文摘
Changes in protein glycosylation profoundly affect proteinfunction. To understand these effects of altered proteinglycosylation, we urgently need high-throughput technologies to analyze glycan expression and glycan-proteininteractions. Methods are not available for amplificationof glycans; therefore, highly efficient sample preparationis a major issue. Here we present a novel strategy thatallows flexible and sequential incorporation of variousfunctional tags into oligosaccharides derived from biological samples in a practical manner. When combined witha chemoselective glycoblotting platform, our analysisenables us to complete sample preparation (from serumto released, purified, methyl-esterified, and labeled glycans) in 8 h from multiple serum samples (up to 96samples) using a 96-well microplate format and a standard de-N-glycosylation protocol that requires reductivealkylation and tryptic digestion prior to PNGase F digestion to ensure maximal de-N-glycosylation efficiency.Using this technique, we quantitatively detected more than120 glycans on human carcinoembryonic antigens for thefirst time. This approach was further developed to includea streamlined method of purification, chromatographicfractionation, and immobilization onto a solid support forinteraction analysis. Since our approach enables rapid,flexible, and highly efficient tag conversion, it will contribute greatly to a variety of glycomic studies.
