Analysis of sulfated sialic acids by liquid chromatography coupled with high-resolution mass spectrometry and fluorescence detection.

Authors

Sota Yamashita, Takayuki Omoto, Nao Yamakawa, Naoko Komura, Hiromune Ando, Di Wu, Ken Kitajima, Masaya Hane, Chihiro Sato

Published in

BBA advances. Volume 10. Pages 100203. Epub Sep 15, 2026.

Abstract

Sialic acid is localized at the outermost glycan, and involved in regulatory roles as one of the most important glycotopes in various biological phenomena. Recently, we established a highly sensitive and selective method for the fine differentiation of 1,2-diamine-4,5-methylenedioxybenzene (DMB)-labeled sialic acids, including N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), deaminoneuraminic acid (Kdn), and their O-acetyl derivatives. This approach enables unambiguous identification of these three sialic acids based on characteristic C-5 diagnostic fragment ions obtained by high-resolution accurate-mass spectrometry and fluorescence detection. In the present study, we extended this analytical platform to sulfated sialic acids (SiaSs) to evaluate its applicability to sialic acids bearing an additional negatively charged sulfate group. We first assessed the liquid chromatographic separation of DMB-labeled SiaSs using authentic standards. Using a CAPCELL PAK ADME column, we successfully separated six authentic SiaS standards and three previously unreported SiaS species in sea urchins. We identified multiple O-sulfated substitutions on Neu5Ac and Neu5Gc species, which were further confirmed by characteristic collision-induced dissociation (CID) fragmentation. In addition, electron-activated dissociation (EAD) was employed to evaluate the fragmentation behavior of SiaS species. EAD analyses efficiently generated characteristic diagnostic ions that enable reliable discrimination of the sulfation site, allowing clear differentiation between 8-O- and 9-O-sulfated isomers. Liquid chromatography-based fragment analysis led to the identification of a novel O-sulfated and O-acetylated sialic acid, Neu5Gc8SOAc1, which had never been reported before. These findings underscore the complementary nature of fluorescence detection and high-resolution mass spectrometry techniques for the identification and quantification of DMB-labeled sialic acids.

PMID:
42834900
Bibliographic data and abstract were imported from PubMed on 06 Oct 2026.

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