Cellular O‐Glycome Reporter/Amplification (CORA): Analytical and Preparative Tools to Study Mucin‐Type O‐Glycans of Living Cells

Abstract Mucin‐type O‐glycosylation (O‐glycans, O‐glycome) is among the most biologically important post‐translational modification in glycoproteins but O‐glycan structural diversity and expression are poorly understood due to the inadequacy of current analytical methods. We recently developed a new tool termed cellular O‐glycome reporter/amplification (CORA), which uses O‐glycan precursors, benzyl‐α‐GalNAc (Bn‐α‐GalNAc) or azido‐Bn‐α‐GalNAc (N3‐Bn‐α‐GalNAc), as surrogates of protein O‐glycosylation. Living cells metabolically convert these precursors to all types of O‐GalNAc glycans representative of the cells’ capabilities. The amplification and secretion of the O‐glycome products greatly facilitates their analysis and functional studies. Here we describe protocols for analytical and preparative applications. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Cellular O‐glycome reporter/amplification for the analysis of mucin‐type O‐glycans from living cells Basic Protocol 2: Preparation of cellular O‐glycans from living cells for functional glycomics and glycan microarrays Basic Protocol 3: Conjugation of cellular O‐glycans with a bifunctional fluorescent tag Basic Protocol 4: 2D‐HPLC purification and MALDI‐TOF/MS identification of individual PYAB‐Bn‐O‐glycan