一种新型凝集素液相芯片系统的建立及其在肝癌血清肽组学糖谱中的应用

Glycomics is the study of comprehensive structural elucidation and characterization of all glycoforms found in nature and their dynamic spatiotemporal changes that are associated with biological processes. Relative to genomics and proteomics, glycomics is just growing out of infancy with great potential in biomedicine for biomarker discovery, diagnosis, and treatment. However, the immense diversity and complexity of glycan structures and their multiple modes of interactions with proteins pose great challenges for development of analytical tools for delineating structure function relationships and understanding glycocode. Several tools are being developed for glycan profiling based on chromatography and mass spectrometry, which was limited in high-throughput methodologies to study the structural and functional aspects of glycan structures. Lectins, which have long been used in glyco-immunology, printed on a microarray provide a versatile platform for rapid high throughput analysis of glycoforms of biological samples. A novel sensitivity lectin suspension microarray and a suspension array assay using dendrimer signal amplification allowed rapid, sensitivity, high-throughput are needed for glycan profiling analyses and clinical application. The peptidome and peptidomics as logical extension to proteome and proteomics were caught more attention by scientists and provided diagnostic and prognostic information on cancer and other diseases. However, few reports on glycan profiling of peptidome can be found although the awareness on the importance of the peptidome has greatly increased. Therefore, preparation of a novel sensitivity, high-throughput and fast lectin suspension microarray system was urgent need for overall and real-time glycan profiling analyses of sample. The serum peptidome glycan profiling of hepatocellular carcinoma will be established and have great potential in biomarker discovery, diagnosis, and prognostic evaluation.

继基因组学、蛋白质组学之后，糖组学在分子标志物发现、疾病诊断以及预后具有潜在的价值。面对多样和复杂的糖链结构研究，凝集素芯片作为糖的解码器成为一种高灵敏、高通量的糖链分析平台，可以快速筛查并区分糖蛋白质的糖谱，在糖组学的研究中具有广阔的生物学、临床应用价值。因而建立一种新型高密度凝集素液相芯片，有效降低固定探针微珠的团聚，并通过信号放大检测方式提高凝集素液相芯片的检测灵敏度，为糖谱研究及其在临床诊断等方面的应用奠定基础。作为蛋白质组学的重要分支，多肽组学在诸多生物过程中具有举足轻重的地位。但目前针对肽组学糖谱的研究却无人涉足，建立一种高灵敏、高通量、高速的凝集素芯片，对肝癌血清中内源性肽或低分子量蛋白质的糖谱进行总体、即时的观察，建立肽组学糖谱，为糖组学的研究提供一个新的分析方法，为肝癌的发生、诊断以及预后评估奠定基础。

本项目通过多聚赖氨酸有机长链分子辅助，将凝集素成功键合到荧光磁性微球上，成功建立了新型凝集素液相芯片。由于亲水性多直链辅助，降低了凝集素液相芯片微珠的团聚现象；同时，提高了凝集素的固载量，从而提高了凝集素液相芯片的灵敏度。我们首次将新型凝集素芯片用于糖肽糖基化修饰的定量分析，其中以标准糖蛋白的Lys酶切肽段为分析物，进行了糖肽糖基化芯片分析方法的建立和评价。该项目首次将新型凝集素芯片应用到肝内胆管细胞癌（ICC）血清糖肽谱建立，以含有24种特异性新型凝集素芯片为基础，检测到ICC血清中糖肽谱中识别Con A、EBL、PSA、s-WGA、WFL、PHA-E、DSL、RCA的糖肽有明显升高趋势，而识别UEA-Ⅰ的糖肽却又相反趋势，其余15种凝集素响应信号没有显著变化。我们对其中部分凝集素响应进行了Dot-blot验证。通过LC-MS进一步的对低分子量糖肽对应蛋白进行了鉴定。从而最终为ICC的临床诊断与发病机制提供了理论依据。