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

Abstract

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.

Efficient utilization of complex N-linked glycans is a selective advantage for Bacteroides fragilis in extraintestinal infections

“…Purified human transferrin was suspended in PBS to 2 mg/mL, and 100 μL used in deglycosylation assays as described above except that following incubation, the assay supernatants were filter sterilized and frozen at −80°C. Samples were analyzed by Ezose Sciences Inc. to quantify N-linked glycans using previously reported methods (33,34). Briefly, samples were denatured, digested with trypsin and then heat-inactivated.…”

Qualitative and Quantitative Cellular Glycomics of Glycosphingolipids Based on Rhodococcal Endoglycosylceramidase-assisted Glycan Cleavage, Glycoblotting-assisted Sample Preparation, and Matrix-assisted Laser Desorption Ionization Tandem Time-of-flight Mass Spectrometry Analysis

“…Glycoblotting-Samples treated with EGCases were subjected to glycoblotting as previously described with minor modifications (13). In brief, the sample solution (50 l) was directly applied to the well of a filterplate (MultiScreen Solvinert 0.45 m Low Binding Hydrophilic PTFE, Millipore, MA) containing BlotGlyco beads (5.0 mg; Sumitomo Bakelite Co. Ltd., Tokyo).…”

Threshold in Stage-specific Embryonic Glycotypes Uncovered by a Full Portrait of Dynamic N-Glycan Expression during Cell Differentiation

“…Mouse ESCs were differentiated under conditions reported previously (30,31). Basic Protocol of Glycoblotting-based Quantitative Cellular N-Glycomics-A preliminary trial for the cellular N-glycomics was reported previously in human prostate cancer cells (PC-3) and normal human prostate epithelial cells on the basis of the protocol designed for human serum N-glycomics using the BlotGlyco TM ABC bead, a prototype bead prepared by conjugating N-(2-aminobenzoyl)cysteine hydrazide and thiopropyl-Sepharose 6B (26). However, we had to re-examine and establish a comprehensive protocol feasible for mammalian cellular glycomics using BlotGlyco H, a commercially available synthetic polymer bead (Sumitomo Bakelite Co., Ltd., Tokyo, Japan) (see Fig.…”

“…Glycoblotting of the sample mixtures containing whole cell Nglycans by means of BlotGlyco H bead was performed according to the procedure described previously (26). BlotGlyco H beads (500 l) (10 mg/ml suspension; Sumitomo Bakelite Co., Ltd.) were aliquoted onto a well of a MultiScreen Solvinert filter plate (Millipore, Billerica, MA).…”

“…We report herein that the glycoblotting method (24), a PCR-like technology developed for rapid and large scale enrichment analysis of human serum glycans (25,26), can be used for rapid and quantitative cellular glycomics to monitor dynamic glycoform alteration during differentiation of mouse embryonic carcinoma cells (P19CL6 and P19C6 cells) and mouse ESCs.…”