基于色谱法与质谱法分析唾液酸的衍生方法的研究进展

唾液酸是一类拥有9碳核心结构的单糖,广泛存在于脊椎动物体内,以及部分无脊椎动物、真菌和细菌中。唾液酸在生物体内可以以游离形式存在,也经常作为重要的组成部分连接于糖缀合物的末端,这使得其能够参与到多项生理活动中,且与炎症、癌症等疾病密切相关。以色谱法、质谱法为核心的分析方法,是表征生物样品中唾液酸的最主要研究方法。为了提高检测灵敏度或色谱分离度,在分析之前通常需要对唾液酸进行衍生化处理。经过几十年的发展,研究者们逐步建立了多种衍生化方法。该文从单糖、自由唾液酸、N/O-聚糖、糖脂的角度综述了用于色谱与质谱分析唾液酸的衍生方法,并展望了该领域的应用及发展趋势。     

Cellular and Molecular Engineering of Glycan Sialylation in Heterologous Systems

Glycans have been shown to play a key role in many biological processes, such as signal transduction, immunogenicity, and disease progression. Among the various glycosylation modifications found on cell surfaces and in biomolecules, sialylation is especially important, because sialic acids are typically found at the terminus of glycans and have unique negatively charged moieties associated with cellular and molecular interactions. Sialic acids are also crucial for glycosylated biopharmaceutics, where they promote stability and activity. In this regard, heterogenous sialylation may produce variability in efficacy and limit therapeutic applications. Homogenous sialylation may be achieved through cellular and molecular engineering, both of which have gained traction in recent years. In this paper, we describe the engineering of intracellular glycosylation pathways through targeted disruption and the introduction of carbohydrate active enzyme genes. The focus of this review is on sialic acid-related genes and efforts to achieve homogenous, humanlike sialylation in model hosts. We also discuss the molecular engineering of sialyltransferases and their application in chemoenzymatic sialylation and sialic acid visualization on cell surfaces. The integration of these complementary engineering strategies will be useful for glycoscience to explore the biological significance of sialic acids on cell surfaces as well as the future development of advanced biopharmaceuticals.     

Communication: Synthetic Studies on Sialoglycoconjugates 25: Reactivity of Glycosyl Promoters in α-Glycosylation of N-Acetyl-Neuraminic Acid with the Primary and Secondary Hydroxyl Groups in the Suitably Protected Galactose and Lactose Derivatives

Development of an efficient α-glycoside synthesis of sialic acids is critically significant for the syntheses of sialoglycoconjugates, especially gangliosides which carry important biological functions¹ in biological systems. Previously, we demonstrated² a new α-glycosylation of sialic acids by use of dimethyl(methylthio)sulfonium triflate (DMTST)³ as the glycosyl promoter, the suitably protected glycosyl acceptors and the methyl 2-thioglycoside 1 of N-acetylneuraminic acid (Neu5Ac) as the donor in acetonitrile under kinetically controlled conditions, and accomplished⁴ the syntheses of a variety of gangliosides and their analogs.     

Fluorous derivatization combined with liquid chromatography/tandem mass spectrometry: a method for the selective and sensitive determination of sialic acids in biological samples

We have developed a novel method for selective and sensitive analysis of sialic acids (N‐acetylneuraminic, N‐glycolylneuraminic, and 2‐keto‐3‐deoxy‐D ‐glycero‐D ‐galactonononic acid) utilizing liquid chromatography/tandem mass spectrometry (LC/MS/MS) combined with a fluorous derivatization technique. In this method, the carboxylic groups in the sialic acids are derivatized via amidation with heptadecafluoroundecylamine, a commercially available perfluoroalkylamine reagent. This reaction proceeds rapidly and readily at room temperature in the presence of a condensation reagent. Subsequently, the derivatives are retained specifically on an LC column with a perfluoroalkyl stationary phase by means of a fluorophilic or 'fluorous' interaction, and detected by positive electrospray ionization MS/MS. The detection limits of the examined sialic acids are in the range of 60–750 amol on column. We show that the proposed method can be used to analyze trace amounts of sialic acids in biological samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination and distribution of N-acetyl- and N-glycolylneuraminic acids in culture media and cell-associated glycoconjugates from human malignant mesothelioma and adenocarcinoma cells

