Figure 1. BST/ICAT work flow for the quantification of Trx1 transnitrosylase and denitrosylase target SNO-peptides.

There are nine steps involved in this procedure; A: Free thiols in proteins are first blocked with MMTS; B: Nitrosothiol reduction by ascorbate; C: Labeling of the newly formed free thiols with ICAT; D: Mixing ICAT light and heavy reagents-labeled proteins; E: Trypsin digestion of the protein mixtures; F: Enrichment of ICAT-labeled peptides using an avidin cartridge; G: TFA cleavage of biotins from ICAT-labeled peptides; H: Desalting of the cleaved peptides; I: Identification of SNO-Cys site and quantification of SNO-peptides by LC/MS/MS. J: An example MS spectrum of SNO-GAPDH [235-248] quantification by ICAT. K: An example MS spectrum of the identification of SNO-GAPDH [235-248] and localization of SNO-Cys²⁴⁷ by MS/MS. Modified from Wu et al., 2011 (6).