NEW METHODS FOR QUANTITATIVE GLYCOSPHINGOLIPIDOMICS

定量定量鞘糖脂的新方法

• 批准号: 6853430
• 负责人: STEVEN B LEVERY
• 金额: $ 17.84万
• 依托单位: UNIVERSITY OF NEW HAMPSHIRE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-27 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6853430
• 关键词: affinity labeling; cell membrane; ceramides; chemical property; chemical structure function; gene expression; glycosphingolipids; high throughput technology; lipid structure; mass spectrometry; membrane lipids; method development; physical property; physical separation; stable isotope

DESCRIPTION (provided by applicant): Glycosphingolipids (GSLs: 1-O-glycosides of the N-acyl-sphingosines, or ceramides) are a structurally and functionally diverse class of biological compounds distributed among all eukaryotes and some bacteria. Along with their biosynthetic intermediates, metabolic products, and simple non-glycosylated sphingosine derivatives, they are believed to be essential components of all eukaryotic cell membranes. A wide diversity in headgroup structures exists, contributing to the wide range of physical/chemical properties observed for GSLs. This, along with the complex sample matrix generally encountered in membrane lipid extracts, can render the quantitative extraction, accurate profiling and/or complete structure elucidation of all GSL components present in a tissue or organism daunting tasks. "Universal" analytical strategies for GSL analysis have been proposed, but these are often highly labor intensive or address only a limited subset of GSL components or tissue types. Herein, development of a novel methodology for GSL analysis is proposed, incorporating goals of wide applicability, quantitative extraction, accurate representation of all components independent of headgroup properties, minimal loss of sensitive headgroup functional modifications, and compatibility with a variety of subsequent analytical instrumental and derivatization strategies. Further goals are ease of use and minimal sample handling for high-throughput applications, increased ionization of molecular species in mass spectrometric profiling, and adaptation to isotope-coding strategies enabling sensitive, reliable quantitative comparisons of GSL expression between tissues or cell types obtained, e.g., from different strains or developmental stages, or grown under different conditions. The proposed methodology is based on enzymatic removal of the ceramide N-acyl chain, followed by replacement with a suitable amino-reactive affinity tag. Biotin-containing tags will be used, enabling quantitative removal and subsequent release of all GSL components via immobilized streptavidin affinity systems. Initial development will be based on commercially available biotinylating reagents, but synthesis of reagents more specifically tailored for GSL applications, incorporating functional groups improving solubility, and promoting higher ionization yields, is proposed. In a later phase, synthesis of isotope coded affinity tags (sphingolipid ICAT, or SL-ICAT) is proposed, in order to facilitate quantitative glycosphingolipidomic profiling, analogous to the "ICAT" methodology already developed for proteomic analysis.

描述（由申请人提供）：糖磷脂脂（GSL：N-酰基 - 闪氨酸或神经酰胺的1-O-糖苷）是在所有真核生物和一些细菌中分布的结构和功能上多样的生物化合物。除了它们的生物合成中间体，代谢产物和简单的非糖基化鞘氨酸衍生物，它们被认为是所有真核细胞膜的必不可少的成分。头部组结构的多样性存在，有助于GSL观察到的广泛的物理/化学特性。这与通常在膜脂质提取物中遇到的复杂样品基质一起，可以阐明定量提取，准确的分析和/或完整的结构阐明组织或有机体艰巨任务中所有GSL成分的完整结构。已经提出了用于GSL分析的“通用”分析策略，但是这些策略通常是高度劳动密集型的，或仅解决了GSL组件或组织类型的有限子集。在此，提出了用于GSL分析的新方法的开发，它结合了广泛的适用性，定量提取的目标，与头组属性无关的所有组件的准确表示，敏感头组功能修饰的最小损失以及与各种后续分析工具和衍生化策略的兼容性最小。进一步的目标是易用性和用于高通量应用的样本处理，质谱分析中分子物种的电离增加，以及适应同位素编码策略，从而实现敏感的，可靠的定量比较，从而在不同的菌株中获得的组织或细胞类型之间的GSL表达进行比较，例如，从不同的菌株或开发阶段或不同的条件下，可以进行不同的条件。所提出的方法基于神经酰胺N-酰基链的酶促去除，然后用合适的氨基反应性亲和力标签代替。将使用含生物素的标签，可以通过固定的链霉亲和素亲和力系统释放定量删除，并随后释放所有GSL组件。最初的开发将基于市售的生物素化试剂，但是提出了更专门针对GSL应用定制的试剂的合成，提出了提高溶解度的功能组以及促进更高的电离产量。在以后的阶段中，提出了同位素编码的亲和力标签（鞘脂ICAT或SL-ICAT）的合成，以促进定量糖磷脂型分析，类似于已经开发用于蛋白质组学分析的“ ICAT”方法。