. 45. Troy, F. A., II (1992) Glycobiology 2, 5–23. 46. Lin, S-L., Inoue, Y., and Inoue, S. (1999) Glycobiology 9, 807– 814. 47. Warren, L. (1959) J. Biol. Chem. 234, 1971–1975. 48. Davis, M. W. (1998) J. Wood Chem. Technol. 18, 235–252. 49. Varki, A., and Diaz, S. (1984) Anal. Biochem. 137, 236 –247. 50. Kiang, J., Shousun, C. S., Wang, L. X., Tang, M., and Lee, Y. C. (1997) Anal. Biochem. 245, 97–101. 51. Reuter, G., and Schauer, R. (1994) Methods in Enzymology 230, 168 –199. 52. Rocklin, R. D., Clarke, A. P., and Weitzhandler, M. (1998) Anal. Chem. 70, 1496 –1501. 53. Technical Note 21 (1998) Dionex Corporation.