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Moran, A.B.; Gardner, R.A.; Wuhrer, M.; Lageveen-Kammeijer, G.S.M.; Spencer, D.I.R. (2022)

Sialic acid derivatization of fluorescently labeled N-glycans allows linkage differentiation by reversed-phase liquid chromatography-fluorescence detection-mass spectrometry

Article / Letter to editor
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 mono-saccharides 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)...Show moreSialic acids have diverse biological roles, ranging from promoting up to preventing protein and cellular recognition in health and disease. The various functions of these mono-saccharides 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, alpha 2,3- and alpha 2,6-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.Show less
All authors
Moran, A.B.; Gardner, R.A.; Wuhrer, M.; Lageveen-Kammeijer, G.S.M.; Spencer, D.I.R.
Date
2022-05-10
Journal
Analytical Chemistry
Volume
94
Issue
18
Pages
6639 - 6648
DOI
doi:10.1021/acs.analchem.1c02610
Link
https://pubs.acs.org/doi/10.1021/acs.analchem.1c02610