Tissue N‐Glycan Analysis Using LC‐MS, MS/MS, and MSⁿ

Abstract

Tissue glycans usually contain various structures, from simple to highly complicated, in different quantities. N-Glycans are particularly heterogeneous, with up to pentaantennary structures, different branch sequences, and several isomeric structures. 2-Aminopyridine (PA) tagging on released N-glycans is useful for separating isomers and to quantitatively analyze both the major and minor glycan structures in tissues using reversed-phase liquid chromatography (LC)−mass spectrometry (MS) and MS/MS analysis. Because the structural differences of PA-N-glycans influence their retention on a reversedphase C18 column, it is easy to deduce the core structure, including core Fuc and bisecting GlcNAc as well as the branching pattern of each PA-Nglycan, based on the results of elution position, full MS, and MS/MS analysis. If more detailed structural analysis is required, combining sequential exoglycosidase digestions, sialic acid linkage−specific alkylamidation (SALSA), and/or SALSA/permethylation is useful for determining glycosidic linkages of branches. This article includes detailed protocols for the preparation of N-glycans released from glycoproteins/glycopeptides by glycoamidase F or hydrazinolysis, PA-tagging of N-glycans, fractionation with anion-exchange chromatography, and chemical or enzymatic modifications of PA-N-glycans, as well as reversed-phase LC-MS, MS/MS, and MS n analysis of PA-N-glycans from tissues.