生物组织中脂质双键异构体的质谱成像分析

Compared with other spectrometric imaging techniques, the high speed, high sensitivity, multiplexed analysis ability and high spatial resolution (up to 10 nm) underlie the wide application of mass spectrometry imaging (MSI) in the field of disease diagnosis. Both disease markers such as proteins, and other small molecular metabolites, such as lipids and carbohydrates, can be effectively detected by MS. As a basic component of cell membrane, energy source and signalling molecules, the relationship between the lipid composition and the disease state has attracted increased attentions from researchers in recent years. In previous studies, we have found for the first time that there are significant differences in the composition of lipid C=C isomers in normal and diseased mice or human tissues. In this project, we will develop a MS technique for the highly specific imaging of lipid C=C isomers in biological tissues, and explore the feasibility of applying this method to define the boundary betweeen normal and diseased tissues, as well as for disease diagnosis. A large class of carbonyl reagents will be screened in terms of the reaction rate, selectivity, degree of side reactions and efficiency of product fragmentation to produce diagnostic ions. The reaction will be carried out in a custom-designed reaction chamber with a uniform LED to promote the reaction. By using the developed MS imaging system, we will analyze the lipid C=C isomer compositions in a variety of human tissues, and compare them with those from healthy controls. It is hoped that a connection between the tissue type and state (normal/diseased) and the isomer composition can be established, which will serve as the foundation for the development of novel disease diagnostic techniques.

与光谱成像技术相比，质谱成像技术以其快速、高灵敏度、多通道质量分析能力和高空间分辨率（可达10 nm）目前正被越来越广泛地用于疾病诊断中。脂类作为细胞膜的基本组成分子、能量来源和信号分子，其组成的变化与疾病间的密切关系近年来日益引起研究者的关注。在此前研究中，我们首次发现脂类异构体的组成在正常和病变小鼠和人体组织中有着显著的差异，有用于疾病诊断的巨大潜力。本课题中，我们将发展可用于生物组织中脂类异构体成像的质谱分析技术，并探索将该方法用于正常-病变组织边界区分和疾病诊断的可行性。将以反应速率、选择性、副反应程度和产物碎裂产生特征离子的效率等指标优化不饱和脂质的衍生反应。反应在温湿度恒定的反应腔中进行，以平面LED光源提供均一的反应条件。在此基础上，利用所搭建的装置系统分析各种正常和病变人体组织中的脂质异构体组成，建立脂质异构体组成与组织类型和“正常或病变”间的联系，发展新型疾病诊断技术。

质谱成像以其高灵敏度、高空间分辨率和多组分同时分析能力，已在基础生物学研究、毒理研究和疾病诊断等方面获得广泛应用。长期以来，质谱成像主要关注分析物的质量测定，结构表征能力较弱。例如，脂类物质的数量目前已超过40,000种，其中异构体数目不在少数。脂质碳碳双键（C=C）和sn异构体化学组成和质量完全相同，难以通过MS/MS直接区分，但往往有着截然不同甚至相反的生物学效应。因此，发展可分辨生物异构体的质谱成像技术，既能填补质谱成像领域的空白，也是深入研究脂质等生物异构体的功能所必需的。本研究的思路是通过组织中脂质的化学衍生和活化，利用串联质谱成像实现生物组织中脂质异构体的成像。具体研究内容包括：（1）脂质异构体结构表征方法和衍生剂筛选；（2）无基质激光解吸附离子化成像研究；（3）生物组织中不饱和脂质异构体的质谱成像。首先，通过脂质衍生反应和衍生化试剂筛选，深入研究了反应机理和产物的碎裂行为，更准确、更快速地实现不饱和脂质C=C定位和脂质C=C异构体定量，并发现一种新的衍生试剂（2-乙酰吡啶）与MS3分析结合（PB-MS3）可获得除磷脂C=C顺反异构外的所有结构信息。其次，发展了新的质谱成像策略，利用金属微纳阵列吸光材料及其表面涂覆聚多巴胺的复合材料，替代小分子基质，消除了低质量端的基质干扰，显著提高了脂类、糖类等代谢物的质谱成像检测灵敏度。最后，在上述研究基础上，利用微萃取和离子化技术实现对鼠脑样品中不饱和脂质C=C异构体和sn异构体的质谱成像。研究结果表明，脂质及其异构体在病变和正常组织中存在差异性空间分布。因此，本项目发展了具备脂质异构体分辨能力的质谱成像技术，为进一步研究脂质生物学功能、寻找新的疾病标记物和实现基于脂质异构体分析的疾病诊断提供了重要的分析手段。