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、细胞黏附和信号传递，都是由多糖介导的。AS 因此，多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记物。 疾病和癌症。尽管它们很重要，但我们目前对不同的多糖结构的看法有限 复杂性存在于正常健康或疾病中。这是因为缺乏现成的方法来实现 糖链结构的广泛鉴定和定量。质谱学的最新进展 相关的软件工具有助于分析多聚糖结构。这些措施的广泛实施 方法一直受限于缺乏可接近的、高通量的样品制备策略，缺乏 统一实验室间数据采集的标准化方法，以及缺乏工具 糖链结构的自动峰分配。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、集成、独一无二的分析平台 Glycomics平台支持基于定量质谱学的多糖结构研究，而无需 特定蛋白质的预纯化。GlyThyra超越了经典凝集素的深度、特异性和速度- 基于方法或其他质谱学方法，通过实现快速、定量的血糖分析 &gt；300个N-和O-连接的多糖结构的丰度在3个数量级范围内。GlyThyra结合了 我们新的批量多聚糖定量和质量前总多糖含量归一化方法 光谱分析，以消除与不均匀加载相关的误差。内部和外部标准启用 技术变异的标准化和低实验室间变异的标准化结果的产生。 我们的新平台产生的初步数据提供了证据，证明新的潜在生物标志物对心脏，肺， 仅需1份μL血清或血浆即可快速识别血液病。拟议的研究将严格执行 验证GlyThyra以鼓励其在生物标记物开发工作中的下游利用和采用。 具体地说，我们将使用国家标准与技术研究所的非侵入性参考材料 样本类型(血清、血浆、尿液)，以进一步细化和评估绩效措施以验证样本 编制和数据采集(目标1)和数据分析模块(目标2)。目标3将建立参考 在100个健康的成人对照血清、血浆和尿样中检测&gt；100个糖链结构。结果 这项拟议的研究将通过使生物标记物能够 研究人员无法获得糖链技术来精确分析多聚糖 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 独家使用商用试剂和免费提供的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.