Sialic acids containing glycoconjugates are very common in human neoplasias and their expression frequently correlates with malignant phenotype and the tumor grade. The majority of tumor markers containing sialic acids in man involve changes in the amount of total sialic acids and in the presence of the two main sialic acid types, Neu5Ac and Neu5Gc, and their derivatives. The aim of the present study was to examine whether malignant mesothelioma cell lines synthesize sialic acid containing glycoconjugates at both the extracellular and cell membrane levels and particularly whether the type and the content of Neu5Ac and Neu5Gc are of biological importance for mesothelioma cell differentiation and evaluation of its prognosis. The study was performed in three human malignant mesothelioma cell lines, two with a fibroblast like phenotype (STAV-FCS and Vester) and one of epithelial differentiation (STAV-AB), which developed from the pleural effusions of patients with malignant mesothelioma and in one human adenocarcinoma cell line (Wart). Neu5Ac and Neu5Gc were determined following a mild hydrolysis step and a sample clean-up procedure. The determination was performed by reversed-phase HPLC after the NeuAc and NeuGc had been converted to per-O-benzoylated derivatives. It was found that Neu5Gc is the major sialic acid in the culture media of all cell lines examined. Molar ratios of Neu5Ac to Neu5Gc showed that Neu5Gc is the predominant sialic acid in the culture medium of the fibroblast-like mesothelioma cells. Neu5Ac is almost undetectable in the cell membrane, whereas Neu5Gc is present in considerable amounts. The obtained results suggest that the type and the content of Neu5Ac and Neu5Gc in culture media are of biological importance for mesothelioma cell differentiation and may be of value in the evaluation of prognosis.     

Determination of sialic acids in human serum by reversed-phase liquid chromatography with fluorimetric detection.

A simple, rapid and highly sensitive reversed-phase liquid chromatographic method has been developed for the determination of sialic acids in human serum. The sialic acids, released by hydrolysis of serum, are converted in borate buffer with malononitrile to highly fluorescent compounds. The reaction mixture is separated isocratically within 5 min using an octadecyl-bonded silica column and a mobile phase of methanol and ammonium acetate buffer (15:85, v/v; pH 5.5). Measurement of the fluorescence intensity of the reaction mixture at 434 nm with irradiation at 357 nm allowed determination of 30-1000 ng/ml of sialic acids with high reproducibility. The limit of detection was 2 ng/ml. Intra-day and inter-day coefficients of variation for assaying 300 ng/ml N-acetylneuraminic acid (NANA) were 1.5% (n = 9) and 2.6% (n = 7), respectively. The recoveries of NANA were 98.5-101.1% for serum. The method has been used for clinical determinations.     

唾液酸糖苷化方法学研究*

唾液酸是一类酸性九碳糖,通过α-糖苷键的方式广泛分布于生物体系内糖缀合物和多聚唾液酸中而发挥着重要的生物学功能。如何有效地构建唾液酸α-糖苷键,合成天然的含有唾液酸的糖缀合物、多聚唾液酸及其衍生物,是糖化学研究的热点和难点。近年来,人们基于唾液酸的结构特点,一方面通过在C2位引入易离去的基因,发展了直接成苷的方法,显著提高成苷的产率;另一方面,通过对C1和C3位引入辅助基因,发展了间接成苷的方法,提高了成苷的α-选择性。本文主要从直接成苷和间接成苷两个方面对目前研究的唾液酸糖苷化的化学方法学进行综述。     

Application of a sensitive fluorometric HPLC assay to determine the sialic acid content of infant formulas

The developing human brain requires high amounts of sialic acids. While human milk is very rich in sialic acids, cow’s milk based infant formulas provide lower amounts of sialic acids, and sialic acids are absent in soy milk based formulas. This has prompted interest in the supplementation of formulas with sialic acids, either free or bound to glycoconjugates. In order for fortification of infant formulas with sialic acids to be appropriate for the developmental needs of the infant, an accurate quantitation of sialic acid content of infant formulas through a reliable and easy-to-use method is, therefore, of great interest to industry. In the present method, we describe the application of one of the most widely used analytical techniques to the quantitation of sialic acids in infant formulas. Briefly, sialic acids are hydrolyzed from glycoconjugates, derivatized using 1,2-diamino-4,5-methylenedioxybenzene dihydrochloride (DMB), and separated using reversed-phase high-performance liquid chromatography. The method fulfilled the established criteria for validation, with an interassay standard deviation of less than 5%, accuracy greater than 97%, and surrogate recovery between 98 and 104%. An investigation of the ruggedness of the method identified two key criteria: both standards and samples must be subjected to the same temperature and pH conditions for an accurate quantitation; and prolonged storage (more than 2 days) of the DMB reagent and derivatives must be avoided. In conclusion, this method is specific, straightforward, and accurate and can be easily performed in a quality-assurance laboratory to track the level of sialic acid in formulas that contain both inherent and fortified amounts of sialic acids. Figure Infant formula and HPLC vials used for the sialic acid quantitation     

MALDI-MS analysis of sialylated N-glycan linkage isomers using solid-phase two step derivatization method

