Figure 1.5.

A proposed proteomic strategy for efficient capture of 3-NT-containing peptides in tryptic digests of complex protein mixtures to enable broad proteomic identification of 3-NT modification sites using MS/MS (Nuriel and Gross, unpublished). The schematized approach is exemplified for a representative dipeptide, NO₂-Tyr-Lys. The overall strategy is an extension of the biotin-switch method (Jaffrey and Snyder, 2001) adapted for high-throughput identification of S-nitrosylation sites on protein cysteine residues (Hao et al., 2006). After complete blocking of free amino groups (by dimethylation), peptides are treated with dithionite to chemically reduce NO₂-Tyr to NH₂-Tyr. This newly formed amine is then modified by reaction with sulfo-NHS-SS-biotin, a thiol-cleavable and amine-selective biotinylating agent (note that an acid-cleavable biotinylating agent can similarly be employed). The biotinylated peptides are affinity captured on streptavidin-Sepharose and washed extensively, and nonbiotinylated peptides are eluted by treatment with β-mercaptoethanol for LC-MS/MS-based amino acid sequence analysis. This approach is expected to provide a more confident and comprehensive identification of protein 3-NT-modification sites than obtained previously.