Mass Spectrometric Analysis Of The N-Glycoproteins And Its Glycans Induced By Oxidized-LDL In Endothelial Cells

Atherosclerosis(AS)is the leading cause of the cardiovascular disease(CVD),which has led to the development of ischemic heart disease,ischemic stroke and peripheral arterial disease.The incidence and mortality of CVD in China are increasing,and its mortality rate ranks first currently.Prevention and treatment of AS is essential for improving the prognosis of patients with CVD.The formation of AS is a complicated process involving a lot of mechanisms,such as endothelial dysfunction,angiogenesis,autophagy and apoptosis,oxidative stress,non-coding RNA,matrix degradation,cholesterol,inflammation and thrombosis,among which inflammation and immune system play an important role in its development.The pathogenesis and development of AS are not c.learly understood.A large number of research have demonstrated that endothelial cell dysfunction or injury induced by ox-LDL is an initial step of AS formation.Endothelial cells has recently became important therapeutic target for the treatment of CVD.Stimulating endothelial cells with ox-LDL to develop AS modules is an effective method for the mechanism study of AS formation.Glycosylation serves as a critical functional group in regulating cellular function and compolicated biological processes.Glycosylation abnormalities are associated with a variety of diseases,including autoimmune diseases,inflammatory diseases and infectious diseases.Many glycoproteins or glycans have been identified as potential disease biomarkers for diagnostic and therapeutic of a lot of deseases.Studies have shown that glycosylation can inhibit the formation of AS by affecting the adhesion molecules and chemokine receptors to leukocyte aggregation,which suggests that abnormal glycosylation plays a significant role in the formation of AS.The structural characterization of glycosylated sites,glycoprotein and glycan is a robust and comprehensive method to study the role of glycosylation in AS formation and develop new biomarkers.In this paper,ox-LDL was used as stimulant to develop the endothelial cells damage module in AS.The glycoproteinomics and glycomics of endothelial cells damaged by ox-LDL studied.At first,enrichment of N-glycopeptides was accomplished by filter aided sample preparation(FASP)and lectin affinity enrichment protocol.And then,PNGase F was applied to release the N-glycan in H218O dissolved buffer.Finally,the LC-MS was used to identify the glycoproteins,glycosylation sites and glycans,and the identified glycoproteins with the differential glycosylation were further analyzed using bioinformatics analysis.A total of 1170 N-glycosylated peptides were identified in this experiment,containing 1248 N-glycosylation sites distributed on 768 N-glycoproteins.A total of 120 glycopeptides distributed on 92 glycoproteins with a fold change in N-glycosylation site occupancy ratio greater than 1.3 times were found.The results of Gene Ontology(GO)enrichment indicate that these differential glycoproteins were mainly distributed in membrane,cell surface,extracellular,vacuole and lysosome,and these differential glycoproteins play an important role in protein complex binding,extracellular matrix binding,receptor binding and oxidoreductase activity.In addition,these glycoproteins are also involved in some important biological processes such as cell adhesion,matrix adhesion,cell migration and localization,and stress response.The protein-protein interaction network was centered on the subnetwork formed by the integrin family which have high degree nodes.Pathway enrichment analysis showed that the most prominent signaling pathways was PI3K-Akt signaling pathway,followed by lysosomal,focal adhesion and extracellular matrix receptor interaction.Bioinformatics analysis indicated that integrins play an important role in AS induced by ox-LDL.After analysis of N-glycans in endothelial cells by PGC-LC-MS,100 N-glycans with different molecular weights were detected.And two unique N-glycans were detected in the ox-LDL treated group.The relative quantitation result show that the variation ratio of 52 N-glycan chains was more than 1.3 times.The overall analysis of all N-glycans revealed a significant increase in the content of high sialylated N-glycans.