Evaluation of high-performance anion-exchange chromatography with pulsed electrochemical and fluorometric detection for extensive application to the analysisof homologous series of oligo- and polysialic acids in bioactive molecules.

Our previous studies have shown extensively diverse structures in oligo/polymers of sialic acid (oligo/polySia) that are expressed often in developmentally regulated manner on animal glycoconjugates. The aim of this study was to establish highlysensitive and specific methods that can be used to identify diverse types of oligo/polySia and thus can be applied to studies of biological phenomena associated with the differential expression of oligo/polySia chains with different degree of polymerization (DP). As model compounds, we analyzed five different homologous series of oligo/polySia, (-->8Neu5Acalpha2-->)(n), (-->9Neu 5Acalpha2-->)(n), (-->8Neu5Gcalpha2-->)(n), (-->5-O(glycolyl)-Neu5Gcalpha2-->)(n), and Neu5Gc9SO(4)alpha2-->(-->5-O(glycolyl)-Neu5Gcalpha2--> )(n), ()expressed in various biopolymers. The latter two structures have recently been identified in sea urchin egg receptor for sperm. First we examined application of high-performance anion-exchange chromatography (HPAEC) on a CarboPac PA-100 column with pulsed electrochemical detection (PED) to new types of oligo/polySiacompounds and confirmed that resolution of high polymers (DP >70) of sialic acids was remarkable as reported previously. However, there are limitations in sensitivity and selectivity in PED that become significant when material is available only in a minute amount or material contained a large proportion of protein. These limitations can be circumvented by fluorometric detection of oligo/polySia tagged with 1,2-diamino-4, 5-methyl-enedioxybenzene (DMB) at the reducing terminal residues after separation on a MonoQ HR5/5 column. The latter method can be applied to any type of oligo/polySia we examined if we choose the derivatization conditions and is more sensitive and specific than the method with PED for analysis of oligo/polySia with DP up to 25.     

Five-minute glycoprotein sialic acid determination by high-performance anion exchange chromatography with pulsed amperometric detection

Glycoprotein sialylation analysis is a common analytical step in characterizing biotherapeutic products and expression experiments to optimize production. In this article, a high-throughput (5-min) high-performance anion exchange chromatography with pulsed amperometric detection (HPAE–PAD)-based analytical method for glycoprotein sialic acid determination is described. Results from this method are compared with both published HPAE–PAD and 1,2-diamino-4,5-methylenedioxybenzene (DMB) derivatization followed by ultra high-performance liquid chromatography fluorescence detection (UHPLC–FLD) assays. The quantified sialic acid amounts agree with prior HPAE–PAD analyses within replicate error and with UHPLC–FLD within an average of 24%, which are equivalent results based on assay reproducibility.     

Glycomic strategy for efficient linkage analysis of di-, oligo- and polysialic acids

Sialic acid polymers of glycoproteins and glycolipids are characterized by a high diversity in nature and are involved in distinct biological processes depending inter alia on the glycosidic linkages between the present sialic acid residues. Though suitable protocols are available for chain length and sialic acid determination, sensitive methods for linkage analysis of di-, oligo-, and polysialic acids (di/oligo/polySia) are still pending. In this study, we have established a highly sensitive glycomic strategy for this purpose which is based on permethylation of di/oligo/polySia after tagging their reducing ends with the fluorescent dye 1,2-diamino-4,5-methylenedioxybenzene (DMB). Using DMB-labeled sialic acid di/oligo/polymers glycosidic linkages could be efficiently determined and, optionally, the established working procedure can be combined with HPLC for in depth characterization of distinct di/oligo/polySia chains. Moreover, the outlined approach can be directly applied to mammalian tissue samples and linkage analysis of sialic acid polymers present in biopsy samples of neuroblastoma tissue demonstrating the usefulness of the outlined work flow to screen, for example, cancer tissue for the presence of distinct variants of di/oligo/polySia as potentially novel biomarkers. Hence, the described strategy offers a highly sensitive and efficient strategy for identification of glycosidic linkages in sialic acid di/oligo/polymers of glycoproteins and glycolipids.     

