High‐Throughput Protein Glycomics: Combined Use of Chemoselective Glycoblotting and MALDI‐TOF/TOF Mass Spectrometry

Abstract

Gängige Oligosaccharide und Aldehyd‐gebundene, von Enzymmodifikationen abgeleitete Glycoproteine können durch ein Glycoblotting abgefangen werden, das auf Oxylamino‐haltigen Polymeren basiert. Die Kombination dieses Glycoblottings mit MALDI‐TOF/TOF‐Massenspektrometrie führte zu einer Hochdurchsatz‐Analysemethode.

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

“…Consequently, it was concluded that changes in the expression level of detected proteins are not helpful for identifying or monitoring the processes of cellular differentiation of both P19 and P19C6 cells. Compared with the results of proteomebased analysis, the high potential of the glycome-based approach is clear because our results revealed for the first time that 28% of glycoforms (19 N-glycans of 67 total N-glycans; peak numbers 13,20,21,22,25,28,29,32,34,37,38,39,40,41,47,48,54,60, and 63) were increased, and 10% (7 of 67) were decreased during P19CL6 cell differentiation to car- 1) and sialyl-Lewis X tetrasaccharide moieties in major N-glycans as shown in Fig. 3C, whereas monofucosylation occurred specifically in the above 31 glycoforms at the GlcNAc residue involved in core chitobiose moiety after differentiation, suggesting that loss of cell adhesion through the interaction with selectins may be key to the differentiation of P19CL6 cells toward cardiomyocytes.…”

“…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. 1), because our recent studies on the functional glycomics using BlotGlyco H bead have demonstrated improved performance of this new platform in terms of quantification, reproducibility, and application (25).…”

“…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.…”

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.…”

Fluorescence polarization-based assay using N-glycan-conjugated quantum dots for screening in hemagglutinin blockers for influenza A viruses

“…The ao-PC-QDs were prepared from the above Qdot 545 ITK, trioctylphosphine oxide (TOPO)-coated QDs (100 L of 1 M solution, 5.8 nm, CdSe/ZnS, λ em = 545 nm), by a general protocol for coating with ao-SH (50%) and PC-SH (50%). Sialylated biantennary N-glycan (A2) prepared by treating A2 glycopeptide with PNGase F (Roche Diagnostics Japan, Tokyo, Japan) was subjected to coupling with ao-PC-QDs by the "glycoblotting" (Nishimura et al, 2005;Ohyanagi et al, 2011) reaction and gave PC-QDs displaying A2 glycans (A2-PC-QDs, Figure 1). Characterization of A2-PC-QDs was performed by 5 direct MALDI-TOFMS (Nagahori et al, 2009;Ohyanagi et al, 2011) and the results showed the advent of an expected molecular ion signal at m/z 3155.7 corresponding to the heterodisulfide A2-S-S-PC instead of disappearance of the signal at m/z 908.7 due to ao-S-S-PC corresponding to free aminooxy-functional groups.…”