Dual Labeling Biotin Switch Assay to Reduce Bias Derived From Different Cysteine Subpopulations: A Method to Maximize S-Nitrosylation Detection.
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.
['Chung|Heaseung Sophia|HS|', 'Murray|Christopher I|CI|', 'Venkatraman|Vidya|V|', 'Crowgey|Erin L|EL|', 'Rainer|Peter P|PP|', 'Cole|Robert N|RN|', 'Bomgarden|Ryan D|RD|', 'Rogers|John C|JC|', 'Balkan|Wayne|W|', 'Hare|Joshua M|JM|', 'Kass|David A|DA|', 'Van Eyk|Jennifer E|JE|']
D000426:Alcohol Dehydrogenase D000818:Animals D001710:Biotin D003545:Cysteine D005260:Female D057809:HEK293 Cells D006801:Humans D008297:Male D008810:Mice, Inbred C57BL D018345:Mice, Knockout D015335:Molecular Probes D009206:Myocardium D015538:Nitrosation D009603:Nitroso Compounds D011499:Protein Processing, Post-Translational D040901:Proteomics D015203:Reproducibility of Results D053719:Tandem Mass Spectrometry
proteomics S-nitrosothiols nitric oxide oxidation-reduction