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 Lex, sialyl Lex, sulfated LacNAc, LacNAc repeat, and LacdiNAc. The results demonstrated that our method is useful for comparing N-glycomes among various tissue samples.
Highlights
We first confirmed that PA-N-glycans could be clearly separated based on their core structures and branching patterns using a C18 column adopted for water-rich mobile phases
This system allowed the eluted PA-glycans to be directly analyzed with an online LC–MS and MS/MS system
The resultant elution patterns were similar to those obtained by conventional methods, using mobile phases containing 1-butanol[15,16], regardless of differences in end capping of the columns
Summary
Trypsin and chymotrypsin were purchased from Sigma-Aldrich (St. Louis, MO, USA), and guanidine hydrochloride, iodoacetamide, and dithiothreitol were purchased from Fujifilm Wako Pure Chemical Co. Recombinant glycoamidase F from Flavobacterium meningosepticum (GAF, aka N-glycosidase F, PNGase F) was purchased from Roche (Mannheim, Germany). Neuraminidase (α2-3,6,8,9 neuraminidase) from Arthrobacter ureafacience was purchased from Nacalai Tesque (Kyoto, Japan). Α1-3,4 Fucosidase from the sweet almond tree and β1-4 galactosidase S from Streptococcus pneumonia were purchased from New England BioLabs (Ipswich, MA, USA). Columns for LC, and PA-N-glycans from human γ-globulin, α1-AGP, and bovine fetuin were obtained as described p reviously. Α2,6-Monosialylated PA-N-glycans were prepared from human transferrin and human γ-globulin, and α2,3-monosialylated PA-N-glycans were prepared by treatment of asialo-biantennary PA-N-glycans with recombinant α2,3-sialyltransferase from Photobacterium phosphoreum[39] Columns for LC, and PA-N-glycans from human γ-globulin, α1-AGP, and bovine fetuin were obtained as described p reviously39,40. α2,6-Monosialylated PA-N-glycans were prepared from human transferrin and human γ-globulin, and α2,3-monosialylated PA-N-glycans were prepared by treatment of asialo-biantennary PA-N-glycans with recombinant α2,3-sialyltransferase from Photobacterium phosphoreum[39]
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