Hideyuki Shimaoka

Comprehensive Approach to Structural and Functional Glycomics Based on Chemoselective Glycoblotting and Sequential Tag Conversion

Changes in protein glycosylation profoundly affect protein function. To understand these effects of altered protein glycosylation, we urgently need high-throughput technologies to analyze glycan expression and glycan-protein interactions. Methods are not available for amplification of glycans; therefore, highly efficient sample preparation is a major issue. Here we present a novel strategy that allows flexible and sequential incorporation of various functional tags into oligosaccharides derived from biological samples in a practical manner. When combined with a chemoselective glycoblotting platform, our analysis enables us to complete sample preparation (from serum to released, purified, methyl-esterified, and labeled glycans) in 8 h from multiple serum samples (up to 96 samples) using a 96-well microplate format and a standard de-N-glycosylation protocol that requires reductive alkylation and tryptic digestion prior to PNGase F digestion to ensure maximal de-N-glycosylation efficiency. Using this technique, we quantitatively detected more than 120 glycans on human carcinoembryonic antigens for the first time. This approach was further developed to include a streamlined method of purification, chromatographic fractionation, and immobilization onto a solid support for interaction analysis. Since our approach enables rapid, flexible, and highly efficient tag conversion, it will contribute greatly to a variety of glycomic studies.

BlotGlycoABC™, an Integrated Glycoblotting Technique for Rapid and Large Scale Clinical Glycomics

Recent progress in mass spectrometry has led to new challenges in glycomics, including the development of rapid glycan enrichment techniques. A facile technique for exploration of a carbohydrate-related biomarker is important because proteomics research targets glycosylation, a posttranslational modification. Here we report an “all-in-one” protocol for high throughput clinical glycomics. This new technique integrates glycoblotting-based glycan enrichment onto the BlotGlycoABC™ bead, on-bead stabilization of sialic acids, and fluorescent labeling of oligosaccharides in a single workflow on a multiwell filter plate. The advantage of this protocol and MALDI-TOF MS was demonstrated through differentiation of serum N-glycan profiles of subjects with congenital disorders of glycosylation and hepatocellular carcinoma and healthy donors. The method also permitted total cellular glycomics analysis of human prostate cancer cells and normal human prostate epithelial cells. These results demonstrate the potentials of glycan enrichment/processing for biomarker discovery.

Alteration of the N‐glycome of bovine milk glycoproteins during early lactation

Milk provides nutritional, immunological and developmental components for newborns. Whereas identification of such components has been performed by targeting proteins and free oligosaccharides, structural and functional analyses of the N‐glycome of milk glycoproteins are scarce. In this study, we investigated, for the first time, the alterations of the bovine milk N‐glycome during early lactation (1 day, 1, 2, 3 and 4 weeks postpartum), characterizing more than 80 N‐glycans. The glycomic profile of colostrum on day 1 after calving differed substantially from that in other periods during early lactation. The proteins in colostrum obtained 1 day postpartum were more highly sialylated than milk samples obtained at other time points, and the N‐glycolylneuraminic acid (Neu5Gc)/N‐acetylneuraminic acid (Neu5Ac) ratio was significantly higher on day 1, showing a gradual decline with time. In order to dissect the N‐glycome of colostrum, alterations of the N‐glycosylation profile of major bovine milk proteins during the early lactation stage were elucidated, revealing that the alteration is largely attributable to qualitative and quantitative N‐glycosylation changes of IgG, the major glycoprotein in colostrum. Furthermore, by preparing and analyzing IgGs in which the N‐glycan structure and subtypes were well characterized, we found that the interaction between IgG and FcRn was not affected by the structure of the N‐glycans attached to IgG. We also found that bovine FcRn binds IgG2 better than IgG1, strongly suggesting that the role of FcRn in the bovine mammary gland is to recycle IgG2 from the udder to blood, rather than to secrete IgG1 into colostrum.