基于液相色谱-质谱的血清糖蛋白质组定量分析

Quantitative and Functional Analysis of Glycoprotein Based on Liquid Chromatography-Mass Spectrometry

糖尿病作为最常见的代谢疾病之一,是一种受环境和遗传因素影响的多因素疾病。糖基化可导致蛋白功能多样性,对重大疾病、免疫系统有着深远影响。本实验通过两性离子亲水相互作用色谱(zwitterionic hydrophilic interaction liquid chromatography, ZIC-HILIC)富集技术结合微升级高效液相色谱-四极杆飞行时间质谱法,对6例糖尿病人和6例正常人的血清中的蛋白和糖蛋白进行了非标记定量分析,定量了65个差异蛋白和24个差异表达的糖蛋白。通过DAVID软件对这些差异蛋白和差异糖蛋白进行功能富集分析,进一步了解它们的细胞定位、分子功能和生物过程。该研究揭示了糖尿病发病过程中糖蛋白的变化,可为疾病的发病机制和临床诊断提供数据参考。

Diabetes mellitus as one of the most prevalent metabolic disease is a multifactorial disease which is influenced by environmental and genetic factors. It can cause complications in most of organs such as heart, eye, kidney and nervous system which has resulted in high economic cost and burden. Proteomics is a comprehensive strategy to analyze all proteins in a biological system. High performance liquid chromatography-mass spectrometry(LC-MS/MS) is powerful in peptides and proteins detection. Protein post translational modifications such as N-Glycosylation is widely implicated as a common modification in numerous blood proteins and impacts the in-vivo protein functions. Abnormal glycosylation is associated with many human diseases and the study of glycosylation has attracted more and more attention. In this study, serum samples were collected from 6 healthy people and 6 patients with diabetic diseases for comprehensive proteome and N-glycosylation modification analysis. All the proteins were digested fully with Trypsin and Zwitterionic hydrophilic interaction liquid chromatography(ZIC-HILIC) was used to enrich glycosylated peptides. The platform equipped with high performance liquid chromatography and time-of-flight mass spectrometry was applied to acquire the whole proteome as well as the glycopeptide data of the serum samples. Furthermore, the raw data were searched and analyzed by Maxquant software. Student’s T-test was used to determine the significantly changed proteins and glycoproteins with P less than 0.05, and the fold change ratio was calculated based on the average abundance of the proteins in the diabetic serum and normal serum sample. In order to find more differentially expressed proteins for subsequent analysis, the expression level of ±20% was used as the criteria which means a fold change greater than 1.2 or less than 0.8 as the criteria for reference value. A total of 291 proteins and 181 glycoproteins were identified. 65 altered proteins and 24 differentially expressed glycoproteins in the diabetic serum and normal serum were quantified by Label-free quantitation. Altered expressed proteins and differential expressed glycoproteins were analyzed by Gene Ontologhy enrichment analysis to further understand their cellular components, molecular functions and biological processes, and compared their differences in these aspects. IgA binding and blood coagulation are more enriched in altered glycoproteins other than proteins. The data reveal changes in glycoproteins during the onset diabetes mellitus, providing a detailed data reference for the occurrence mechanism and clinical diagnosis of the diabetes mellitus.