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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.
Highlights
Glycosylation is one of the most complex post-translational modifications of proteins that affects protein folding, stability, half-life, protein-protein interactions, signaling and trafficking[1,2,3]
A sheathless capillary electrophoresis (CE)–electrospray ionization (ESI)-mass spectrometry (MS) platform was used for the analysis of Fragment crystallizable (Fc) glycopeptides derived from pooled intravenous immunoglobulin G (IgG) (IVIgG) and IgG1 monoclonal antibody (IgGmAb) produced in Chinese Hamster Ovary (CHO) cells
IgG Fc glycopeptides derived from CHO cells are well-known to contain only α2,3-linked sialic acids while IgG Fc glycopeptides derived from human plasma are expected to only contain α2,6-linkages[20, 30, 31]
Summary
Glycosylation is one of the most complex post-translational modifications of proteins that affects protein folding, stability, half-life, protein-protein interactions, signaling and trafficking[1,2,3]. The characterization of protein glycosylation often requires information about site occupancy as well as the glycan structures This information is often obtained via proteolytic cleavage and subsequent glycopeptide analysis by mass spectrometry (MS)[26, 27]. The differentiation between the differently linked sialic acids of immunoglobulin G (IgG) Fragment crystallizable (Fc) glycopeptides has been achieved using a linkage-specific derivatization method prior to MS analysis[29]. Such methods are not broadly accessible and there is a clear need for the differentiation of sialic acid isomers in conjunction with MS detection
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