Proteomic Study Of S-nitrosylation In Endothelial Cells

Protein S-nitrosylation, which is one kind of post-translational modifications, is formed through reaction between the free thiol group of cysteine and nitric oxide (NO). As a messenger molecular of cardiovascular system, NO is closely related to cardiovascular physiological and pathological processes, for example, blood pressure regulation, thrombus prevention, atherosclerosis and coronary heart disease occurrence and development. NO can regulate protein activity and function through S-nitrosylation, which belongs to redox signaling transduction pathway. Various kinds of S-nitrosylated proteins have been found including enzymes, membrane receptors, ion channels, transcriptional factors, metalloproteins and so on. S-nitrosylation level influences protein structure and function within target sites and participates in a series of physiological processes and diseases’occurrence and development. Research on S-nitrosylation proteomics is fundamental to understanding NO-mediated signal transduction, and revealing S-nitrosylation irregular mechanisms in related diseases for new potential drug targets.Protein S-nitrosylation is characterized by labile bond and low level. The approach derived from classical biotin switch technique (BST) was adopted and optimized herein to identify S-nitrosylated sites. There are three main steps in the experimental operation:blocking the free thiols of cysteines, specifically reducing the S-nitrosylated sites by ascorbic acid and labeling the new free thiols by biotin reagent. After these three procedures, labile S-nitrosylated sites are converted into stable biotinylated sites, on the other hand, enrichment method of avidin-biotin system helps to lower sample complexity and detect low abundant peptides. High performance liquid chromatography (HPLC) coupling with high resolution mass spectrometry (MS) was used to obtain high quality mass spectra. The information on S-nitrosylated sites can be obtained by mascot software processing and data filtration.Human umbilical vein endothelial cells (HUVECs) have the potential of stem cells and vascular endothelial cells can generate NO. Oxidative stress, referring to the imbalance of NO and reactive oxygen species (ROS), may contribute to endothelial dysfunction and further cardiovascular complications. At present there is no systematic research on the significant role of oxidative stress in S-nitrosylated site in human endothelial cells. In the paper presented herein, a synthetic peptide and bovine serum albumin were used to examine the efficiency of labeling reaction and enriching method. Experimental conditions were optimized with human plasma proteins in order to establish an S-nitrosylated site identification approach applicable to proteomics. HUVECs were divided into two groups as normal control group and drug treated group. The normal control group grew in complete culture medium while the other group grew in culture medium added NOC-18, who provided an nitroso stress environment to alter S-nitrosylation level. In normal control group, a total of 92 S-nitrosylated proteins including 111 S-nitrosyated sites were identified while 241 S-nitrosylated proteins including 309 S-nitrosyated sites in drug treated group. By comparing S-nitrosylated sites between these two groups,97 S-nitrosylated sites were presented in both groups and other 212 sites were unique when endothelial cells were subjected to oxidative stress. The research will contribute to construct modulating network of endothelial cells related to irregular S-nitrosylation, which leads to understanding aberrant regulation mechanism of S-nitrosylation level in physiological and pathological processes.