NEW METHODS FOR QUANTITATIVE GLYCOSPHINGOLIPIDOMICS

描述（由申请人提供）：鞘糖脂（GSL：N-酰基鞘氨醇的1-O-糖苷或神经酰胺）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。沿着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，它们被认为是所有真核细胞膜的必需组分。头基结构存在广泛的多样性，有助于观察到的GSL的广泛的物理/化学性质。这沿着膜脂质提取物中通常遇到的复杂样品基质，可以使得组织或生物体中存在的所有GSL组分的定量提取、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，但是这些通常是高度劳动密集型的或者仅针对GSL组分或组织类型的有限子集。在此，提出了一种新的GSL分析方法的发展，结合广泛的适用性，定量提取，准确表示的所有组件独立的头基属性，最小的损失敏感的头基功能性修饰，并与各种后续的分析仪器和衍生化策略的兼容性的目标。进一步的目标是易于使用和用于高通量应用的最小样品处理，在质谱分析中分子种类的电离增加，以及适应同位素编码策略，使得能够在获得的组织或细胞类型之间灵敏、可靠地定量比较GSL表达，例如，来自不同的菌株或发育阶段，或在不同的条件下生长。所提出的方法是基于神经酰胺N-酰基链的酶促去除，然后用合适的氨基反应性亲和标签替换。将使用含生物素的标签，从而能够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组分。最初的开发将基于市售的生物素化试剂，但提出了更专门针对GSL应用的试剂合成，将官能团引入改善溶解度，并促进更高的电离产率。在稍后的阶段中，提出了同位素编码的亲和标签（鞘脂ICAT，或SL-ICAT）的合成，以促进定量鞘糖脂组学分析，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.