Glycoproteins are ubiquitous components of extracellular matrices and cellular surfaces, and have been 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 glycan expression in glycoprotein have been associated with many diseases, such as cancers, diabetes, and immune disorders. Meanwhile, the identification of glycosylation site is also crucial for deciphering physiological relevance and potential value as a diagnostic. Therefore characterization the disease-associated glycoproteins is essential for the understanding of their functions at a molecular level, and thus benefits the identification of diagnostic glycoprotein and/or glycan biomarkers and the better design of therapeutic drugs. Over the past decade, mass spectrometry has become the pivotal technique for glycan and glycoprotein characterization. However, 1) the structural complexity of glycan moiety, 2) difficulty in determining glycosylation sites, and 3) the low- abundance of native glycoproteins render the mass spectrometric characterization of glycoprotein especially challenging. Therefore, efficient enrichment of glycoprotein and/or glycans prior to mass spectrometry analysis, and facial mass spectrometric analysis of glycoprotein are desirable for the comprehensive characterization of glycoproteins. Noting that free radicals excel in the deconstruction 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) for glycan moiety enrichment and characterization, and free radial activated glycoprotein structure elucidation (FRAGPSE) reagent for glycoprotein characterization, especially the protein sequencing and glycosylation determination. The proposed research activities will bring an 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 proposed research includes (1) design and synthesis of SSFRP and FRAGPSE, (2) examination of the capability of SSFRP to elucidate glycan structure, (3) examination of the capability of the SSFRP to enrich and characterize glycans released from glycoproteins, and (4) examination of the capability of the FRAGPSE to elucidate the structures of glycoproteins. Overall, the successful completion of proposed 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.
The proposed research will develop an innovative tool to permit facile and high-throughput structure elucidation of glycans and glycoproteins from complex biological samples. The outcomes will set the stage for the development of glycan and/or glycoprotein biomarkers for glycosylation-related diseases and monitoring of patient response to therapy. This study will also benefit the investigation of the molecular mechanism of cancer disease, such as the modifications to signal transduction related to ?aberrant? glycan expression, and thus inform new approaches to cancer therapy.