Sialic acids usually locate at the terminal of many glycan structures in either α(2,3) or α(2,6) linkage, playing different roles in various biological and pathological processes. Several linkage specific carboxyl derivatization methods have been reported to discriminate between α(2,3) and α(2,6)-linked sialic acids by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Among them, ethyl esterification was recently reported to achieve linkage specific derivatization between α(2,3) and α(2,6)-linked sialic acids with good selectivity. However, the method suffered from the instability of the generated lactones and byproducts of the derivatives. To overcome these shortcomings, a solid-phase two step derivatization method was introduced to convert the α(2,6)-linked sialic acid into ethyl esters and the α(2,3)-inked counterparts into N-methyl amides, respectively. Under the optimized derivatization conditions, our method was able to achieve accurate relative quantification of N-glycan as well as their corresponding sialylated linkage types, superior to the ethyl esterification method. The solid phase derivatization strategy was further applied to investigate N-glycans from biosimilar antibody drug and human serum from patients and healthy volunteers. This method has the potential to be used in the biomarker discovery and pharmaceutical industry.     

Probe sialidase substrate specificity using chemoenzymatically synthesized sialosides containing C9-modified sialic acid

A library of α2-3- and α2-6-linked sialyl galactosides containing C9-modified sialic acids was synthesized from C6-modified mannose derivatives using an efficient one-pot three-enzyme system. These sialosides were used in a high-throughput sialidase substrate specificity assay to elucidate the importance of C9-OH in sialidase recognition.     

Capillary electrophoretic separation of recombinant granulocyte-colony-stimulating factor glycoforms

Free zone capillary electrophoresis separated recombinant human granulocyte-colony-stimulating factor, expressed in Chinese hamster ovary cells, into two well-resolved species. Following incubation with neuraminidase, these species comigrated, eluting earlier than either of the original two species. This indicated that the observed heterogeneity was caused by different amounts of sialic acid present on the carbohydrate portion of the protein. It was determined that optimum separation occurred in the buffer pH range 7–9. Evidence is also presented to show that these glycoforms migrate in order of increasing numbers of sialic acids present.     

Derivatization of sialic acids for stabilization in matrix‐assisted laser desorption/ionization mass spectrometry and concomitant differentiation of α(2 → 3)‐ and α(2 → 6)‐isomers

Sialylated carbohydrates usually decompose by loss of sialic acid when ionized by matrix‐assisted laser desorption/ionization (MALDI) as the result of the labile carboxylic proton. Stabilization has previously been achieved by forming methyl esters with methyl iodide, a procedure that eliminates the labile proton. In this paper, we describe an alternative procedure for methyl ester formation that provides information on the sialic acid linkage directly from the MALDI spectrum. The sugars were desalted, dissolved in methanol, and treated with 4‐(4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl)‐4‐methylmorpholinium chloride (DMT‐MM). After removal of the solvent, the products were transferred directly to the MALDI target and examined from 2,5‐dihydroxybenzoic acid. Small amounts of N‐glycans derived from biological sources benefited from an additional clean‐up stage involving Nafion 117. α(2 → 6)‐Linked sialic acid produced only methyl esters whereas α(2 → 3)‐linked sialic acids were converted into their lactones providing a 32 Da difference in mass. Negative ion collision‐induced decomposition (CID) mass spectra of these neutralized glycans provided information, in many cases, on the antenna of N‐linked glycans to which the variously linked sialic acids were attached. The method was applied to N‐linked glycans released from bovine fetuin and porcine thyroglobulin. Copyright © 2008 John Wiley & Sons, Ltd.     

Two-step derivatization and mass spectral distinction of α2,3 and α2,6 sialic acid linkages on N-glycans by MALDI-TOF

Sialic acid linkages on N-glycans were distinguished by MALDI-TOF MS after two steps derivatization by dimethylamine and ammonium hydroxide. By using this method, more than 20 kinds of sialic acid with detailed linkage information were detected on A549 cells.     

Metabolic Glycoengineering with N‐Acyl Side Chain Modified Mannosamines

In metabolic glycoengineering (MGE), cells or animals are treated with unnatural derivatives of monosaccharides. After entering the cytosol, these sugar analogues are metabolized and subsequently expressed on newly synthesized glycoconjugates. The feasibility of MGE was first discovered for sialylated glycans, by using N‐acyl‐modified mannosamines as precursor molecules for unnatural sialic acids. Prerequisite is the promiscuity of the enzymes of the Roseman–Warren biosynthetic pathway. These enzymes were shown to tolerate specific modifications of the N‐acyl side chain of mannosamine analogues, for example, elongation by one or more methylene groups (aliphatic modifications) or by insertion of reactive groups (bioorthogonal modifications). Unnatural sialic acids are incorporated into glycoconjugates of cells and organs. MGE has intriguing biological consequences for treated cells (aliphatic MGE) and offers the opportunity to visualize the topography and dynamics of sialylated glycans in vitro, ex vivo, and in vivo (bioorthogonal MGE).     

