基于表面辅助激光解吸电离质谱的肝癌组织糖蛋白质组成像分析方法研究

糖链结构的异常变化与肝癌的发生发展和侵袭转移密切相关，系统开展糖蛋白/糖链表达的研究对深入理解肝癌发病机制和开展肝癌精准诊疗具有重要意义。基于生物质谱的蛋白质组学策略尽管可以获得糖蛋白/糖链的结构信息，但无法精准反映糖蛋白/糖链的组织空间分布和动态变化。于此，本项目拟构建一种集原位酶解、原位富集、原位鉴定于一体的规模化高通量的肝癌组织糖蛋白质组质谱成像分析平台，具体包括：开发酶-无机杂化纳米复合材料，发展糖蛋白原位酶解新策略；开发富集型纳米探针，发展糖肽/糖链原位富集新技术；开发SALDI纳米基质，建立糖链原位鉴定及其质谱成像新方法。在此基础上，整合各系统并构建适用于糖蛋白/糖链的SALDI-MSI分析平台。利用该平台有望全面获取肝癌组织中低丰度糖蛋白/糖链的空间分布和动态变化信息，筛选具有表达差异和空间分布差异的生物标志物，阐明蛋白质糖基化异常表达对肝癌发生发展与侵袭转移的影响及其机制。

Glycosylation is one of the most important posttranslational modifications of proteins, involved in many biological pathways and molecular function. Recent studies have demonstrated that aberrant alterations of glycan structure and glycosylation site are closely related to occurrence, development, invasion, and metastasis processes of hepatocellular carcinoma (HCC). Therefore, comprehensive analysis of glycoprotein/glycans expression is of great significance for in-depth understanding the pathogenic mechanism and conducting precise diagnosis and treatment of HCC. Although mass spectrometry (MS)-based glycoproteomics can realize analysis of thousands of glycoproteins to obtain detailed information concerning protein glycosylation in both qualitative and quantitative manners, the site-specific spatial distributions and dynamic changes of glycoprotein/glycans in tissues cannot be visualized precisely, which hinders advances in precise diagnosis and treatment of HCC. To address this issue, a highly integrated platform of surface assisted laser desorption ionization mass spectrometry imaging (SALDI-MSI), which includes in situ proteolytic digestion, enrichment and MS identification of glycoprotein/glycans, is proposed for large-scale, high-throughput, and spatial profiling analysis of glycoproteome in HCC tissues. To be more specific, this proposal in the project is to perform, (1) a series of enzyme-inorganic hybrid nanocomposites will be developed and served as novel immobilized-enzyme microreactors (IMERs) for highly efficient in situ proteolytic digestion of glycoproteins in tissues; (2) a series of hydrophilic and affinity interaction-based functionalized nanomaterials will be exploited and employed as new probes for in situ selective enrichment of low-abundance glycopeptides/glycans from tissues; (3) a series of low dimensional nanomaterials will be explored and applied as novel SALDI matrices for interference-free in situ analysis of glycans in tissues. On that basis, the SALDI-MSI platform fit for glycoprotein/glycan analysis will be constructed by integrating the aforementioned units. With this platform, it is expected to obtain more comprehensive identification of low-abundance glycoproteins/glycans, profile their spatial distributions and dynamic changes, screen the potential biomarkers with differential expression and spatial distributions, and further explain the effect and mechanism of aberrant alteration of glycans on the development, invasion and metastasis of HCC.