Determination of mono-O-acetylated N-acetylneuraminic acids in human and rat sera by fluorometric high-performance liquid chromatography

A simple, rapid, and highly sensitive fluorometric high-performance liquid chromatographic method for the determination of N-acetylneuraminic acid and its mono-O-acetyl derivatives in human and rat sera is described. The neuraminic acids, released by hydrolysis of serum in 2 M acetic acid, are converted with 1,2-diamino-4,5-methylenedioxybenzene, a fluorogenic reagent for alpha-keto acids, to highly fluorescent derivatives without the occurrence of O-acetyl migration and de-O-acetylation. The derivatives are separated isocratically within 25 min by reversed-phase chromatography using a TSK gel ODS-120T column. The limits of detection are 57-192 fmol in a 10-microliters injection volume at a signal-to-noise ratio of 3. This sensitivity permits precise determination of the neuraminic acids in 5 microliters of human and rat sera.     

Quantitation of the glycation intermediate 3-deoxyglucosone by oxidation with rabbit liver oxoaldehyde dehydrogenase to 2-keto-3-deoxygluconic acid followed by high-performance liquid chromatography

A simple and sensitive method for the detection of 3-deoxyglucosone was developed using oxidation with crude oxoaldehyde dehydrogenase to 2-keto-3-deoxygluconic acid followed by high-performance liquid chromatography (HPLC). Oxoaldehyde dehydrogenase was prepared from rabbit liver and partially characterized. 2-Keto-3-deoxygluconic acid produced from 3-deoxyglucosone by oxoaldehyde dehydrogenase was derivatized with 1,2-diamino-4,5-methylenedioxybenzene, and the fluorescent products were detected and quantitated by HPLC using a solvent containing borate. In the presence of borate, 2-keto-3-deoxygluconic acid was completely separated from N-acetylneuraminic acid. The detection limit of 3-deoxyglucosone was 2.5 pmol/injection (10 μl) at a signal-to-noise ratio of 3. This method was used to confirm the inhibitory effect of aminoguanidine on glycation.     

Comparative studies of HPLC-fluorometry and LC/MS method for the determination of N-acetylneuraminic acid as a marker of deteriorated ophthalmic solutions

Methods for determining the deterioration of ophthalmic solutions using both high-performance liquid chromatography (HPLC) with fluorescence detection and liquid chromatography coupled with selected ion monitoring mass spectrometry (LC/MS) are described. The methods are based on the determination of N-acetylneuraminic acid (NeuAc) released by the hydrolysis of foreign bodies that contaminate eye drops during use. The released NeuAc was either labeled with 1,2-diamino-4,5-methylenedioxybenzene (DMB) for fluorometric detection or detected without derivatization by mass spectrometry. The calibration curves for NeuAc showed good linearity between 1.2 ng/mL and 39 ng/mL for fluorometric HPLC and 5.0 ng/mL and 100 ng/mL for LC/MS, respectively. Detection limits for fluorometric HPLC and LC/MS were 0.20 ng/mL and 0.88 ng/mL, respectively. The NeuAc content of some model glycoproteins determined by LC/MS method were 62-78% of those determined by fluorometry. The differences are attributed to matrix effects but the LC/MS method afforded sufficiently high sensitivity that NeuAc in the foreign bodies could be determined in eight of nine test samples.     

Polysialic acid profiles of mice expressing variant allelic combinations of the polysialyltransferases ST8SiaII and ST8SiaIV

