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

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.