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

Abstract

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 dataset 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 datasets for wild-type and S-nitrosoglutathione reductase (GSNOR) knock-out mouse hearts (GSNOR is a negative regulator of GSNO production) and NO-induced human embryonic kidney cell using two 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, while the remaining SNO sites were only labeled by one reagent. Characterization of the two distinct subpopulations of SNO residues indicated that pyridyldithiol reagent preferentially labels cysteine residues that are more basic and hydrophobic. Based on this observation, we proposed a parallel dual labeling strategy followed by an optimized proteomics workflow. This enabled the profiling of 493 SNO-sites in GSNOR knock-out hearts.

Conclusions

Using a protocol comprising two tags for dual labeling maximizes overall detection of SNO by reducing the previously unrecognized labeling bias derived from different cysteine subpopulations.