Mass Defect-based Chemical Tags for Multiplex Glycan Quantitation

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ABSTRACT Protein glycosylation plays critical roles in many biological processes as one of the most common and the most complex posttranslational modifications. Alterations in glycosylation profiles are known to be associated with many diseases, including cancer, neurodegenerative disorders, immunological diseases and cardiovascular problems. Advances in mass spectrometry (MS)-based glycoproteomics and glycomics are increasingly enabling qualitative and quantitative approaches for site-specific structural analysis of protein glycosylation. However, quantitative analysis of native glycans remains extremely challenging due to high complexity and heterogeneity and diversity of glycan structures, the low response in both optical and mass spectrometric detection methods, and the wide dynamic range of glycans in clinically relevant samples. Analytical strategies for N-linked glycans from glycoproteins usually involve releasing glycans from the glycoproteins via PNGase F digestion and labeling the released glycans with reagents that facilitate detection. The primary goal of this proposal is to develop a novel and cost effective chemical labeling technology via the custom synthesis and development of a set of mass defect-based tags that enable both sensitive fluorescent detection and high-throughput quantification of complex glycans with high resolution mass spectrometry (MS) platforms. We propose the following specific aims: Specific Aim 1 – To synthesize and develop novel mass defect-based multiplex dimethyl pyrimidinyl ornithine (DiPyrO) tags for cost effective and high-throughput MS- based relative quantification of N-glycans released from biological samples. Specific Aim 2 – To optimize the resolution of isomeric glycan structures and maximize the glycan ionization efficiency, nanoHILIC will be coupled with a novel sub-ambient pressure ionization nanospray (SPIN) source and high resolution Orbitrap MS platform for ultrasensitive detection of the multiplex DiPyrO labeled N-glycans released from complex biological samples such as serum and cell lysates. Specific Aim 3 – To synthesize and develop new mass defect-based multiplex dimethyl quinazolinyl ornithine (DiQuniO) tags for improved optical detection and develop capillary electrophoresis-UV/LIF-MALDI-ion mobility-mass spectrometric imaging platform for multi-dimensional glycan characterization and quantification. Collectively, our proposed experiments will develop new enabling tools and will generate cost-effective and novel mass defect-based labeling reagents for robust, sensitive and accurate glycan analysis with enhanced quantitative performance and structural elucidation capabilities.