The post-translational modification of the neural cell adhesion molecule (NCAM) by polysialic acid (polySia) represents a remarkable example of dynamic modulation of homo- and heterophilic cell interactions by glycosylation. The synthesis of this unique carbohydrate polymer depends on the polysialyltransferases ST8SiaII and ST8SiaIV. Aiming to understand in more detail the contributions of ST8SiaII and ST8SiaIV to polySia biosynthesis in vivo, we used mutant mouse lines that differ in the number of functional polysialyltransferase alleles. The 1,2-diamino-4,5-methylenedioxybenzene method was used to qualitatively and quantitatively assess the polySia patterns. Similar to the wild-type genotype, long polySia chains (>50 residues) were detected in all genotypes expressing at least one functional polysialyltransferase allele. However, variant allelic combinations resulted in distinct alterations in the total amount of poly-Sia; the relative abundance of long, medium, and short polymers; and the ratio of polysialylated to non-polysialylated NCAM. In ST8SiaII-null mice, 45% of the brain NCAM was non-polysialylated, whereas a single functional allele of ST8SiaII was sufficient to polysialylate approximately 90% of the NCAM pool. Our data reveal a complex polysialylation pattern and show that, under in vivo conditions, the coordinated action of ST8SiaII and ST8SiaIV is crucial to fine-tune the amount and structure of polySia on NCAM.     

Sialic acids in gastropods.

The occurrence of N-acetylneuraminic acid and N-glycolylneuraminic acid residues in preparations of the slug Arion lusitanicus (Gastropoda) was determined by sodium dodecyl sulphate electrophoresis of the proteins followed by lectin blots stained with the sialic acid specific lectin from Maackia amurensis, by the sensitivity of this binding to sialidase from Clostridium perfringens, by specific fluorescent labelling of sialic acids with 1,2-diamino-4,5-methylenedioxybenzene, by the determination of the sensitivity to sialate-pyruvate-lyase, by co-migration with standards on high performance anion exchange chromatography with pulsed amperometric detection and by identification of the typical masses in the fragmentation patterns of the trimethylsilyl derivatives after gas chromatography. It is the first time sialic acids are identified in gastropods.     

A comparative study of monosaccharide composition analysis as a carbohydrate test for biopharmaceuticals

The various monosaccharide composition analysis methods were evaluated as monosaccharide test for glycoprotein-based pharmaceuticals. Neutral and amino sugars were released by hydrolysis with 4–7 N trifluoroacetic acid. The monosaccharides were N-acetylated if necessary, and analyzed by high-performance liquid chromatography (HPLC) with fluorometric or UV detection after derivatization with 2-aminopyridine, ethyl 4-aminobenzoate, 2-aminobenzoic acid or 1-phenyl-3-methyl-5-pyrazolone, or high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Sialic acids were released by mild acid hydrolysis or sialidase digestion, and analyzed by HPLC with fluorometric detection after derivatization with 1,2-diamino-4,5-methylenedioxybenzene, or HPAEC-PAD. These methods were verified for resolution, linearity, repeatability, and accuracy using a monosaccharide standard solution, a mixture of epoetin alfa and beta, and alteplase as models. It was confirmed that those methods were useful for ensuring the consistency of glycosylation. It is considered essential that the analytical conditions including desalting, selection of internal standards, release of monosaccharides, and gradient time course should be determined carefully to eliminate interference of sample matrix.Various HPLC-based monosaccharide analysis methods were evaluated as a carbohydrate test for glycoprotein pharmaceuticals by an inter-laboratory study.     

Determination of sialic acids in milks and milk-based products

Sialic acids are becoming recognized as important components of milk-based products for infants and young children. As such, many companies now label the sialic acid content of their products. To control the labeling, suitable methods are required for this analysis. The objective of this work was to set up a rapid and sensitive method for the determination of the two most commonly occurring sialic acids, N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), using high-performance liquid chromatography (HPLC). The sialic acids were released from their parent oligosaccharides, glycoproteins, or glycolipids by mild acid hydrolysis using formic acid. They were then derivatized using 1,2-diamino-4,5-methylenedioxybenzene (DMB) and subsequently separated on a Zorbax SB-Aq Rapid Resolution column in less than 2 min. The method developed was validated on various milk-based products and ingredients containing sialic acid at levels from 0.3 to 900 mg/100 g. Spiking experiments indicate that the sialic acid recoveries ranged from 87% to 108%. The expanded measurement uncertainty was typically below 15% for Neu5Gc and typically below 10% for Neu5Ac or the sum of the sialic acids, with a few exceptions. The proposed method is fast, specific, and easy to set up for compliance analysis in a routine laboratory.     

