Convenient pre-separation method depends on negative charges suitable for glycan sequencing analysis.

For precise structural analysis of complex glycans, it is necessary to fractionate glycans based on acidity prior to liquid chromatography-mass spectrometry analysis. Conventional fractionation using high-performance liquid chromatography is time consuming and not high throughput. Therefore, we developed a rapid method for the fractionation using a small cartridge column. This enables rapid multi-sample processing for glycan sequencing.

Microheterogeneity and Individual Differences of Human Urinary N-Glycome under Normal Physiological Conditions.

Urine is considered an outstanding biological fluid for biomarker discovery, reflecting both systemic and urogenital physiology. However, analyzing the N-glycome in urine in detail has been challenging due to the low abundance of glycans attached to glycoproteins compared to free oligosaccharides. Therefore, this study aims to thoroughly analyze urinary N-glycome using LC-MS/MS. The N-glycans were released using hydrazine and labeled with 2-aminopyridine (PA), followed by anion-exchange fractionation before LC-MS/MS analysis. A total of 109 N-glycans were identified and quantified, of which 58 were identified and quantified repeatedly in at least 80% of samples and accounted for approximately 85% of the total urinary glycome signal. Interestingly, a comparison between urine and serum N-glycome revealed that approximately 50% of the urinary glycome could originate from the kidney and urinary tract, where they were exclusively identified in urine, while the remaining 50% were common in both. Additionally, a correlation was found between age/sex and the relative abundances of urinary N-glycome, with more age-related changes observed in women than men. The results of this study provide a reference for human urine N-glycome profiling and structural annotations.

Increased levels of acidic free-N-glycans, including multi-antennary and fucosylated structures, in the urine of cancer patients.

We recently reported increased levels of urinary free-glycans in some cancer patients. Here, we focused on cancer related alterations in the levels of high molecular weight free-glycans. The rationale for this study was that branching, elongation, fucosylation and sialylation, which lead to increases in the molecular weight of glycans, are known to be up-regulated in cancer. Urine samples from patients with gastric cancer, pancreatic cancer, cholangiocarcinoma and colorectal cancer and normal controls were analyzed. The extracted free-glycans were fluorescently labeled with 2-aminopyridine and analyzed by multi-step liquid chromatography. Comparison of the glycan profiles revealed increased levels of glycans in some cancer patients. Structural analysis of the glycans was carried out by performing chromatography and mass spectrometry together with enzymatic or chemical treatments. To compare glycan levels between samples with high sensitivity and selectivity, simultaneous measurements by reversed-phase liquid chromatography-selected ion monitoring of mass spectrometry were also performed. As a result, three lactose-core glycans and 78 free-N-glycans (one phosphorylated oligomannose-type, four sialylated hybrid-type and 73 bi-, tri- and tetra-antennary complex-type structures) were identified. Among them, glycans with α1,3-fucosylation ((+/- sialyl) Lewis X), triply α2,6-sialylated tri-antennary structures and/or a (Man3)GlcNAc1-core displayed elevated levels in cancer patients. However, simple α2,3-sialylation and α1,6-core-fucosylation did not appear to contribute to the observed increase in the level of glycans. Interestingly, one tri-antennary free-N-glycan that showed remarkable elevation in some cancer patients contained a unique Glcß1-4GlcNAc-core instead of the common GlcNAc2-core at the reducing end. This study provides further insights into free-glycans as potential tumor markers and their processing pathways in cancer.

Structural analysis of N-glycans in chicken trachea and lung reveals potential receptors of chicken influenza viruses.

