Navigating the glycomics landscape with CE-MS: advances in sample preparation and analytical strategies

Glycosylation is one of the most structurally diverse and biologically consequential co- and post-translational modifications, yet its analytical characterisation remains challenging due to extensive isomerism, microheterogeneity, branching structure and the presence of labile residues. Among the available analytical platforms, capillary electrophoresis (CE), particularly when coupled to mass spectrometry (CE-MS), offers exceptional separation efficiency at nanolitre sample loadings and can resolve glycan variants that remain obscured in conventional LC- or MALDI-based workflows. This review provides a comprehensive overview of recent advances that have expanded the utility of CE and CE-MS in glycomics. We discuss practical considerations in enzymatic and chemical glycan release and highlight how the workflow format and clean-up influence recovery, quantitative precision and downstream compatibility. A major section is dedicated to the critical evaluation of major reducing-end derivatisation chemistries, including reductive amination, hydrazide and Michael-addition labelling, stable isotope, isobaric, and emerging instant-labelling strategies as well as permethylation, focusing on how labelling modulates electrophoretic mobility, isomer resolution, ionisation efficiency and MS/MS fragmentation. We outline current CE-MS methodologies, focusing on background electrolyte design, capillary coatings, sample injection modes, and the latest developments in sheath-flow, sheathless, nanoflow, and microfluidic interfaces. Performance benchmarks, including sensitivity, isomer resolution, robustness, and quantitative precision, are evaluated alongside recent innovations such as dopant enriched gases and integrated CE-MS cartridges. Finally, we assess the opportunities and remaining barriers for the broader adoption of CE-MS in biomedical, clinical, and biopharmaceutical glycomics. Continued advances in MS interface design, automation, and MS-compatible labelling chemistries are expected to further transform CE-MS into a routinely and widely deployable platform for high-resolution glycan characterisation.