The assay of methylglyoxal in biological systems by derivatization with 1,2-diamino-4,5-dimethoxybenzene.

A procedure for the assay of methylglyoxal in biological systems is described, together with sample storage, sample processing procedures, and statistical evaluation. Specimen data are presented. Methylglyoxal was assayed by derivatization with 1,2-diamino-4,5-dimethoxybenzene and high-performance liquid chromatography (HPLC) of the resulting quinoxaline, 6,7-dimethoxy-2-methylquinoxaline, with spectrophotometric or fluorescence detection. Derivatization, solid-phase extraction, and HPLC were performed under acid conditions to prevent the spontaneous formation of methylglyoxal from glyceraldehyde 3-phosphate and dihydroxyacetone phosphate during the assay. The limits of detection in the biological matrix were 45 pmol (absorbance detection) and 10 pmol (fluorimetric detection), the recovery was 58%, and the intra- and interbatch coefficients of variance were 7.7 and 30.0%, respectively. The concentration of methylglyoxal in whole blood from normal healthy human individuals was (mean +/- SE, nM) 256 +/- 92 (n = 12) and that from diabetic patients was 479 +/- 49 (n = 55), showing a significant increase in diabetes mellitus (P < 0.01; Mann-Whitney U test). Sample processing under acidic conditions was essential to avoid interferences. Previous estimates of the concentration of methylglyoxal in biological samples require re-evaluation.     

Acid-base properties of the reaction product of sialic acid with fluorogenic reagent, 1,2-diamino-4,5-methylenedioxybenzene (DMB)

N-Acetylneuraminic acid (Neu5Ac) forms the highly fluorophoric quinoxalinone derivative (Q) when treated with 1,2-diamino-4,5-methylenedioxybenzene (DMB). Effects of protonation and deprotonation on the fluorescence of Q were examined at room temperature. The strong fluorescence was found to be caused by the neutral form Q but not the protonated form of its excited state [Q]* and at pH below 1 the emission was completely quenched. The deprotonated singlet form [Q-]* was a less efficient fluorescer than [Q]*.     

Simultaneous determination of D-amino acids by the coupling method of D-amino acid oxidase with high-performance liquid chromatography

An enzymatic assay system of D-amino acids was established using the D-amino acid oxidase of Schizosaccharomyces pombe. In this method, the enzyme converts the D-amino acids to the corresponding α-keto acids, which are then reacted with 1,2-diamino-4,5-methylenedioxybenzene (DMB) in an organic solvent. The resultant fluorescent compounds are separated and quantified by high-performance liquid chromatography (HPLC). Use of an organic solvent following the α-keto acid modification with DMB prevents the non-enzymatic deamination of L-amino acids, which are generally present at much higher concentrations than D-amino acids in biological samples. With this method, D-Glu, D-Asn, D-Gln, D-Ala, D-Val, D-Leu, D-Phe, and D-Ile can be quantified in the order of micromolar, and other D-amino acids except D-Asp can be assayed within a sensitivity range of 50-100 μM. The established enzymatic method was used to analyze the d-amino acid contents in human urine. The concentration of D-Ser obtained using this enzymatic method (223 μM) was in good agreement with that obtained using the conventional HPLC method (198 μM). The enzymatic method also demonstrated that the human urine contained 5.45 μM of d-Ala and 0.91 μM of D-Asn. Both D-amino acids were difficult to be identified using the conventional method, because the large signals from L-amino acids masked those from d-amino acids. The enzymatic method that we have developed can circumvent this problem.     

