CHARACTERIZATION AND ENRICHMENT OF GLYCOPROTEIN/GLYCAN VIA MULTI-FUNCTIONAL FREE RADICAL REAGENTS

Project SummaryGlycoproteins are ubiquitous components of extracellular matrices and cellular surfaces, and havebeen reported to have unique structural and functional roles in cell-cell and cell-matrix interactions.Over 50% of human proteins are estimated to be potentially glycosylated. Alterations of glycanexpression in glycoprotein have been associated with many diseases, such as cancers, diabetes,and immune disorders. Meanwhile, the identification of glycosylation site is also crucial fordeciphering physiological relevance and potential value as a diagnostic. Therefore characterizationthe disease-associated glycoproteins is essential for the understanding of their functions at amolecular level, and thus benefits the identification of diagnostic glycoprotein and/or glycanbiomarkers and the better design of therapeutic drugs. Over the past decade, mass spectrometryhas become the pivotal technique for glycan and glycoprotein characterization. However, 1) thestructural complexity of glycan moiety, 2) difficulty in determining glycosylation sites, and 3) the low-abundance of native glycoproteins render the mass spectrometric characterization of glycoproteinespecially challenging. Therefore, efficient enrichment of glycoprotein and/or glycans prior to massspectrometry analysis, and facial mass spectrometric analysis of glycoprotein are desirable for thecomprehensive characterization of glycoproteins. Noting that free radicals excel in thedeconstruction of glycan and glycoprotein in a systematic and predictable fashion (preliminary data),the goal of this research is to develop solid-supported free radical probes (SSFRP) forglycan moiety enrichment and characterization, and free radial activated glycoproteinstructure elucidation (FRAGPSE) reagent for glycoprotein characterization, especially theprotein sequencing and glycosylation determination. The proposed research activities will bringan easily accessible tool to simultaneously enrich and characterize glycoproteins, including 1)enrichment of glycoprotein and glycans from complex mixtures, 2) analysis of protein sequences, 3)elucidation of glycan moiety structure, and 4) identification of the glycosylation sites. The proposedresearch includes (1) design and synthesis of SSFRP and FRAGPSE, (2) examination of thecapability of SSFRP to elucidate glycan structure, (3) examination of the capability of the SSFRP toenrich and characterize glycans released from glycoproteins, and (4) examination of the capabilityof the FRAGPSE to elucidate the structures of glycoproteins. Overall, the successful completion ofproposed approach will result in high-throughput characterization of trace amount of glycoproteins,allow the characterization of glycoproteins broadly accessible and cost-effective for the end-user.The proposed research will move the frontier of glycan study forward significantly.>