We use mass spectrometry to achieve detailed characterization and quantification of glycan structures, including N and O-glycans released from proteins. Chromatography using porous graphitized carbon achieves separation of structurally similar glycans with the same composition, allowing each glycan structure to be individually quantified and characterized. Normalized retention times provide the first level of information regarding glycan structure and is orthogonal to glycan detection and characterization by mass-spectrometry. Using an LTQ Velos Orbitrap, accurate masses for glycans and corresponding MS/MS spectra for each structure allow orthogonal characterization of glycan structures to normalized retention time values. Data analysis has been automated using KNIME and the Skyline platform.
We are currently applying this technology to provide the first dynamic view of glycan structures within hPSC-CM differentiation cultures and human primary cells isolated from normal and diseased hearts. These data compliment our cell surface glycoproteomics work, as the glycan libraries we are building serve as a reference for our intact glycopeptide analyses.