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 reversed-phase 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-N-glycan, 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 MSn analysis of PA-N-glycans from tissues. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of released N-glycans from tissue samples using glycoamidase F. Alternate Protocol: Preparation of released N-glycans from tissue samples by hydrazinolysis. Basic Protocol 2: PA-tagging of N-glycans and sample cleanup. Support Protocol 1: Monitoring of PA-N-glycans using normal-phase HPLC. Basic Protocol 3: Anion-exchange chromatography of PA-N-glycans. Basic Protocol 4: Sequential exoglycosidase digestions. Basic Protocol 5: Determination of Sia-linkages by SALSA. Support Protocol 2: Cotton-HILIC solid-phase extraction to remove reagents for alkylamidation. Basic Protocol 6: Sequential modifications of glycans with SALSA and permethylation. Basic Protocol 7: LC-MS and MS/MS analysis of PA-N-glycans (before permethylation). Basic Protocol 8: LC-MS, MS/MS, and MSn analysis of PA-N-glycans (after permethylation).