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

Circ Res. 2015 Oct 23;117(10):846-57. doi: 10.1161/CIRCRESAHA.115.307336. Epub 2015 Sep 3.


Rationale: S-nitrosylation (SNO), an oxidative post-translational modification of cysteine residues, responds to changes in the cardiac redox-environment. Classic biotin-switch assay and its derivatives are the most common methods used for detecting SNO. In this approach, the labile SNO group is selectively replaced with a single stable tag. To date, a variety of thiol-reactive tags have been introduced. However, these methods have not produced a consistent data set, which suggests an incomplete capture by a single tag and potentially the presence of different cysteine subpopulations.

Objective: To investigate potential labeling bias in the existing methods with a single tag to detect SNO, explore if there are distinct cysteine subpopulations, and then, develop a strategy to maximize the coverage of SNO proteome.

Methods and results: We obtained SNO-modified cysteine data sets for wild-type and S-nitrosoglutathione reductase knockout mouse hearts (S-nitrosoglutathione 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.

Keywords: S-nitrosothiols; nitric oxide; oxidation-reduction; proteomics.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alcohol Dehydrogenase / deficiency
  • Alcohol Dehydrogenase / genetics
  • Animals
  • Biotin / metabolism*
  • Cysteine / metabolism*
  • Female
  • HEK293 Cells
  • Humans
  • Male
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Molecular Probes*
  • Myocardium / metabolism*
  • Nitrosation
  • Nitroso Compounds / metabolism*
  • Protein Processing, Post-Translational
  • Proteomics / methods*
  • Reproducibility of Results
  • Tandem Mass Spectrometry


  • Molecular Probes
  • Nitroso Compounds
  • Biotin
  • Adh5 protein, mouse
  • Alcohol Dehydrogenase
  • Cysteine