An enhanced dual detection of DMB-labeled sialic acids using high-resolution accurate mass spectrometry and fluorescence detection

Abstract

Sialic acids are important glycan components not only in the conformation of the structure but also in functions of many biological activities. Detection methods for sialic acid have been developed, and fluorescent labeling of sialic acids with 1,2-diamino-4,5-methylenedioxybenzene (DMB) is the best method for its analysis. However, identifying and quantifying sialic acids using mass spectrometry (MS) remains difficult because of the diversity of sialic acids, including O-acetylation. In this study, we initially focused on enhancing the liquid chromatography (LC) separation of DMB-labeled sialic acids in bovine submaxillary mucin (BSM). We successfully achieved highly resolutive separation of 14 types of sialic acids, including multiply O-acetylated species, using a CAPCELL CORE C18 column. These structures were subsequently confirmed through collision-induced dissociation (CID) fragmentation. We then assessed MS and fluorescence detection (FLD) sensitivity using Neu5Ac, Neu5Gc, and Kdn standards, determining detection limits of 32 fmol for MS and 320 amol for FLD, with 320 fmol needed for CID-based structural analysis. These findings highlight the complementary nature of fluorescence and mass spectrometry techniques for DMB-labeled sialic acids identification and quantification. We also examined both CID and electron-activated dissociation (EAD) on the ZenoTOF 7600. CID fragment analysis revealed C5-specific diagnostic fragment ions for Neu5Ac, Neu5Gc, and Kdn. EAD spectra predominantly induced fragmentation of the fluorescent DMB core, regardless of applied variable kinetic energies. This study also led to the discovery of a new structure, Neu5Gc7,8,9Ac3, alongside the previously reported Neu5,7,8,9Ac4 in BSM.

Keywords

• Sialic acid
• High-resolution mass spectrometry
• Fluorescence detection
• Collision-induced dissociation
• Electron-activated dissociation