Dual Labeling Biotin Switch Assay to Reduce Bias Derived From Different Cysteine Subpopulations A Method to Maximize S-Nitrosylation Detection

reductase is a negative regulator of S-nitrosoglutathione production) and nitric oxide–induced human embryonic kidney cell using 2 labeling reagents: the cysteine-reactive pyridyldithiol and iodoacetyl based tandem mass tags. Comparison revealed that <30% of the SNO-modified residues were detected by both tags, whereas the remaining SNO sites were only labeled by 1 reagent. Characterization of the 2 distinct subpopulations of SNO residues indicated that pyridyldithiol reagent preferentially labels cysteine residues that are more basic and hydrophobic. On the basis of this observation, we proposed a parallel dual-labeling strategy followed by an optimized proteomics workflow. This enabled the profiling of 493 SNO sites in S-nitrosoglutathione reductase knockout hearts. Conclusions: Using a protocol comprising 2 tags for dual-labeling maximizes overall detection of SNO by reducing the previously unrecognized labeling bias derived from different cysteine subpopulations. ( Circ Res . 2015;117:846-857. DOI: 10.1161/CIRCRESAHA.115.307336.) sites per protein. The majority of the 1008 SNO-modified proteins had a single SNO site, whereas 24% of them had ≥ 2 SNO-modified cysteine residues. B and C , Subcellular locations and molecular functions of the proteins were analyzed based on Gene Ontology: Subcellular location of SNO-modified proteins ( B ) and functional specificity of the SNO-modified proteins ( C ). These features showed similar distributions to those of other studies for the identification of SNO cysteine 20 or data from our previous study with single cysTMT 18 (data not shown). This indicates that our increased number of SNO proteins is derived from overall proteome, not from a certain class of proteins (eg, localized or functionalized exclusively); further supporting that SNO is able to impact a broad number of subproteomes and various functional classes of proteins.