Protein glycosylation plays a role in a wide range of biological functions and disease processes, from protein folding, cell adhesion and signalling, to pathogen recognition and immune responses. However, our current view of protein glycosylation is incomplete because current technologies offer limited throughput and accuracy. To address these challenges, we developed a new analytical platform, GlyThyra, integrating new preparation and analysis workflows. The semi-automated sample preparation workflow exploits solid-phase PNGase-F digestion with reduced handling time. To ensure accuracy, we developed a released glycan quantitation assay to enable normalized on-column loading, improving quality control over current protein normalization approaches. The data analysis burden is reduced by using an N-glycan library of over 300 structures covering all major biosynthetic classes.
GlyThyra was compared to a widely used manual-intensive glycomics method for the analysis of monoclonal antibodies, plasma, and human primary tissue. Repeatability, throughput, and total glycan yield were assessed for each sample type. Our new fluorescent glycan quantitation method significantly reduces LC-MS variation for both the classic and newly developed methods and is accurate over 3 orders of magnitude with a limit of detection of 6 pmol. Automated glycan assignment is informed by three dimensions of LC-MS values, high-resolution MS1, MS21 and normalized retention time2, enabling over 95% of structures to be automatically assigned correctly and the remaining 5% of peaks highlighted for manual inspection. Overall, GlyThyra enables complete N-glycan release, LC-MS acquisition and data analysis to be completed within 24 hours, compared to >3 days with the classic method. Moreover, as two of the samples are NIST standard materials, we expect these glycan libraries will be useful references for future QC efforts. Overcoming throughput and accuracy challenges by generating glycomic data with improved precision, the GlyThyra platform will facilitate future translational glycomics across a broad range of sample and disease types.