Although avian influenza A viruses (avian IAVs) bind preferentially to terminal sialic acids (Sia) on glycans that possess Siaα2-3Gal, the actual glycan structures found in chicken respiratory tracts have not been reported. Herein, we analyzed N-glycan structures in chicken trachea and lung, the main target tissues of low pathogenic avian IAVs. 2-Aminopyridine (PA)-labeled N-glycans from chicken tissues were analyzed by combined methods using reversed-phase liquid chromatography (LC), electrospray ionization (ESI)-mass spectrometry (MS), MS/MS, and multistage MS (MSn), with or without modifications using exoglycosidases, sialic acid linkage-specific alkylamidation (SALSA), and/or permethylation. The results of SALSA indicated that PA-N-glycans in both chicken trachea and lung harbored slightly more α2,6-Sia than α2,3-Sia. Most α2,3-Sia on N-glycans in chicken trachea was a fucosylated form (sialyl Lewis X, sLex), whereas no sLex was detected in lung. By contrast, small amounts of N-glycans with 6-sulfo sialyl LacNAc were detected in lung but not in trachea. Considering previous reports that hemagglutinins (HAs) of avian IAVs originally isolated from chicken bind preferentially to α2,3-Sia with or without fucosylation and/or 6-sulfation but not to α2,6-Sia, our results imply that avian IAVs do not evolve to possess HAs that bind preferentially to α2,6-Sia, regardless of the abundance of α2,6-Sia.

Toward robust N-glycomics of various tissue samples that may contain glycans with unknown or unexpected structures.

Glycans in tissues are structurally diverse and usually include a large number of isomers that cannot be easily distinguished by mass spectrometry (MS). To address this issue, we developed a combined method that can efficiently separate and identify glycan isomers. First, we separated 2-aminopyridine (PA)-derivatized N-glycans from chicken colon by reversed-phase liquid chromatography (LC) and directly analyzed them by electrospray ionization (ESI)-MS and MS/MS to obtain an overview of the structural features of tissue glycans. Next, we deduced the structures of isomers based on their elution positions, full MS, and MS/MS data, before or after digestions with several exoglycosidases. In this method, the elution position differed greatly depending on the core structure and branching pattern, allowing multiantennary N-glycan structures to be easily distinguished. To further determine linkages of branch sequences, we modified PA-N-glycans with sialic acid linkage-specific alkylamidation and/or permethylation, and analyzed the products by LC-MS and multistage MS. We determined the relative abundances of core structures, branching patterns, and branch sequences of N-glycans from chicken colon, and confirmed presence of characteristic branch sequences such as Lex, sialyl Lex, sulfated LacNAc, LacNAc repeat, and LacdiNAc. The results demonstrated that our method is useful for comparing N-glycomes among various tissue samples.

Sequential modifications of glycans by linkage-specific alkylamidation of sialic acids and permethylation.

Permethylation is useful for glycosidic linkage analysis, but is often accompanied by a large proportion of by-products, especially for glycans containing sialic acids (Sia). Unlike hydroxyl groups of glycans, which are converted to stable methyl ethers by permethylation, the carboxylic acids on Sia are converted to methyl esters, which are easily reversible to carboxylate under alkaline conditions. To overcome this problem, we used linkage-specific alkylamidation to protect Sia prior to the permethylation. This method not only decreased the levels of by-products, but also enabled us to distinguish isomers of α2,3- and α2,6-Sia while simultaneously determining other glycosidic linkages.

Characterisation of N-glycans in the epithelial-like tissue of the rat cochlea.

Membrane proteins (such as ion channels, transporters, and receptors) and secreted proteins are essential for cellular activities. N-linked glycosylation is involved in stability and function of these proteins and occurs at Asn residues. In several organs, profiles of N-glycans have been determined by comprehensive analyses. Nevertheless, the cochlea of the mammalian inner ear, a tiny organ mediating hearing, has yet to be examined. Here, we focused on the stria vascularis, an epithelial-like tissue in the cochlea, and characterised N-glycans by liquid chromatography with mass spectrometry. This hypervascular tissue not only expresses several ion transporters and channels to control the electrochemical balance in the cochlea but also harbours different transporters and receptors that maintain structure and activity of the organ. Seventy-nine N-linked glycans were identified in the rat stria vascularis. Among these, in 55 glycans, the complete structures were determined; in the other 24 species, partial glycosidic linkage patterns and full profiles of the monosaccharide composition were identified. In the process of characterisation, several sialylated glycans were subjected sequentially to two different alkylamidation reactions; this derivatisation helped to distinguish α2,3-linkage and α2,6-linkage sialyl isomers with mass spectrometry. These data should accelerate elucidation of the molecular architecture of the cochlea.

Quantitative LC-MS and MS/MS analysis of sialylated glycans modified by linkage-specific alkylamidation.

