NEW METHODS FOR QUANTITATIVE GLYCOSPHINGOLIPIDOMICS

定量定量鞘糖脂的新方法

• 批准号: 6951395
• 负责人: STEVEN B LEVERY
• 金额: $ 21.47万
• 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 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6951395
• 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）： 鞘糖脂（GSL：N-酰基鞘氨醇或神经酰胺的 1-O-糖苷）是一类结构和功能多样的生物化合物，分布于所有真核生物和一些细菌中。连同其生物合成中间体、代谢产物和简单的非糖基化鞘氨醇衍生物，它们被认为是所有真核细胞膜的重要组成部分。头基结构存在广泛的多样性，导致 GSL 具有广泛的物理/化学性质。这与膜脂提取物中通常遇到的复杂样品基质一起，可以使组织或生物体中存在的所有 GSL 成分的定量提取、准确分析和/或完整结构阐明成为一项艰巨的任务。已经提出了用于 GSL 分析的“通用”分析策略，但这些策略通常是高度劳动密集型的，或者仅涉及 GSL 成分或组织类型的有限子集。在此，提出了一种新的 GSL 分析方法的开发，其目标包括广泛的适用性、定量提取、独立于头基特性的所有组分的准确表示、敏感头基功能修饰的最小损失以及与各种后续分析仪器和衍生化策略的兼容性。进一步的目标是高通量应用的易用性和最少的样品处理，在质谱分析中增加分子种类的电离，以及适应同位素编码策略，从而能够对从不同菌株或发育阶段或在不同条件下生长的组织或细胞类型之间获得的 GSL 表达进行灵敏、可靠的定量比较。所提出的方法基于酶法去除神经酰胺 N-酰基链，然后用合适的氨基反应性亲和标签替换。将使用含生物素的标签，通过固定化链霉亲和素亲和系统定量去除和随后释放所有 GSL 成分。最初的开发将基于市售的生物素化试剂，但建议合成更适合 GSL 应用的试剂，掺入提高溶解度的官能团，并促进更高的电离产率。在后期阶段，建议合成同位素编码的亲和标签（鞘脂 ICAT 或 SL-ICAT），以促进定量鞘糖脂组分析，类似于已经开发用于蛋白质组分析的“ICAT”方法。