Versatile Glycoblotting Nanoparticles for High‐Throughput Protein Glycomics

Abstract

We have developed an effective and practical trap-and-release method based on chemoselective ligation of carbohydrates with reactive aminooxyl groups attached to the surface of nanoparticles (referred to as glycoblotting nanoparticles). These glycoblotting nanoparticles were synthesized by UV irradiation of diacetylene-functionalized lipids that contain the aminooxyl group. The glycoblotting nanoparticles captured carbohydrates in aqueous solution under mild conditions and were collected by simple centrifugation. The trapped carbohydrates were effectively released from the nanoparticles under acidic conditions to give pure oligosaccharides. This glycoblotting process reduced the time required for the purification process of carbohydrates to less than 6 h, compared to the several days needed for conventional chromatographic techniques. The oligosaccharides (N-glycan) were released from ovalbumin (glycoprotein) by PNGase F after tryptic digestion. MALDI-TOF mass spectra before purification did not show any significant signals corresponding to N-glycans because these signals were hidden by the large signals of the abundant peptides. However, after purification with the glycoblotting nanoparticles, only signals corresponding to oligosaccharides appeared. We also demonstrated a clear analysis of the oligosaccharides contained in the mice dermis by means of glycoblotting.

Quantitative Glycomics of Human Whole Serum Glycoproteins Based on the Standardized Protocol for Liberating N-Glycans

“…Our finding, however, indicates that the quantitative serum glycomic profile could be severely affected by the digestion condition used, and therefore care should be taken when handling the quantitative glycomics data. As an efficient tool to rapidly purify oligosaccharides from highly complicated biological matrices, we recently developed a chemoselective glycoblotting platform utilizing synthetic polymers displaying aminooxy functionality (31)(32)(33). The combination of the described standardized sugar-liberating protocol and glycoblotting technique for high throughput, large scale disease-related serum glycomics is currently in progress in our laboratory.…”

High Throughput Quantitative Glycomics and Glycoform-focused Proteomics of Murine Dermis and Epidermis

“…Because of its high ionization efficiency of aoWR on MALDI-TOF analysis, aoWR derivatives could be highly selectively detected with minimum purification steps. We have recently reported that polymers displaying oxylamino groups provide a useful platform for "glycoblotting," a high throughput oligosaccharide purification procedure in a crude mixture (22,23). Derivatization with aoWR is therefore regarded as an analogue of glycoblotting where chemoselective ligation is done using functional low molecular weight compounds instead of polymers.…”

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

“…Our recent efforts have been directed to the establishment of practical methods for glycan enrichment, namely "glycoblotting" (15)(16)(17)(18)(19)(20). The optimized protocol requires only 5 l of human serum for the quantitative profiling of 30 -40 kinds of major glycoforms within 5-8 h (19).…”