Site-Mapping of In Vitro S-nitrosation in Cardiac Mitochondria: Implications for Cardioprotection

Abstract

S-nitrosation (SNO) of mitochondrial protein cysteines can be cardioprotective. Several targets have been implicated, yet the scope and identification of specific residues has not been fully assessed. To address this, a comprehensive assessment of mitochondrial SNO-modifiable cysteines was performed to determine nitric oxide (NO) susceptible pathways and identify novel mechanisms of oxidative cardioprotection. The biotin switch assay and mass spectrometry were used on rat cardiac mitochondrial lysates treated with the nitric oxide donor, S-nitrosoglutathione, and controls (n ‫؍‬ 3) to map 83 SNO-modified cysteine residues on 60 proteins. Of these, three sites have been reported, 30 sites are new to 21 proteins previously known to be S-nitrosated but which lacked sitespecific information and 50 sites were found on 39 proteins not previously implicated in SNO pathways. The SNO-modifications occurred in only a subset of available cysteines, indicating a specific targeted effect. Functional annotation and site-specificity analysis revealed a twofold greater nitric oxide-susceptibility for proteins involved in transport; including regulators of mitochondrial permeability transition suggesting SNO-regulation and a possible protective mechanism. Additionally, we identified many novel SNO-modified proteins with cardioprotective potential involved in the electron transport chain, tricarboxylic acid cycle, oxidative stress defense, fatty acid and amino acid metabolism. These findings suggest that SNO-modification may represent a novel mechanism for the regulation of oxidative phosphorylation and/or cell death. S-

Identification and Quantification of S-Nitrosylation by Cysteine Reactive Tandem Mass Tag Switch Assay

“…An early report of SNO-modified cysteine residues suggested that a linear acid/ base motif surrounding the target cysteine facilitated modification (46). Since then, there has not been agreement on the utility of this motif; although there have been some confirmatory reports (47,48), it has not been identified in all data sets (13,19,28). We had hoped that thiol-reactivity would provide some additional insight into this question.…”

“…This results in unlabeled cysteine residues for MS analysis making it difficult to assess nonspecific binding in the capture and make site assignments for peptides containing multiple cysteines. Previous studies have used the number of noncysteine containing peptides present in the analysis as an estimation of the mis-assigned sites (13,19). The cysTMT 6 switch assay removes this ambiguity by utilizing a low pH elution from the TMT affinity resin, instead of reduction, preserving the 304 Da mass tag on each modified cysteine residue.…”

“…Each of these concerns has the potential to increase the incidence of false-positive results. Different variations of the assay offer several accommodations for these issues (13,(17)(18)(19)(20)(21)(22); however, there is currently no unified solution.…”

The Expanding Landscape of the Thiol Redox Proteome

“…wide analyses of protein S-nitrosylation (23)(24)(25)(26)(27). The original BST also can be easily adapted to study other distinct reversible thiol modifications by changing the specific reducing agents.…”

S-Nitrosylation: Specificity, Occupancy, and Interaction with Other Post-Translational Modifications

“…To help explain SNO specificity, the existence of a SNO motif has been explored (39,58,82,117). An acid-base motif within eight angstroms of a cysteine is thought to act as a preferential SNO motif (74).…”