Mass Defect-based Chemical Tags for Multiplex Glycan Quantitation

摘要 蛋白质糖基化在许多生物学过程中起着关键作用，是最常见和最重要的生物学过程之一。 复杂的翻译后修饰已知糖基化谱的改变与 许多疾病，包括癌症、神经退行性疾病、免疫性疾病和心血管疾病 问题基于质谱（MS）的糖蛋白质组学和糖组学的进展越来越多 使得能够进行蛋白质糖基化的位点特异性结构分析的定性和定量方法。 然而，天然聚糖的定量分析由于其高度复杂性和复杂性而仍然极具挑战性。 聚糖结构的异质性和多样性，光学和质谱的低响应， 检测方法和临床相关样品中聚糖的宽动态范围。分析战略 对于来自糖蛋白的N-连接聚糖，通常涉及通过PNGase F从糖蛋白释放聚糖 消化并用便于检测的试剂标记释放的聚糖。这个的主要目标 建议是通过定制合成开发一种新颖且具有成本效益的化学标记技术 并开发了一套基于质量缺陷的标签， 高分辨率质谱（MS）对复杂聚糖进行高通量定量 平台我们提出以下具体目标：具体目标1 -合成和开发新的质量 基于缺陷的多重二甲基嘧啶基鸟氨酸（DiPyrO）标签，用于成本有效和高通量的MS- 基于从生物样品释放的N-聚糖的相对定量。具体目标2 -优化 异构体聚糖结构的分辨率和最大限度地提高聚糖电离效率，nanoHILIC将耦合 采用新型亚环境压力电离纳米喷雾（SPIN）源和高分辨率Orbitrap MS平台 用于从复杂生物样品中释放的多重DiPyrO标记的N-聚糖的超灵敏检测 例如血清和细胞裂解物。具体目标3 -合成和开发新的基于质量缺陷的多路复用 二甲基喹唑啉基鸟氨酸（DiQuuniO）标签，用于改进光学检测和开发毛细管 一种多维聚糖的荧光-UV/LIF-MALDI-离子迁移率-质谱成像平台 表征和定量。总的来说，我们提出的实验将开发新的使能工具， 将产生具有成本效益和新型的基于质量缺陷的标记试剂， 具有增强的定量性能和结构解析能力的聚糖分析。

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.