Sialic acid linkage differentiation of glycopeptides using capillary electrophoresis – electrospray ionization – mass spectrometry

Abstract

Sialylation is a glycosylation feature that occurs in different linkages at the non-reducing end of a glycan moiety, the linkage isomers are often differentially associated with various biological processes. Due to very similar physico-chemical properties, the separation of isomeric sialylated glycopeptides remains challenging but of utmost importance in the biomedicine and biotechnology, including biomarker discovery, glyco-engineering and biopharmaceutical characterization. This study presents the implementation of a high-resolution separation platform based on capillary electrophoresis – mass spectrometry (CE–MS) allowing for the selective analysis of α2,3- and α2,6-sialylated glycopeptides. These differentially linked glycopeptides showed an identical fragmentation pattern (collision induced dissociation) but different electrophoretic mobilities, allowing for baseline separation of the different linkages without the need for an extensive sample preparation. The different migration behavior between the two moieties was found to correlate with differences in pKa values. Using a novel methodology adapted from the so-called internal standard CE approach, a relative difference of 3.4·10−2 in pKa unit was determined. This approach was applied for the analysis of tryptic glycopeptides of prostate specific antigen, which shows highly complex and heterogeneous glycosylation. The developed platform therefore appears attractive for the identification of differentially linked sialic acids that may be related to pathological conditions.

Recent advances in capillary electrophoresis‐mass spectrometry: Instrumentation, methodology and applications

“…In this regard, Kammeijer et al. developed a CZE‐ESI‐MS method for glycopeptide analysis, including the separation of glycopeptides exhibiting different sialic acid linkages . The applicability of this platform was demonstrated by the analysis of N ‐glycopeptides derived from prostate specific antigen (PSA), detecting 75 different glycopeptides attributed to one single N ‐linked glycosylation site.…”

“…The ‘PEP’ label illustrates the tryptic peptide sequence N 69 K to which the glycan is attached. Reprinted with permission . Copyright © 2018 Springer Nature Limited.…”

Hydrophilic interaction liquid chromatography in the separation of glycopeptides and their isomers

“…Specific fragmentation features have been reported for anionic glycans, enabling the distinction between isomeric structures [7, 8]. However, positive ionisation mode of MS usually does not allow comprehensive characterization of isomeric glycopeptide structures due to similar fragmentation patterns using collision induced dissociation [9, 10]. Therefore, it is essential to connect MS with chromatographic separation to achieve efficiently resolution of the analytes and to allow their quantification in complex samples [6, 11, 12].…”

Recent progress in analytical capillary isotachophoresis

“…Kammeijer and co‐workers proposed the implementation of a high‐resolution separation platform based on CE‐MS allowing selective analysis of alpha 2,3‐ and alpha 2,6‐sialylated glycopeptides. These differentially linked glycopeptides showed an identical fragmentation pattern (collision induced dissociation) but different electrophoretic mobilities, allowing for baseline separation of the different linkages without the need for extensive sample preparation.…”