Fig. 3.

A generalized schematic of the biotin switch technique. The technique entails irreversible alkylation of free thiols, selective reduction of SNO, and biotinylation of SNO sites. Biotinylated proteins may be subject to blotting with anti-biotin or enriched with streptavidin followed by specific protein blotting. Bands can be excised, trypsinized, and subjected to mass spectrometry. Common variations include choice of thiol alkylating agent or replacement of biotin with an agent such as iodoTMT. Methods based on the biotin switch technique typically utilize modifications to the labeling and enrichment strategies, such as replacing biotin/streptavidin with a thiol-active resin (SNO-RAC) or direct tagging of SNO without prior selective reduction (SNOTRAP).