Site-Specific Proteomics Approach for Study Protein S-Nitrosylation

Here we present a novel and robust method for the identification of protein S-nitrosylation sites in complex protein mixtures. The approach utilizes the cysteinyl affinity resin to selectively enrich S-nitrosylated peptides reduced by ascorbate followed by nanoscale liquid chromatography tandem mass spectrometry. Two alkylation agents with different added masses were employed to differentiate the S-nitrosylation sites from the non-S-nitrosylation sites. We applied this approach to MDA-MB-231 cells treated with Angeli's salt, a nitric oxide donor that has been shown to inhibit breast tumor growth and angiogenesis. A total of 162 S-nitrosylation sites were identified and an S-nitrosylation motif was revealed in our study. The 162 sites are significantly more than the number reported by previous methods, demonstrating the efficiency of our approach. Our approach will further facilitate the functional study of protein S-nitrosylation in cellular processes and may reveal new therapeutic targets.