基于交联剂跨膜靶向转运载体的细胞内蛋白质复合物原位靶向分析方法

Chemical cross-linking coupled with mass spectrometry (CXMS) has become an important tool for the analysis of protein complexes. In CXMS, chemical crosslinkers can not be transported to the intracellular targets, which lead to the difficulty of in situ intracellular analysis of protein complexes in subcellular organelles. In this project, the crosslinker transmembrane targeted carrier will be designed through active controllable polymerization and self-assembly technology with boric monomers, chitosan and cholesterol monomers as the skeleton monomers, environmental sensitive monomers as the response unit, and subcellular localization reagents as the post-modification ligands. Thermodynamics and kinetics of crosslinker transmembrane targeted carrier-based intracellular transmission is going to be studied to build the structure-activity relationship between the transport carrier and the targeting transport capacity. We are going to build the relationship between the physical stimulation and the release rate of the carrier in order to fabricate the model and reveal the mechanism of the controllable releasement of carriers in cells. Combing the cross-linked peptide enrichment technique with high selectivity and high recovery rate and protein complex data mining technique with high accuracy, crosslinker transmembrane targeted carrier-based in situ intracellular analysis method of the protein complex is going to be built. Finally, as the demonstration, the crosslinker transmembrane targeted carrier will be applied in the drug resistant tumor cells for the in situ dynamic analysis toward protein complex on the subcellular level. This would provide technical support for the further study of tumor resistance.

化学交联结合质谱技术（CXMS）已成为当前蛋白质复合物分析的重要工具。针对CXMS中由于化学交联剂无法靶向输运而导致的亚细胞器内蛋白质复合物的原位分析困难的问题，以硼酸类单体、壳聚糖以及胆固醇类单体为骨架单体，以环境敏感单体为响应单体，以亚细胞器定位试剂为后修饰配基，通过活性可控聚合和聚合物自组装技术，制备蛋白质复合物化学交联剂跨膜靶向转运载体；通过转运载体在细胞内传输的热力学和动力学研究，建立转运载体结构与靶向转运能力的构效关系；通过建立物理刺激与跨膜靶向转运载体释放速度之间的关系，构建化学交联剂的体内原位释放模型，揭示转运载体在细胞内原位靶向释放的机理。结合高选择性、高回收率的交联肽段富集技术以及高准确性蛋白质复合物解析技术，建立基于转运载体的细胞内蛋白质复合物原位分析方法。将靶向转运载体示范性应用于肿瘤耐药细胞内亚细胞层面上蛋白质复合物的原位动态分析，为肿瘤耐药的研究提供技术支撑。

针对化学交联质谱技术（CXMS）中化学交联剂无法靶向输运而导致细胞内蛋白质复合物原位分析困难的问题，制备了具有高效跨膜和原位可控快速释放的化学交联剂跨膜靶向转运载体；并基于此，建立了基于化学交联剂跨膜靶向转运载体的细胞内蛋白质复合物原位分析方法；从蛋白冠水平，构建了载体跨膜靶向递送的模型；研究了转运载体的胞内释放方式和效率；并将研制的化学交联剂跨膜转运载体用于肿瘤细胞内亚细胞层面（线粒体）蛋白质复合物的原位动态分析，为活细胞中参与细胞能量代谢的TCA 酶、OXPHOS 超复合物和动态蛋白质的结构信息提供了证据。并且交联信息还从实验角度，匹配了AlphaFold 2预测模型，为超复合物（SCI1III2IV1）的冷冻电镜结构提供了补充信息。为活细胞内亚细胞器蛋白质结构和相互作用原位分析提供了关键技术支撑。已发表SCI文章4篇，另1篇稿件在Nat. Commun.修稿，申请发明专利6项；在高水平国际会议上发表论文3篇，培养博士研究生3名，完成了《资助项目计划书》中的研究目标。