具有光交联和蛋白“质谱标签”转化功能的寡糖探针构建及其在"糖链-活体膜蛋白"相互作用研究中的应用

在活体条件下开展糖链膜蛋白受体的寻找与鉴定研究极具挑战性，至今尚无有效的方法。本课题拟以申请人在执行青年基金中建立的新型糖脂探针及其富集技术为基础，针对活体条件下由于“糖链—蛋白复合体”作用力极其微弱造成糖链—蛋白复合体难于分离和鉴定的问题，利用已确立的b1,3-葡寡糖十三糖与Dectin-1蛋白特异识别，通过葡寡糖糖脂探针与NHS-SS-Diazirine反应实现双吖丙啶和二硫键基团的引入，双吖丙啶通过与Dectin-1光交联，有效解决 “糖链-蛋白”在分离中的“脱离”难题；通过用二硫苏糖醇来打断“糖链-Dectin-1”交联臂上的二硫键，实现糖脂探针上的“S-Diazirine”在蛋白上的“质谱标签” 转化；然后结合MALDI-MS/MS分析和Mascot软件实现S-Diazirine标记Dectin-1蛋白的高灵敏、高通量鉴定；最后再在表达Dectin-1的THP-1细胞株上进行验证

Detection and characterization of oligosaccharide ligands of membrane protein receptors in living cells have been very challenging in glycobiology and cell biology. there is still no an effective method. To address this, we will develop a novel oligosaccharide probe, based on the recent success in the neoglycolipid (NGL) and its enrichment method developed in the recent NSFC funded project. The proposal will be centred on the key issue that the affinity of the carbohydrate-protein interaction is very weak and therefore it is difficult to separate and enrich the “carbohydrate-protein” complex for characterization. Our plan is to use the well documented recognition system beta1,3-glucooligosaccharides-13 mer binding to Dectin-1 as a model to develope a novel NGL probe using beta1,3-glucooligosaccharides-13 mer NGL conjugated to photocrosslinker NHS-SS-Diazirine, to realize the glucan oligosaccharide NGL probe co-valently linking to Dectin-1 protein, effectively solving the problem of isolating the complex. Dithiothreitol is then used to break the “-SS-” bond. The resulting “S-Diazirine" group conjugated in Dectin-1 can be used as a unique mass-tag for mass spectrometry. High sensitivity and high throughput identification of the tagged peptide by MALDI-MS/MS combined with Mascot software will achieve peptide sequencing analysis and protein identification. Finally, the THP-1 cell line expressing Dectin-1 is treated and verified with the probes and technology developed in this work.