Selective Engineering of Linkage‐Specific α2,6‐N‐Linked Sialoproteins Using Sydnone‐Modified Sialic Acid Bioorthogonal Reporters

The metabolic oligosaccharide engineering (MOE) strategy using unnatural sialic acids has recently enabled the visualization of the sialome in living systems. However, MOE only reports on global sialylation and dissected information regarding subsets of sialosides is missing. Described here is the synthesis and utilization of sialic acids modified with a sydnone reporter for the metabolic labeling of sialoconjugates. The positioning of the reporter on the sugar significantly altered its metabolic fate. Further in vitro enzymatic assays revealed that the 9‐modified neuraminic acid is preferentially accepted by the sialyltransferase ST6Gal‐I over ST3Gal‐IV, leading to the favored incorporation of the reporter into linkage‐specific α2,6‐N‐linked sialoproteins. This sydnone sugar presents the possibility of investigating the roles of specific sialosides.     

An improved method for preparation of perbenzoylated ganglioside-derived sialic acids and nanogram detection of N-acetyl- and N-glycolylneuraminic acid by high performance liquid chromatography.

An improved method for the preparation of perbenzoylated ganglioside-derived sialic acids is described. After mild acid hydrolysis, isolation of sialic acids can be achieved by Folch partition (Method A) or by anion exchange chromatography (Method B). Perbenzoylated sialic acids were freed from benzoylation reagents by a second Folch partition. Total recoveries of both methods were found to be greater than or equal to 90%, calculated from metabolically labelled gangliosides. Derivatized N-acetylneuraminic and N-glycolylneuraminic acids were separated and quantified by isocratic high performance liquid chromatography using a RP18 column as the stationary phase and methanol:water (8:2) as the mobile phase. Both sialic acids were completely separated and eluted as single peaks within 15 min, monitored by UV detection. As little as 20 ng of neuraminic acid could be detected, the detector being linear up to 5 micrograms tested.     

Sialoside analogue arrays for rapid identification of high affinity siglec ligands

The siglec family of sialic acid binding proteins participates in diverse cell surface biology that includes regulation of immune cell signaling and the interaction of neuronal cells with glial cells. The weak intrinsic affinity of the natural sialoside ligands has hampered the development of synthetic ligand based probes needed to elucidate their roles in siglec function. In this report, we describe a glycan microarray comprising a library of 9-acyl-substituted sialic acids incorporated into sialosides containing the Neu5Acalpha2-3Gal and Neu5Acalpha-6Gal linkages commonly recognized by the siglecs. The array is demonstrated to exhibit utility for detecting 9-acyl substituents that increase the affinity of siglecs for their ligands. Substituents that increase affinity are anticipated to be useful for the design of high affinity ligand based probes of siglec function.     

基因重组糖蛋白-人尿激酶原糖基化修饰的质谱测定

酶法结合生物质谱技术测定了基因重组糖蛋白-人尿激酶原(rhProUK)的N-糖含量、唾液酸含量分别为9％和5％。糖蛋白／肽先经Con A凝集素亲和富集,肽：N-糖苷酶F(PNGaseF)对N-糖基化位点进行特异性质量标记,然后利用Lc-MS/MS技术测定出N-糖基化位点在第302位天冬酰胺残基上。     

Analysis of N-acetyl and N-glycolylneuraminic acid in rat serum and tissues with Walker 256 carcinoma by high-performance liquid chromatography

Serum and tissue specimens from healthy Wistar rats and from rats with Walker 256 carcinoma were analysed for N-acetyl and N-glycolylneuraminic acid by high performance liquid chromatography (HPLC) as per-O-benzoylated derivatives. Both neuraminic acids were identified, while N-acetylneuraminic acid was the predominant sialic acid. Samples from rats with generalized metastasis showed a significant increase (45-80%) of total sialic acids. This phenomenon in serum is caused by the overproduction of sialic acids, as a result of synthesis of both types of neuraminic acids to a similar molar ratio. The increase of sialic acids in rat bones with metastatic cancer is mainly because of increased N-acetylneuraminic acid synthesis. These results suggest that the molecular mechanisms responsible for cancer metastasis in different tissues may be closely associated with increased synthesis of dominating neuraminic acid.     

Structural analysis of underivatized sialic acids by combined high-performance liquid chromatography-mass spectrometry

Mass spectra of chemically ionized, positive ions of underivatized N,O-acylated sialic acids, 2-deoxy-2,3-didehydro-N-acetylneuraminic acid and sialyl-alpha(2-3)-lactose were obtained by combined high-performance liquid chromatography--mass spectrometry, using a direct liquid inlet system. The mass spectra of the different compounds for which fragmentation schemes are proposed enable the differentiation between sialic acids, although the localization of O-substituents is not possible. However, since the various sialic acids separated well on high-performance liquid chromatography, combined high-performance liquid chromatography-mass spectrometry allowed their unequivocal characterization.