Cellular O-Glycome Reporter/Amplification to Explore O-Glycans of Living Cells

Abstract

Protein O-glycosylation plays key roles in many biological processes, but the repertoire of O-glycans synthesized by cells is difficult to determine. Here we describe a new approach termed Cellular O-Glycome Reporter/Amplification (CORA), a sensitive method to amplify and profile mucin-type O-glycans synthesized by living cells. Cells incubated with peracetylated benzyl-α-N-acetylgalactosamine (GalNAc-α-Benzyl) convert it to a large variety of modified O-glycan derivatives that are secreted from cells, allowing easy purification for analysis by HPLC and mass spectrometry (MS). CORA results in ~100–1000-fold increase in sensitivity over conventional O-glycan analyses and identifies a more complex repertoire of O-glycans in more than a dozen cell types from Homo sapiens and Mus musculus. Furthermore, CORA coupled with computational modeling allows predictions on the diversity of the human O-glycome and offers new opportunities to identify novel glycan biomarkers for human diseases.