A universal fluorescent acceptor for high-performance liquid chromatography analysis of pro- and eukaryotic polysialyltransferases

Polysialyltransferases (polySTs) play critical roles in diverse biological processes, including neural development, tumorigenesis, and bacterial pathogenesis. Although the bacterial enzymes are presumed to have evolved to provide molecular mimics of the host-specific polysialic acid, no analytical technique is currently available to facilitate a direct comparison of the bacterial and vertebrate enzymes. Here we describe a new fluorescent acceptor, a 1,2-diamino-4,5-methylenedioxybenzene (DMB)-labeled trimer of α2,8-linked sialic acid (DMB-DP3), which primes both pro- and eukaryotic polySTs. High-performance liquid chromatography separation and fluorescence detection (HPLC-FD) of reaction products enabled the sensitive and quantitative detection of polyST activity, even using cell lysates as enzyme source, and revealed product profiles characteristic of each enzyme. Single product resolution afforded by this assay system revealed mechanistic insights into a kinetic lag phase exhibited by the polyST from Neisseria meningitidis serogroup B during chain elongation. DMB-DP3 is the first fluorescent acceptor shown to prime the mammalian polySTs. Moreover, product profiles obtained for the two murine polySTs provided direct biochemical evidence for enzymatic properties that had, until now, only been inferred from the analysis of biological samples. With DMB-DP3, we introduce a universal acceptor that provides an easy, fast, and reliable system for the comprehensive mechanistic and comparative analysis of polySTs.     

Sialic acids inTrichoplusia ni andDanaus plexippus egg glycoproteins

Baculovirus expression vector insect cell system (BEVIS) is useful for high level production of human therapeutic proteins. However, it has been reported that recombinant glycoproteins produced from this system are, in many cases, biologically inactive or less active than authentic counterparts, due to incomplete glycosylation potential of insect cells used so far, producing recombinant proteins with only high-or paucimannosidic oligosaccharides without sialylation. The presence of sialic acids in insects is still controversial. Egg proteins ofTrichoplusia ni andDanaus plexippus were isolated, and the presence of sialic acids was examined using reverse-phase fluorescent HPLC after derivatization of samples with 1,2-diamino-4,5-methylenedioxybenzene (DMB). The proteins of both eggs were shown to contain 5-N-acetylneuraminic acid. The results suggest that both insects may be able to produce proteins with sialylated complex-type oligosaccharide chains.     

Fluorometric high-performance liquid chromatography of N-acetyl- and N-glycolylneuraminic acids and its application to their microdetermination in human and animal sera, glycoproteins, and glycolipids

A simple, rapid, and highly sensitive high-performance liquid chromatographic method is described for the determination of N-acetyl- and N-glycolylneuraminic acids in human and animal sera, glycoproteins, and glycolipids. The neuraminic acids, released by acid hydrolysis of these biological samples, are converted in dilute sulfuric acid with 1,2-diamino-4,5-methylene-dioxybenzene, a fluorogenic reagent for alpha-keto acids, to highly fluorescent derivatives. The derivatives are separated within 12 min on a reversed-phase column (Radial-Pak cartridge C18) with an isocratic elution and detected fluorometrically. The detection limits are 25 fmol (7.7 pg) for N-acetylneuraminic acid and 23 fmol (7.5 pg) for N-glycolylneuraminic acid in a 10-microliter injection volume at a signal-to-noise ratio of 2. This method permits precise determination of the neuraminic acids in 5 microliter of human and animal sera or in 0.25-2.5 micrograms of glycoproteins and glycolipids.     

Fluorometric determination of 2-oxoadipic acid, a common metabolite of tryptophan and lysine, by high-performance liquid chromatography with pre-chemical derivatization

2-Oxoadipic acid, a key metabolite of tryptophan and lysine, reacted with 1,2-diamino-4,5-methylenebenzene in an acidic solution to produce a fluorescent derivative. The reaction product was separated using a Tosoh ODS-80Ts column with 20 mmol/L of KH?PO?-K?HPO? buffer (pH 7.0) containing 26% methanol at a flow rate 0.8 mL/min. The excitation wavelength of detection was 367 nm, and the emission wavelength was 446 nm. The limit of quantification was 1 pmol per injection, sufficiently sensitive for the determination of 2-oxoadipic acid in human and experimental animal urine.     

