Mitsuhiro Kinoshita

Minor acidic glycans, such as sulfated and phosphorylated glycans, constitute only a small fraction of biological glycome, making their analysis a considerable challenge. In this study, we developed a technique to analyze minor acidic O-glycans in biological samples. First, efficient reaction conditions for the release of O-glycans from the proteins were determined. Next, a high-throughput method was established for the recovery of minor acidic glycans using NH₂ spin columns. The performance of the established method was evaluated using mucin samples, and sulfated O-glycans were successfully detected in bovine submaxillary gland mucin and porcine stomach mucin. We also analyzed the minor acidic O-glycans in cultured cancer cells. In addition to trifucosylated sulfated O-glycans and disulfated O-glycans, sulfated O-glycans with KDN were detected in LS174T cells. The relative amount of sulfated glycans in LS174T cells was almost 10-fold higher than that in the other cells. Moreover, a large polylactosamine-type sulfated O-glycan with a molecular weight >3500 was detected in MKN45 cells. Interestingly, phosphorylated ribose, possibly bound to serine/threonine, was observed in all the cells used in this study. Thus, our established analytical method allows for the analysis of minor acidic O-glycans that cannot be detected using existing glycomics methods.

An improved method for the online preconcentration, derivatization, and separation of phosphorylated compounds was developed based on the affinity of a Phos-tag acrylamide gel formed at the intersection of a polydimethylsiloxane/glass multichannel microfluidic chip toward these compounds. The acrylamide solution comprised Phos-tag acrylamide, acrylamide, and N,N-methylene-bis-acrylamide, while 2,2′-azobis[2-methyl-N-(2-hydroxyethyl)propionamide] was used as a photocatalytic initiator. The Phos-tag acrylamide gel was formed around the channel crossing point via irradiation with a 365 nm LED laser. The phosphorylated peptides were specifically concentrated in the Phos-tag acrylamide gel by applying a voltage across the gel plug. After entrapment of the phosphorylated compounds in the Phos-tag acrylamide gel, 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF) was introduced to the gel for online derivatization of the concentrated phosphorylated compounds. The online derivatized DTAF-labeled phosphorylated compounds were eluted by delivering a complex of phosphate ions and ethylenediamine tetraacetic acid as the separation buffer. This method enabled sensitive analysis of the phosphorylated peptides.

N-Glycosylation of therapeutic antibodies is a critical quality attribute (CQA), and the micro-heterogeneity affects the biological and physicochemical properties of antibodies. Therefore, the profiling of N-glycans on antibodies is essential for controlling the manufacturing process and ensuring the efficacy and safety of the therapeutic antibodies. To monitor N-glycosylation in recombinant proteins, a high-throughput (HTP) methodology for glycan analysis is required to handle bulk samples in various stages of the manufacturing process. In this study, we focused on the HTP methodology for N-glycan analysis using a commercial microchip electrophoresis-based DNA analyzer and demonstrated the feasibility of the workflow consisting of sample preparation and electrophoretic separation. Even if there is a demand to analyze up to 96 samples, the present workflow can be completed in a day without expensive instruments and reagent kits for sample preparation, and it will be a promising methodology for cost-effective and facile HTP N-glycosylation analysis while optimizing the manufacturing process and development for therapeutic antibodies.

O-Acetylation of sialic acids has been widely found in eukaryotic cells. Such modifications of sialic acids are tissue-specific and seem to be developmentally regulated. In this study, we performed comprehensive analysis of age-related changes in the serum N-glycans of male rats using capillary electrophoresis (CE) and investigated the changes in the O-acetylation of sialic acids bound to N-glycans with aging and different diets. The present method offered sufficient resolution to assess the degree of O-acetylation of the N-glycans and allowed for the determination of the age-related changes in O-acetylation of sialic acids. Using the CE-based method, we found that the relative abundance of disialo-biantennary N-glycans modified with 9-O-acetylated N-acetylneuraminic acid (Neu5,9Ac) significantly increased with aging. In addition, the relative abundances of N-glycans with two Neu5,9Ac reversed to those of N-glycans with only Neu5Ac during 12 weeks. Next, we evaluated the influence of high-fat diet and food restriction on age-related changes in O-acetylation. Although the total amount of disialo-biantennary N-glycans increased with aging, age-related O-acetylation of sialic acids was suppressed by a high-fat diet. On the other hand, food restriction enhanced the O-acetylation of sialic acids, and the relative abundance of N-glycans with two Neu5,9Ac residues at 15 weeks of age was higher than that observed in the standard diet group. These findings suggest that the O-acetylation of sialic acids is closely related to changes in energy metabolisms such as glycolysis or fatty acid metabolism.

Prior investigations by our research group focused on the method development for the simultaneous analysis of sulfated and phosphorylated glycans. Herein, the developed method was applied to analyze minor acidic N-glycans including sulfated and phosphorylated N-glycans in human serum. First, 2-aminobenzoic acid-labeled minor acidic N-glycans were enriched from the serum using a serotonin-immobilized column, which were then separated into groups using hydrophilic interaction liquid chromatography, and analyzed by matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry. Phosphorylated hybrid-type and sulfated bi-antennary N-glycans were detected in the serum. In addition, we observed that multiple types of glucuronidated N-glycans were present. These results indicate that the developed method is applicable to the analysis of glucuronidated, as well as sulfated and phosphorylated N-glycans. It was also applied to the sera obtained from 17 healthy subjects and 15 pancreatic cancer patients, and the profiles of sulfated, phosphorylated, and glucuronidated N-glycans were compared. The expressed amount of glucuronidated N-glycans was significantly decreased in some pancreatic cancer patients. Numerous examples for the N-glycan analysis in human serum were reported but phosphorylated and glucuronidated glycans were not investigated. The methods described herein allowed the analysis of minor acidic glycans that are typically difficult to detect.

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