Detection of Protein S-Nitrosation using Irreversible Biotinylation Procedures (IBP)

The biotin switch assay for detection of protein S-nitrosation has been widely used in the field of nitric oxide and redox signaling. However, here we found that there is experimental and theoretical interference of intermolecular disulfide bonds in S-nitrosated protein identification with avidin purification after biotin switch method: proteins linked to S-nitrosated proteins by intermolecular disulfide bonds can be falsely detected as S-nitrosated targets. Then we developed irreversible biotinylation procedures (IBP) to prevent this interference, in which irreversible biotinylation was used to instead of reversible biotinylation, all the intermolecular disulfide bonds were broken before purification of biotinylated proteins added as a new step, and doing elution by denaturation of avidin after the purification. This strategy enables us to specifically detect protein S-nitrosation without the potential interference of intermolecular disulfide bonds. Furthermore, we applied IBP to proteomic approaches and quantitative proteomic approaches for high-throughput studies of protein S-nitrosation.