The elucidation of the mechanisms whereby heparan sulfate (HS) binds proteins remains one of the most challenging problems in glycobiology. The binding between HS and growth factors and growth factor receptors plays an essential role in embryonic patterning and development. Many diseases have been linked to HS-protein binding events, including atherosclerosis. Nearly all mamallian cells express HS on their surfaces to mediate interactions between growth factors and growth factor receptors. Despite the central role HS plays in glycobiology, understanding of its interactions with growth factors and growth factor receptors has been severly limited by the lack of tools that are both rapid and sensitive enough to analyze it at biologically relevant levels. We are seeking to further the understanding of the HS structures that mediate growth factor binding by employing capillary equilibrium size exclusion chromatography (SEC) on-line with electrospray mass spectrometric detection. This technique will be capable of simultaneously determining the structures and binding orders of HS oligosaccharides released from cell surfaces. Equilibrium SEC carries the significant advantage that binding constants are determined purely from elution times and do not require knowledge of the ligand concentration. The use of capillary SEC columns will allow binding measurements to be made from biologically relevant levels of HS and binding proteins. Electrospray mass spectrometric detection will allow direct sequencing of the highly sulfated HS oligosaccharides using modern tandem mass spectrometric scans. The major areas proposed herein include: 1) Development and optimization of an equilibrium SEC interface for electrospray mass spectrometers; 2) Development of a scheme for identification of antithrombin Ill-binding epitopes and flanking sequences from commercial heparin and HS; 3) Determination of the expression of protein-binding sequences on particular proteoglycan gene products as a function of vascular cell type and growth conditions.
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