RUI: Development of Fluorescent Free-Radical Tags for N-Glycan Quantitation and Characterization using UPLC-MS/MS

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With support from the Chemical Measurement and Imaging Program in the Division of Chemistry, Jinshan Gao and his students at Montclair State University are pursuing research that seeks improved capabilities for simultaneous characterization and quantitation of an important class of biomolecules known as glycans – the carbohydrate-modifications that decorate so many proteins in biological systems and that are of great significance in biomedical science. Dr. Gao's approach uses mass spectrometry (MS), one of the most widely used and powerful tools of chemical analysis. Specifically, he is developing ways to label glycan molecules with fluorescent chemical "tags" to achieve absolute quantitation and to facilitate their structural characterization. The impact of proposed research is expected to be enhanced by the involvement of undergraduate students, including first-generation college students and members of underrepresented groups in science - Montclair State University is a Hispanic-Serving Institution and Primarily Undergraduate Institution. Elements of the research are integrated into Dr. Gao's undergraduate and graduate courses, enabling students to become familiar with modern analytical techniques and helping them to develop long-term interests for possible careers in the STEM (Science, Technology, Engineering and Mathematics) fields.The Gao group is developing novel fluorescent free-radical tags for simultaneous glycan quantitation and characterization, and novel multiplexed, isobaric fluorescent free-radical tags for simultaneous characterization and quantitation of each glycan from multiple samples. The tags contain four equally important chemical characteristics: 1) the fluorophore facilitates highly sensitive and reliable quantitation; 2) the free-radical precursor is expected to induce systematic, efficient, and predictable fragmentation for structure characterization; 3) a hydrazide or oxyamine group selectively targets the reducing terminus of the glycan; and 4) a pyridine or tertiary amine group is expected to significantly enhance the MS signal. These tailored tags are designed to enable systematic, efficient, and predictable free-radical-mediated glycan dissociation, allowing efficient and accurate glycan characterization via one-step dissociation. Fluorescence detection enables quantitation with high sensitivity independent of the glycan structure.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.