Sialic Acid Derivatization of Fluorescently Labeled N-Glycans Allows Linkage Differentiation by Reversed-Phase Liquid Chromatography–Fluorescence Detection–Mass Spectrometry

Abstract

Sialic acids have diverse biological roles, ranging from promoting up to preventing protein and cellular recognition in health and disease. The various functions of these monosaccharides are owed, in part, to linkage variants, and as a result, linkage-specific analysis of sialic acids is an important aspect of glycomic studies. This has been addressed by derivatization strategies using matrix-assisted laser desorption/ionization mass spectrometry (MS) or sialidase digestion arrays followed by liquid chromatography (LC)-MS. Despite this, these approaches are unable to simultaneously provide unambiguous assignment of sialic acid linkages and assess further isomeric glycan features within a single measurement. Thus, for the first time, we present the combination of procainamide fluorescent labeling with sialic acid linkage-specific derivatization via ethyl esterification and amidation for the analysis of released plasma N -glycans using reversed-phase (RP)LC-fluorescence detection (FD)-MS. As a result, α2,3- and α2,6-sialylated N -glycans, with the same mass prior to derivatization, are differentiated based on retention time, precursor mass, and fragmentation spectra, and additional sialylated isomers were also separated. Furthermore, improved glycan coverage and protocol precision were found via the novel application using a combined FD-MS quantification approach. Overall, this platform achieved unambiguous assignment of N -glycan sialic acid linkages within a single RPLC-FD-MS measurement, and by improving their retention on RPLC, this technique can be used for future investigations of released N -glycans as an additional or orthogonal method to current analytical approaches.

Egg yolk sialylglycopeptide: purification, isolation and characterization of N-glycans from minor glycopeptide species

“…32 The selectivity of the mass changes due to EEA was verified using glycan standards of known sialylation (Fig S2 †). ESI-MS/ MS fragmentation of α-2,6 linked sialic acid after EEA produced a fragment of 686.35 m/z whereas α-2,3 linked sialic acid produced a fragment of 656.33 which is consistent with ethyl esterification and amidation respectively 33 (Fig S5 †). Fig.…”

Determination of Sialic Acid Isomers from Released N-Glycans Using Ion Mobility Spectrometry

“…Recently, sialic acid derivatization in combination with reversed-phase LC separation showed great potential for this purpose. 37 For an application in routine diagnostics, however, these details are usually not important. Instead, a general quantitative and qualitative assignment is more crucial to monitor pathological changes.…”

“…HILIC columns are described for their potential to separate glycan isomers that differ in the linkage-type of terminal monosaccharides such as sialic acids. , However, branching isomers with the same sialic acid linkage-type seem to be unresolved by HILIC chromatography, probably due to the equal hydrophilicity of these isomers. Recently, sialic acid derivatization in combination with reversed-phase LC separation showed great potential for this purpose . For an application in routine diagnostics, however, these details are usually not important.…”

Gangliosides and Their Role in Multilineage Differentiation of Mesenchymal Stem Cells

“…GGs are the main membrane components expressed on the external cell surface in direct contact with the extracellular environment [ 8 ]. GGs are glycosphingolipids (GSLs) complexes consisting of ceramide and a bulky sugar chain containing one or more sialic acids [ 9 , 10 , 11 ] ( Figure 1 A). They are also involved in the formation of lipid rafts [ 10 , 12 , 13 ].…”