Development of high-throughput workflow for glycosphingolipid analysis and annotation

项目总结 鞘糖脂(GSL)是所有生物膜的基本成分，作为受体和 细胞与细胞、细胞与病原体相互作用的调节因子以及多种受体介导的细胞调节因子 信号事件。用于分析GSL结构的高灵敏、快速和简便的方法 生物来源目前还不存在，但将催化非糖类科学家更广泛地参与 了解GSL功能。GSL结构表征的大多数糖组学方法 利用酶消化法从疏水性脂类中释放出亲水性的多糖。虽然这种方法 简化了分析物的复杂性，使分析员无法检测脂类异质性，从而， 忽视了GSLS中脂质部分的生物学重要性。此外，酶-底物偏好 由消化产生的偏向糖链图谱。释放的GSL偶联多聚糖的液相色谱(LC) 质谱仪(MS)已被广泛应用于GSL谱的定量和表征。 遗憾的是，基于LC-MS的分析方法不能同时分析中性和酸性GSL 这些工作流程通常需要耗时的样品准备和 对数据进行密集的人工解释。纠正酶偏向和层析的后果 复杂性，我们将开发一种灵敏、稳健和全面的糖鞘磷脂组学方法 由半自动批注软件和高度精选的数据库支持，以进一步了解 GSLS的生物学意义。 我们建议开发标准化的方法来制备和鉴定完整的GSL 利用酶从脂类部分释放多聚糖。此外，这些方法将使用 全甲基化以加强结构分析，并支持同时鉴定中性和 酸性GSL结构。作为提议的工作流程的一部分，我们将针对不同的MS分析优化此方法 平台和碎片类型。样品制备和MS方法将由一位 便于分析和注释结果数据集的软件套件。该软件将支持高 通过显著减少注释和解释所需的时间，提高了MS数据分析的吞吐量。 样品制备协议以及针对不同平台和碎片类型的MS方法将包括 在我们的项目网站上向社区公开提供。该软件将免费提供给 也可以下载。

PROJECT SUMMARY Glycosphingolipids (GSL) are essential components of all biological membranes and serve as receptors and modulators of cell-cell and cell-pathogen interactions as well as regulators of diverse receptor-mediated cell signaling events. Highly sensitive, rapid, and facile methods for analyzing GSL structures harvested from biological sources do not currently exist, but would catalyze broader participation by non-glycoscientists in understanding GSL function. Most current glycomics approaches for the characterization of GSL structure utilize enzymatic digestion to release the hydrophilic glycan from the hydrophobic lipid. While this approach simplifies the complexity of the analyte, it blinds the analyst from detecting lipid heterogeneity and, thereby, ignores the biological importance of the lipid portion of the GSLs. Furthermore, enzyme-substrate preferences bias glycan profiles generated by digestion. Liquid chromatography (LC) of released GSL glycans coupled to mass spectrometry (MS) have been broadly applied to quantifying and characterizing GSL profiles. Unfortunately, LC-MS based analytic approaches are not able to profile both neutral and acidic GSL components at the same time and these workflows generally require time-consuming sample preparation and intensive manual interpretation of the data. To remedy the consequences of enzyme bias and chromatographic complexity, we will develop, a sensitive, robust and comprehensive methodology for glycosphingolipidomics supported by a semi-automated annotation software with a highly-curated database to further understand the biological significances of GSLs. We propose to develop standardized methods for the preparation and identification of intact GSL without using enzymatic release of the glycan from the lipid portion. Furthermore, these methods will employ permethylation to enhance structural analysis and to support the simultaneous identification of neutral and acidic GSL structures. As part of the proposed workflow we will optimize this method for different MS analytical platforms and fragmentation types. The sample preparation and MS methods will be complimented by a software suite that facilitates analysis and annotation of the resulting datasets. The software will support high throughput MS data analysis by significantly reducing the time required for annotation and interpretation. Sample preparation protocols as well as MS methods for different platforms and fragmentation types will be made publicly available to the community on our project website. The software will be freely available for download as well.