Simplified cysteine dioxygenase activity assay allows simultaneous quantitation of both substrate and product

A high-performance liquid chromatography (HPLC) method for enzyme activity assays using a hydrophilic interaction liquid chromatography (HILIC) column in combination with an evaporative light scattering detector was developed. The method was used to measure the activity of the non-heme mono-iron enzyme cysteine dioxygenase. The substrate cysteine and the product cysteine sulfinic acid are very weak chromophores, making direct ultraviolet (UV) detection without derivatization rather insensitive; moreover, derivatization of cysteine is often not efficient. Using the system described, underivatized substrate and product in samples from cysteine dioxygenase activity assays could be separated and analyzed. Furthermore, it was possible to quantify cysteic acid, the noncatalytic oxidation product of cysteine sulfinic acid. Acetone was used both to stop the enzymatic reaction by protein precipitation and as an organic mobile phase, making sample preparation very easy and the assay highly reproducible.     

Degree of polymerization (DP) of polysialic acid (polySia) on neural cell adhesion molecules (N-CAMS): development and application of a new strategy to accurately determine the DP of polySia chains on N-CAMS

Alpha2,8-linked polysialic acid (polySia) is a structurally unique antiadhesive glycotope that covalently modifies N-linked glycans on neural cell adhesion molecules (N-CAMs). These sugar chains play a key role in modulating cell-cell interactions, principally during embryonic development, neural plasticity, and tumor metastasis. The degree of polymerization (DP) of polySia chains on N-CAM is postulated to be of critical importance in regulating N-CAM function. There are limitations, however, in the conventional methods to accurately determine the DP of polySia on N-CAM, the most serious being partial acid hydrolysis of internal alpha2,8-ketosidic linkages that occur during fluorescent derivatization, a step necessary to enhance chromatographic detection. To circumvent this problem, we have developed a facile method that combines the use of Endo-beta-galactosidase to first release linear polySia chains from N-CAM, with high resolution high pressure liquid chromatography profiling. This strategy avoids acid hydrolysis prior to chromatographic profiling and thus provides an accurate determination of the DP and distribution of polySia on N-CAM. The potential of this new method was evaluated using a nonpolysialylated construct of N-CAM that was polysialylated in vitro using a soluble construct of ST8Sia II or ST8Sia IV. Whereas most of the oligosialic acid/polySia chains consisted of DPs approximately 50-60 or less, a subpopulation of chains with DPs approximately 150 to approximately 180 and extending to DP approximately 400 were detected. The DP of this subpopulation is considerably greater than reported previously for N-CAM. Endo-beta-galactosidase can also release polySia chains from polysialylated membranes expressed in the neuroblastoma cell line, Neuro2A, and native N-CAM from embryonic chick brains.     

Polysialic Acid Is Present in Mammalian Semen as a Post-translational Modification of the Neural Cell Adhesion Molecule NCAM and the Polysialyltransferase ST8SiaII

Fertilization in animals is a complex sequence of several biochemical events beginning with the insemination into the female reproductive tract and, finally, leading to embryogenesis. Studies by Kitajima and co-workers (Miyata, S., Sato, C., and Kitajima, K. (2007) Trends Glycosci. Glyc, 19, 85–98) demonstrated the presence of polysialic acid (polySia) on sea urchin sperm. Based on these results, we became interested in the potential involvement of sialic acid polymers in mammalian fertilization. Therefore, we isolated human sperm and performed analyses, including Western blotting and mild 1,2-diamino-4,5-methylenedioxybenzene-HPLC, that revealed the presence α2,8-linked polySia chains. Further analysis by a glyco-proteomics approach led to the identification of two polySia carriers. Interestingly, besides the neural cell adhesion molecule, the polysialyltransferase ST8SiaII has also been found to be a target for polysialylation. Further analysis of testis and epididymis tissue sections demonstrated that only epithelial cells of the caput were polySia-positive. During the epididymal transit, polySia carriers were partially integrated into the sperm membrane of the postacrosomal region. Because polySia is known to counteract histone as well as neutrophil extracellular trap-mediated cytotoxicity against host cells, which plays a role after insemination, we propose that polySia in semen represents a cytoprotective element to increase the number of vital sperm.