Toward robust N-glycomics of various tissue samples that may contain glycans with unknown or unexpected structures

Glycans in tissues are structurally diverse and usually include a large number of isomers that cannot be easily distinguished by mass spectrometry (MS). To address this issue, we developed a combined method that can efficiently separate and identify glycan isomers. First, we separated 2-aminopyridine (PA)-derivatized N-glycans from chicken colon by reversed-phase liquid chromatography (LC) and directly analyzed them by electrospray ionization (ESI)-MS and MS/MS to obtain an overview of the structural features of tissue glycans. Next, we deduced the structures of isomers based on their elution positions, full MS, and MS/MS data, before or after digestions with several exoglycosidases. In this method, the elution position differed greatly depending on the core structure and branching pattern, allowing multiantennary N-glycan structures to be easily distinguished. To further determine linkages of branch sequences, we modified PA-N-glycans with sialic acid linkage-specific alkylamidation and/or permethylation, and analyzed the products by LC–MS and multistage MS. We determined the relative abundances of core structures, branching patterns, and branch sequences of N-glycans from chicken colon, and confirmed presence of characteristic branch sequences such as Le(x), sialyl Le(x), sulfated LacNAc, LacNAc repeat, and LacdiNAc. The results demonstrated that our method is useful for comparing N-glycomes among various tissue samples.