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

作者：Chung Heaseung Sophia; Murray Christopher I; Venkatraman Vidya; Crowgey Erin L; Rainer Peter P; Cole Robert N; Bomgarden Ryan D; Rogers John C; Balkan Wayne; Hare Joshua M; Kass David A; Van Eyk Jennifer E^*

来源：Circulation Research, 2015, 117(10): 846-857.

DOI：10.1161/CIRCRESAHA.115.307336

• nitric oxide
• oxidation-reduction
• proteomics
• S-nitrosothiols

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.

• 出版日期2015-10-23