Sialic acids (Sia) are involved in various biological and pathological processes, and are often found attached to non-reducing ends of glycans through either α2,3- or α2,6-linkages. To quantitatively analyze glycan structures with these linkage isoforms by liquid chromatography-mass spectrometry (LC-MS), we established a linkage-specific two-step alkylamidation method for N-glycans. Using this method, carboxyl groups of α2,3- and α2,6-linked Sia are derivatized with two kinds of alkylamines with different mass values in a linkage-specific manner, allowing products to be easily distinguished. The reaction efficiencies for di-, tri-, and tetra-sialyl PA-N-glycans were >94%, with few by-products. Mixtures of 2-aminopyridine (PA)-tagged N-glycans from human α1-acid glycoprotein were subjected to the method, and products were analyzed by LC-MS and MS/MS, and simultaneously monitored with a fluorescence detector. The relative content of Siaα2-3Gal and Siaα2-6Gal was estimated from the integrated fluorescence intensity of each peak. Moreover, MS/MS data clearly indicated characteristic B-ion fragments of N-glycan branches, such as the sialyl Lex sequence, with Sia linkage-specific alkylamidation, suggesting that this method also provides useful information of branch sequences. We optimized the method with the aim of (1) enabling high-throughput analysis and (2) maximizing the analysis of glycans from various types of samples, including highly heterogeneous glycans.

Preparation of a Molecular Library of Branched ß-Glucan Oligosaccharides Derived from Laminarin.

To study the structure of ß-glucans, we developed a separation method and molecular library of ß-glucan oligosaccharides. The oligosaccharides were prepared by partial acid hydrolysis from laminarin, which is a ß-glucan of Laminaria digitata. They were labeled with the 2-aminopyridine fluorophore and separated to homogeneity by size-fractionation and reversed phase high-performance liquid chromatography (HPLC). Branching structures of all isomeric oligosaccharides from trimers to pentamers were determined, and a two-dimensional (2D)-HPLC map of the ß-glucan oligosaccharides was made based on the data. Next, structural analysis of the longer ß-glucan oligosaccharide was performed using the 2D-HPLC map. A branched decamer oligosaccharide was isolated from the ß-glucan and cleaved to smaller oligosaccharides by partial acid hydrolysis. The structure of the longer oligosaccharide was successfully elucidated from the fragment structures determined by the 2D-HPLC map. The molecular library and the 2D-HPLC map described in this study will be useful for the structural analysis of ß-glucans.

Structures and developmental alterations of N-glycans of zebrafish embryos.

Zebrafish is a model organism suitable for studying vertebrate development. We analyzed the N-glycan structures of zebrafish embryos and their alterations during zebrafish embryogenesis to obtain basic data for studying the roles of N-glycosylation. Multiple modes of high-performance liquid chromatography and multistage mass spectrometry were used for structural analysis of N-glycans. The N-glycans from deyolked embryos at 36 hours postfertilization, a mid-pharyngula stage, contained relatively higher amounts of complex- and hybrid-type glycans with LacNAc (Galß1-4GlcNAc) and/or sialyl LacNAc without additional ß1,4-Gal, which are commonly found in mammalian tissues, as well as abundant oligomannose-type glycans. Some of the complex- and hybrid-type glycans possessed various extended LacNAc structures, such as Galß1-4LacNAc, LacNAc-repeat or unique (+/- dHex)-GalNAcα1-GlcNAcß1-LacNAc. In contrast, the yolk of the embryo contains predominant oligomannose-type glycans and complex-type glycans with Galß1-4(Siaα2-3)Galß1-4(Fucα1-3)GlcNAc antennae. N-Glycan profiles obtained from deyolked embryos at different stages showed stage-dependent variation of complex- and hybrid-type glycans. At gastrula and early segmentation stages, complex- and hybrid-type glycans were minor components, and their antenna structures were mainly sialyl LacdiNAc (Siaα2-6GalNAcß1-4GlcNAc). From the mid-segmentation to pharyngula stages, those with LacNAc and/or α2,6-sialyl LacNAc antenna structures increased remarkably, and those with α2,3-sialyl LacNAc antenna, core α1,6-Fuc and bisecting GlcNAc modifications increased gradually. These results suggest the presence of mechanisms for regulating the antenna structures of complex/hybrid N-glycan biosynthesis in the phylotypic stage of vertebrate development.