摘要：Abstract 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.
其他摘要：Abstract 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.
