Accumulating evidence indicates that S-nitrosylation plays an important role in protein function either physiologically or pathologically. In the present study, we describe a modified method for the isolation and enrichment of S-nitrosylated (SNO) proteins from brain tissue based on a biotin labeling system using the biotin switch technique (BST). Various working conditions for the incubation of biotin-labeled samples with streptavidin beads and for the elution of SNO proteins from streptavidin beads were comparatively evaluated. The working conditions were optimized with incubation at a ratio of 1:3 (streptavidin beads/brain homogenates) at 25˚C for 120 min, and the elution conditions were optimized using buffer containing 0.5% sodium dodecyl sulfate. Under these conditions, we found that at least 12 rounds of successive incubation were required in order to recover all the SNO proteins in the human and rodent brain homogenates. Western blot analyses of some of the eluted products confirmed the reliable immunoreactivity of the isolated SNO proteins. iTRAQ-based mass spectrometric (MS) analysis of the eluted products from the brain tissues of a normal healthy subject and patients with various prion diseases identified 1,509 SNO proteins with high confidence [false discovery rate (FDR) <1%]. These data indicate that with this optimized method, the endogenous SNO proteins from the brain tissue of humans and rodents can be sufficiently isolated, which can then be used directly in further assays, such as large-scale analysis of the S-nitrosoproteome in complex backgrounds.