Development of a next-generation glycomics platform to enable glycan structure analyses for precision medicine

生物学的几乎所有方面，包括蛋白质折叠、细胞粘附和信号传导，都由聚糖介导。作为 这些聚糖已被用作血细胞分型、心血管和自身免疫的临床生物标志物 疾病和癌症。尽管它们很重要，但我们目前对不同聚糖结构的认识有限。 在正常健康或疾病中存在的复杂性。这是由于缺乏现成的方法， 聚糖结构的广泛鉴定和定量。质谱学的最新发展 和相关的软件工具促进了聚糖结构的分析。广泛实施这些 由于缺乏可接近的高通量样品制备策略， 统一实验室数据采集的标准化方法，以及缺乏 聚糖结构的自动峰归属。在初步研究中，我们克服了主要的可行性差距 以前的技术。我们的新分析平台GlyThyra是第一个单一的、集成的、独一无二的 糖组学平台，以实现基于定量质谱的聚糖结构研究，而无需 预纯化特定蛋白质。GlyThyra超越了经典凝集素的深度、特异性和速度- 基于质谱的方法或其他质谱方法，通过实现快速、定量的糖组学分析， 在丰度上跨越3个数量级范围的>300个N-和0-连接的聚糖结构。GlyThyra公司 我们的新方法用于批量聚糖定量和质量分析前总聚糖含量的标准化 光谱法，以消除与不等负载相关的误差。内部和外部标准使 技术变异的标准化和产生具有低实验室间变异的标准化结果。 我们的新平台生成的初步数据提供了证据，表明心脏，肺， 血液疾病可以从1μL血清或血浆中快速识别。拟议的研究将严格 验证GlyThyra，以鼓励其在生物标志物开发工作中的下游利用和采用。 具体来说，我们将使用美国国家标准与技术研究所的参考材料进行非侵入性 样本类型（血清、血浆、尿液），以进一步完善和评估验证样本的性能指标 准备和数据采集（目标1）和数据分析模块（目标2）。目标3将建立参考 在100个健康成人对照血清、血浆和尿液样品中>100个聚糖结构的范围。成果 拟议的研究将影响生物标志物的发展，在广泛的疾病， 否则无法使用糖组学技术进行精确的聚糖分析的研究人员 临床相关样本中的结构。所有分析和生物信息学工作流程的无缝集成 独家使用市售试剂和免费软件将确保平台能够 在研究实验室中广泛实施。

Nearly all aspects of biology, including protein folding, cell adhesion and signaling, are mediated by glycans. As such, glycans have been exploited as clinical biomarkers for blood cell typing, cardiovascular and autoimmune disease, and cancer. Despite their importance, we currently have a limited view of the diverse glycan structure complexity present in normal health or disease. This is due to the lack of off-the-shelf approaches that enable the widespread identification and quantitation of glycan structures. Recent developments in mass spectrometry and related software tools facilitate the analysis of glycan structures. Widespread implementation of these approaches has been limited by the lack of approachable, high-throughput sample preparation strategies, lack of standardized methods for harmonization of data acquisition among laboratories, and absence of tools for automated peak assignment of glycan structures. In preliminary studies we have overcome major feasibility gaps of previous technologies. Our new analytical platform, GlyThyra, is the first single, integrated, one-of-a-kind glycomics platform to enable quantitative mass spectrometry-based glycan structure studies without requiring pre-purification of specific proteins. GlyThyra surpasses the depth, specificity, and speed of classical lectin- based approaches or other mass spectrometry approaches by enabling rapid, quantitative glycomic profiling of >300 N- and O-linked glycan structures across 3 orders of magnitude range in abundance. GlyThyra incorporates our novel approach for bulk glycan quantitation and normalization of total glycan content prior to mass spectrometry to eliminate errors associated with unequal loading. Internal and external standards enable normalization of technical variation and the generation of standardized results with low interlab variation. Preliminary data generated by our new platform provide evidence that novel potential biomarkers for heart, lung, and blood disease can be rapidly identified from just 1μL serum or plasma. The proposed studies will rigorously validate GlyThyra to encourage its downstream utilization and adoption in biomarker development efforts. Specifically, we will use National Institute for Standards and Technology reference materials for non-invasive sample types (serum, plasma, urine) to further refine and assess performance measures to validate the sample preparation and data acquisition (Aim 1) and data analysis modules (Aim 2). Aim 3 will establish the reference ranges for >100 glycan structures across 100 healthy adult control serum, plasma, and urine samples. Outcomes of the proposed studies will impact biomarker development across a broad range of diseases by enabling researchers who would not otherwise have access to glycomic technologies to perform precise profiling of glycan structures in clinically relevant samples. The seamless integration of all analytical and bioinformatic workflows and exclusive use of commercially available reagents and freely available software will ensure the platform can be widely implemented among research laboratories.