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.

说明(申请人提供)：鞘糖脂(GSLS：N-酰基鞘氨醇的1-O-糖苷，或神经酰胺)是一类结构和功能不同的生物化合物，分布于所有真核生物和一些细菌中。与它们的生物合成中间体、代谢产物和简单的非糖基化鞘氨醇衍生物一起，它们被认为是所有真核细胞膜的基本成分。头基结构中存在着广泛的多样性，这有助于观察到GSLS的广泛的物理/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、准确剖析和/或完整的结构阐明成为艰巨的任务。已经提出了用于GSL分析的“通用”分析策略，但这些分析策略通常是高度劳动密集型的，或者仅针对GSL成分或组织类型的有限子集。在此，建议开发一种新的GSL分析方法，其目标包括广泛的适用性、定量提取、与头基性质无关的所有组分的准确表示、灵敏的头基功能修饰的最小损失以及与各种后续的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，在质谱学分析中增加分子物种的电离，以及适应同位素编码策略，从而能够对组织或细胞类型之间的GSL表达进行敏感、可靠的定量比较，例如，从不同的菌株或发育阶段获得的，或在不同的条件下生长的。所提出的方法是基于酶去除神经酰胺N-酰基链，然后用合适的氨基反应亲和标记来取代。将使用含有生物素的标签，通过固定化的链霉亲和素亲和系统定量移除和随后释放所有GSL成分。最初的开发将基于商业上可获得的生物素化试剂，但建议合成更专为GSL应用而定制的试剂，加入改善溶解性的官能团，并促进更高的电离产率。在较后的阶段，建议合成同位素编码的亲和标签(鞘磷脂ICAT，或SL-ICAT)，以促进糖鞘脂体的定量分析，类似于已经开发的用于蛋白质组分析的“ICAT”方法。