Development of a next-generation glycomics platform to enable glycan structure analyses for precision medicine

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Nearly all aspects of biology, including protein folding, cell adhesion and signaling, are mediated by glycans. As such, glycans have been exploited as clinical biomarkers for blood cell typing, cardiovascular and autoimmune disease, and cancer. Despite their importance, we currently have a limited view of the diverse glycan structure complexity present in normal health or disease. This is due to the lack of off-the-shelf approaches that enable the widespread identification and quantitation of glycan structures. Recent developments in mass spectrometry and related software tools facilitate the analysis of glycan structures. Widespread implementation of these approaches has been limited by the lack of approachable, high-throughput sample preparation strategies, lack of standardized methods for harmonization of data acquisition among laboratories, and absence of tools for automated peak assignment of glycan structures. In preliminary studies we have overcome major feasibility gaps of previous technologies. Our new analytical platform, GlyThyra, is the first single, integrated, one-of-a-kind glycomics platform to enable quantitative mass spectrometry-based glycan structure studies without requiring pre-purification of specific proteins. GlyThyra surpasses the depth, specificity, and speed of classical lectin- based approaches or other mass spectrometry approaches by enabling rapid, quantitative glycomic profiling of >300 N- and O-linked glycan structures across 3 orders of magnitude range in abundance. GlyThyra incorporates our novel approach for bulk glycan quantitation and normalization of total glycan content prior to mass spectrometry to eliminate errors associated with unequal loading. Internal and external standards enable normalization of technical variation and the generation of standardized results with low interlab variation. Preliminary data generated by our new platform provide evidence that novel potential biomarkers for heart, lung, and blood disease can be rapidly identified from just 1μL serum or plasma. The proposed studies will rigorously validate GlyThyra to encourage its downstream utilization and adoption in biomarker development efforts. Specifically, we will use National Institute for Standards and Technology reference materials for non-invasive sample types (serum, plasma, urine) to further refine and assess performance measures to validate the sample preparation and data acquisition (Aim 1) and data analysis modules (Aim 2). Aim 3 will establish the reference ranges for >100 glycan structures across 100 healthy adult control serum, plasma, and urine samples. Outcomes of the proposed studies will impact biomarker development across a broad range of diseases by enabling researchers who would not otherwise have access to glycomic technologies to perform precise profiling of glycan structures in clinically relevant samples. The seamless integration of all analytical and bioinformatic workflows and exclusive use of commercially available reagents and freely available software will ensure the platform can be widely implemented among research laboratories.