基于新型光衍生化方法和紫外光解离质谱的脂质双键异构体研究

The biological function of unsaturated lipids in organisms is closely related to the locations of their carbon–carbon double bonds (C=C bonds). Photochemical derivatization of C=C bonds in unsaturated lipid can rapidly distinguish C=C location isomers with the aid of CID tandem mass spectrometry (MS/MS), but the selectivity of the derivatization of lipid C=C bonds and the fragmentation of CID MS/MS remains to be improved. Previous studies have demonstrated that photochemical reactions of unsaturated lipids with derivatization reagents without pretreatment generally give rise to a large number of photo-derived by-products, resulting in uninformative fragment ions indicative of C=C bond positions with MS/MS. This project proposes the "complex-derivatization" strategy to achieve the derivatization of lipid C=C bonds, that is, the formation of unsaturated lipid-carbonyl compound precursors by preserving weak non-covalent interaction, followed with [2+2] photocycloadditions between lipid C=C bonds and C=O bonds. An ultraviolet photodissociation mass spectrometry, that is assembled with a high resolution time-of-flight mass spectrometry coupled with a linear ion trap and a wavelength-tunable UV laser, will be used to identify lipid C=C location isomers with diagnostic ions specific for the C=C bond positions. The structure changes of precursors will be traced by trapped ion mobility spectrometry -mass spectrometry during the photochemical derivatization. The theoretical simulations will be used to understand the interaction of unsaturated lipids and carbonyl compounds. With the optimization of the reaction conditions for photo-derivatization and analysis conditions for mass spectrometry, ultraviolet photodissociation mass spectrometry based lipid C=C bonds analysis with a novel photo-derivatization strategy for C=C bonds will be developed to obtain the distribution of lipid C=C bonds isomers in different biological tissues.

不饱和脂质在生物体中的生物功能与其C=C键的位置密切相关。脂质双键光化学衍生法串联CID串级质谱可快速分析不饱和脂质双键异构体，但脂质双键衍生及串级质谱裂解的选择性仍有待提高。以往研究都是不饱和脂质和衍生试剂直接进行光衍生反应，这会生成大量光衍生副产物，MS/MS不可提供C=C键位置信息。本项目拟采用“复合-衍生”的策略实现脂质双键的衍生，即先形成非共价相互作用的脂质-羰基化合物复合物前驱体，再进行脂质双键光化学[2+2]加成衍生。紫外光解离质谱裂解脂质C=C键光衍生物表征脂质双键异构体。通过离子淌度质谱追踪光化学衍生过程中复合物前驱体转化至光衍生物的结构变化，理论模拟认识不饱和脂质和羰基化合物的作用规律。优化光衍生条件和质谱分析条件，旨在建立新型的脂质双键衍生方法和高效的紫外光解离质谱分析方法，获得不同生物组织中脂质双键异构体的分布情况。

不饱和脂质占生物体内所有脂质的40%以上，且在维持生物膜流动性、细胞信号传导和炎症等生理过程发挥着重要作用。不饱和脂质在生物体中的生物功能与其C=C键的位置密切相关，但C=C键位置的鉴定仍是不饱和脂质结构表征的主要挑战。脂质双键光化学衍生法结合串联质谱技术可快速分析不饱和脂质双键异构体，但脂质双键衍生及串联质谱裂解的选择性仍有待提高。本项目针对串联质谱技术和脂质双键衍生方法进行研究，开发质谱离子活化技术和新型脂质C=C键环加成方法，以实现高效的脂质双键异构体分析。主要研究内容及研究结果包括：（1）可见光诱导实现不饱和脂质C=C键[2 + 2]环加成。通过构建不饱和脂质中烯烃结构和羰基底物的非共价相互作用，实现可见光诱导C=C和C=O键发生[2 + 2]环加成产生氧杂环丁烷。密度泛函理论研究表明溶剂系统中的H+促进底物中脂肪酰基链C=C单元和二苯甲酮C=O结构非共价相互作用，在可见光诱导下，复合物内C=C键电子转移至二苯甲酮，进而实现高选择性的[2 + 2]环加成反应。（2）商用质谱搭建具有波长调谐功能的UVPD MS/MS技术。通过硬件设计和电信号改造将商用Q-TOF质谱碰撞池功能升级为线性离子阱，以OPO激光为光源，在离子阱中实现了分析物离子UVPD MS/MS分析。通过波长作用谱研究，可选择利于目标离子的UV解离波长，实现分析物高效结构表征。（3）Paternò-Büchi（PB）反应结合UVPD MS/MS技术表征脂质双键异构体。PB特异性C=C键衍生可将不饱和脂质的UVPD分析波长红移，在≥210 nm下实现脂质双键异构体表征，且双键异构体诊断离子相对丰度明显高于不饱和脂质直接UVPD分析。该技术经“鸟枪法”法可实现对生物混合物样品中不饱和PC、PE和PG的双键位置异构体分析。项目形成的不饱和脂质双键可见光[2 + 2]环加成衍生新方法，及脂质双键异构体PB-UVPD MS/MS分析技术，有望为生物体中大规模不饱和脂质的结构鉴定提供技术支撑，进而助力于脂质组学的发展。