硫酸寡糖的质谱分析新方法研究

Since sulfated oligosaccharides are a group of important bio-information molecules with various biological functions, detection and structural analysis of sulfated oligosaccharides have become imperative to understand their structure-activity relationship and biological roles. However, the analysis of sulfated oligosaccharides are technically challenging due to their sulfate lability, structural diversity, low ionization efficiency in the positive ion mode mass spectrometry (MS) and inefficient chromatographic separation of isomers. Accordingly, improved methods for structural analysis of sulfated oligosaccharides are required to understand their functional roles in various biological processes. The present project is based on our preliminary study to develope a new chemical derivatization strategy, which is highly sensitive for the analysis of sulfated oligosaccharides using mass spectrometry (MS) and high-performance liquid chromatography coupling with mass spectrometry (LC-MS). The method will be developed based on replacing the labile sulfate groups with stable silyl groups, while maintaining sulfation information (the position and number of sulfate groups) of the original sulfated oligosaccharides. In our method, sulfated oligosaccharides are first permethylated and then desulfated with silylating reagents, sulfate groups substituted by silyl groups which are sufficiently stable during MS analysis. Therefore, the number of sulfate groups of a particular sulfated oligosaccharide can be caculated from the mass shift between the MS spectra of the permethylated and silylated compounds, and the position of sulfate groups can be revealed by comparing the prompt fragmentation surviving in multi-stage mass spectrometry (MSn) of silylated compounds, and isomer mixtures of sulfated oligosaccharides can be separated by high-performance liquid chromatography (HPLC) of the derivatized oligosaccharides. In traditional MS analysis, sulfated oligosaccharides are often detected using negative ion mode MS, which usually produces multiple-charged ions depending on the size of the oligosaccharides and the number of sulfate groups, and sulfated oligosaccharides have a tendency to lose labile sulfate groups during MSn analysis. Since our method presented here could overcome the above problems by improving the detection sensitivity of sulfated oligosaccharides in positive ion mode MS and overcoming the obstacle of high polarity of highly sulfated oligosaccharides or highly polymerized sulfated oligosaccharides in HPLC separation, it could be easy and effective to separate and analyze isomer mixtures of sulfated oligosaccharides by LC-MS. The method is expected to provide investigations of high sensitivity, wide applicability and high throughput into sulfated oligosaccharides.

硫酸寡糖有多种生物活性，是重要的生物信息分子。硫酸寡糖分析与结构解析是其构效关系研究的前提。由于极性大、结构多样、不易分离等原因，硫酸寡糖的分析面临很多困难。本课题拟在前期研究的基础上，以质谱和液质联用为主要检测手段发展高灵敏度的基于衍生化方法的硫酸寡糖分析新策略。首先对硫酸寡糖羟基甲基化，然后用硅醚化试剂脱硫酸基，硫酸化位点在脱硫酸基的同时被硅醚化，将不稳定的硫酸基转化为稳定的取代基；研究脱硫酸寡糖在多级质谱中的裂解行为，确定硫酸基的取代位点、硫酸基的数目并进行同分异构体的分离分析。该方法的建立可克服现有的质谱技术分析硫酸寡糖时易形成多电荷离子，对硫酸寡糖进行多级裂解时，硫酸基易丢失的缺点，也可克服目前由于多硫酸基的高亲水性不利于HPLC分离分析的缺点，可以实现与生物质谱联用用于硫酸寡糖分子量的精确测定、序列及连接方式分析及同分异构体的区分，对硫酸寡糖分析鉴定的方法学研究有重要意义。

硫酸寡糖有多种生物活性，是重要的生物信息分子。硫酸寡糖分析与结构解析是其构效关系研究的前提。由于极性大、结构多样、不易分离等原因，硫酸寡糖的分析面临很多困难。本课题以质谱和液质联用为主要检测手段发展高灵敏度的基于衍生化方法的硫酸寡糖分析新策略。首先对硫酸寡糖羟基甲基化，然后用硅醚化试剂脱硫酸基，硫酸化位点在脱硫酸基的同时被硅醚化，将不稳定的硫酸基转化为稳定的取代基；研究脱硫酸寡糖在多级质谱中的裂解行为，确定硫酸基的取代位点、硫酸基的数目并进行同分异构体的分离分析。. 通过该课题的实施，完成了硫酸基取代的蔗糖的合成方法；以合成的硫酸基取代的蔗糖(6, 1’,6’-三硫酸基蔗糖，4, 1’,6’-三硫酸基蔗糖)为标准化合物考察了各种因素包括硅醚化试剂的种类、硅醚化试剂与硫酸寡糖的摩尔比、反应溶剂、反应时间、反应温度、pH值等对脱硫酸基的影响，确定了最佳的脱硫酸基条件使硫酸寡糖能完全定量脱除硫酸基并在脱硫酸基时衍生化上硅醚化试剂；酶解制备硫酸化的κ-卡拉胶寡糖，以其为模型研究脱硫酸寡糖在多级质谱中的裂解行为；建立了硫酸化多糖的硫酸化位点分析的新方法，实现了对硫酸化多糖分子中硫酸基团的定性、定量分析。