NEW METHODS FOR QUANTITATIVE GLYCOSPHINGOLIPIDOMICS

鞘糖脂（GSLs: n -酰基鞘脂的1- o糖苷，或神经酰胺）是一种结构和功能多样化的生物化合物，分布在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生物细胞膜的必需成分。头基结构的多样性使得GSLs具有广泛的物理/化学性质。这一点，以及在膜脂提取物中通常遇到的复杂样品基质，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，或者只处理GSL成分或组织类型的有限子集。本文提出了一种新的GSL分析方法，其目标包括广泛的适用性、定量提取、独立于头基团性质的所有成分的准确表示、敏感头基团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。进一步的目标是在高通量应用中易于使用和最少的样品处理，在质谱分析中增加分子物种的电离，以及适应同位素编码策略，以便在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进行敏感、可靠的GSL表达定量比较。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性亲和标签替代。将使用含生物素的标签，通过固定化链亲和素亲和系统实现所有GSL成分的定量去除和随后释放。最初的开发将基于商业上可获得的生物素化试剂，但建议合成更专门为GSL应用定制的试剂，加入官能团以改善溶解度，并促进更高的电离收率。在后期阶段，提出了同位素编码亲和标签（鞘脂ICAT，或SL-ICAT）的合成，以促进定量鞘脂糖组学分析，类似于已经开发用于蛋白质组学分析的“ICAT”方法。

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.