A Proteomics Workflow for Dual Labeling Biotin Switch Assay to Detect and Quantify Protein S-Nitroylation

S-nitrosylation (or S-nitrosation, SNO) is an oxidative posttranslational modification to the thiol group of a cysteine amino acid residue. There are several methods to detect SNO modifications, mostly based on the classic biotin-switch assay, where the labile SNO sites are replaced with a stable biotin moiety to facilitate enrichment of the modified proteins. As the technique has evolved, new and more advanced thiol-reactive reagents have been introduced in the protocol to improve the identification of modified peptides or to quantify the level of modification at individual cysteine residues. However, the growing diversity of thiol-reactive affinity tags has not produced a consistent set of protein modifications, suggesting incomplete coverage using a single tag. Here, we present a parallel dual labeling strategy followed by an optimized proteomics workflow, which maximizes the overall detection of SNO by reducing the labeling bias derived from the use of a single tag-capture approach.