Label-free strategy for site-specific quantitation of S-nitrosylome

S-nitrosylation, an emerging post-translational modification (PTM) specifically on cysteine residues by nitric oxide (NO), plays important roles to regulate cellular functions and signaling events. Despite its importance, the study of S-nitrosylation remains methodologically challenges because of the lability of S-NO bond and low-abundant level of endogenously S-nitrosylated proteins. In the first part of thesis, a modified biotin switch method was presented to site-specifically identify S-nitrosylated protein and modification site. Using S-alkylating agents, IAM and PEO-biotin, in modified biotin switch method coupled with tandem mass spectrometric analysis, this protocol provided a direct site-specific identification method, allowing unambiguous differentiation of the S-nitrosylated cysteine site. n the second part of thesis, a label-free quantitative approach combining modified biotin switch method and LC-MS/MS analysis was developed for site-specific identification and quantitation of in-vivo S-nitrosylation. For accurate quantitation, S-nitrosylated internal standard proteins were spiked into sample proteins and subjected to modified biotin switch method, digestion, purification, and LC-MS/MS analysis. A reliable linear calibration curve with 400-fold dynamic range (R2=0.9873) for quantitation was established by spiking different amount of S-nitrosylated ovalbumin into SNAP/L-cysteine-treated endothelial cell lysate. The results obtained from duplicate experiments revealed high accuracy and low variation (2 SD = 0.2), demonstrated that this strategy offer a stable and efficient platform for identification and quantitation of S-nitrosylation. The preliminary quantitative results revealed that 308 proteins with over-expressed S-nitrosylation signals were quantified in replicate experiments after stimulation. Some of these proteins have been found to be involved in NO-related processes, such as oxidative stress signaling pathway. Most importantly, the unknown sites of some previously reported S-nitrosylated targets were site-specifically identified by our approach, such as proteasome, etc. Therefore, the date demonstrated that the label-free quantitative approach combining modified biotin switch method was an effective proteomic approach for site-specific identification of the S-nitrosylated targets as well as quantification of the dynamic change of S-nitrosylation. To our knowledge, this study was the first quantitative proteomic for protein S-nitrosylation by label-free strategy.