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

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.

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

“…We previously analyzed N-glycans from chicken colon (Suzuki et al, 2021) groups (Wuhrer et al, 2011, Suzuki et al, 2021Wuhrer et al, 2006). Therefore, there is a risk of incorrectly identifying the branch sequences based only on the MS/MS results.…”

“…Sialic acids are of core α-Man residues, or by the addition of bisecting GlcNAc (orange line). Reproduced from a previous publication with some modifications (Suzuki et al, 2021). gradually removed at room temperature.…”

“…These differences are attributable to the contribution of branching GlcNAc linked to either the C‐6 position of α6‐Man (β6´‐GlcNAc) or the C‐4 position of α3‐Man (β4‐GlcNAc). Given that β6´‐GlcNAc and β4‐GlcNAc exert opposing effects, 2,4,2´,6´‐tetraantennary PA‐ N ‐glycans that contain both β6´‐GlcNAc and β4‐GlcNAc elute at positions similar to those of 2,2´‐biantennary PA‐ N ‐glycans (Suzuki et al., 2021). It has been reported that human α1‐acid glycoprotein contains more 2,4,2´‐triantennary structures than 2,2´,6´‐triantennary structures (Stubbs, Shia, & Rice, 1997).…”

“…( E ) MS 3 spectrum of the B ion fragments Hex 1 HexNAc 1 Fuc 1 at m/z 660. Reproduced from a previous publication with some modifications (Suzuki et al., 2021).…”

“…Because an individual monosaccharide unit at a specific position and linkage on N-glycans contributes to the retention on the column either positively or negatively within a certain range, the presence or absence of the specific monosaccharides on the PA-N-glycans can be assumed from their elution positions. Consequently, it is easy to deduce the core structures, including core Fuc and bisecting GlcNAc, as well as the branching patterns of PA-N-glycans, using a C18 column (Suzuki, Abe, Hanzawa, & Natsuka, 2021). This article starts with methods for preparation of N-glycans released from tissue samples by glycoamidase F (Basic Protocol 1) or hydrazinolysis (Alternate Protocol) and PA-tagging of N-glycans by reductive amination (Basic Protocol 2).…”