Quantitative Proteomics of Nitrosylated Proteins in Melanoma Using the Biotin-Switch Technique Combined With Tandem Mass Tag Labeling.

Protein S-nitrosylation is a critical post-translational modification that regulates diverse cellular functions and signaling pathways. Although various biochemical methods have been developed to detect S-nitrosylated proteins, many suffer from limited specificity and sensitivity. Here, we describe a robust protocol that combines a modified biotin-switch technique (BST) with streptavidin-based affinity enrichment and quantitative mass spectrometry to detect and profile nitrosylated proteins in cultured cells. The method involves blocking free thiols, selective reduction of nitrosothiols, biotin labeling, enrichment of biotinylated proteins, and identification by tandem mass tag (TMT)-based quantitative mass spectrometry. Additionally, site-directed mutagenesis is employed to generate "non-nitrosylable" mutants for functional validation of specific nitrosylation sites. This protocol provides high specificity, quantitative capability, and versatility for both targeted and global analysis of protein nitrosylation. Key features • Specific thiol blocking and labeling: Free thiols are blocked with N-ethylmaleimide, followed by selective reduction and biotinylation of S-nitrosothiols for precise nitrosylation detection. • Quantitative proteomics: TMT-labeling with high-resolution LC-MS/MS enables multiplexed, accurate quantification and comprehensive nitrosylome profiling with faster data acquisition and fewer missing values than label-free proteomics. • Functional mutagenesis: Site-directed mutagenesis of cysteine residues generates "non-nitrosylable" mutants to study nitrosylation's impact on protein function. • Versatile application: The protocol is adaptable for both targeted protein analysis and global nitrosylation profiling across diverse cell types and experimental conditions. This protocol is used in: Cancer Research (2025), DOI: 10.1158/0008-5472.CAN-24-0693.