Abstract
Nitric oxide (NO) is covalently attached to cysteine thiols of proteins resulting in the formation of S-nitrosothiols (RSNOs), and regulation of protein activity and function in a wide range of physiological processes. The objectives of this thesis are development of probes for the enrichment of S-nitrosylated peptides. We conjugated two types of thioester phosphine ligand to either magnetic nanoparticle or agarose bead, respectively for identification and purification of RSNOs. Furthermore, through traceless reductive ligation of S-nitrosothiol mechanism the unstable primary RSNOs were converted to stable disulfide-iminophosphorane products. The S-nitrosylated PTP1B peptide mixed with tryptic BSA mixture was successfully captured by developed probe and identified by MALDI-TOF. In addition, the agarose beads show less non-specific interaction to tryptic BSA peptide. The enrichment of S-nitrosylated peptides from COS-7 cell lysate was achieved by 2nd_TEP@agarose. Thus, This strategy is a potential tool for investigate of S-nitrosylation proteins. Furthermore, we synthesized other type of thioester phosphine ligand by changing the original structure and investigate the effect of probe structure on the reactivity to RSNO.