Strategies for profiling native S-nitrosylation

Abstract

Cysteine is a uniquely reactive amino acid, capable of undergoing both nucleophlilic and oxidative post-translational modifications. One such oxidation reaction involves the covalent modification of cysteine via the gaseous second messenger nitric oxide (NO), termed S-nitrosylation (SNO). This dynamic post-translational modification is involved in the redox regulation of proteins across all phylogenic kingdoms. In mammals, calcium-dependent activation of NO synthase triggers the local release of NO, which activates nearby guanylyl cyclases and cGMP-dependent pathways. In parallel, diffusible NO can locally modify redox active cellular thiols, functionally modulating many redox sensitive enzymes. Aberrant SNO is implicated in the pathology of many diseases, including neurodegeneration, inflammation, and stroke. In this review, we discuss current methods to label sites of SNO for biochemical analysis. The most popular method involves a series of biochemical steps to mask free thiols followed by selective nitrosothiol reduction and capture. Other emerging methods include mechanism-based phosphine probes and mercury enrichment chemistry. By bridging new enrichment approaches with high-resolution mass spectrometry, large-scale analysis of protein nitrosylation has highlighted new pathways of oxidative regulation.

Keywords: nitric oxide; nitrosylation; post-translational modification.

Figure 1

Formation of nitrosothiols from nitric oxide (NO) occurs through distinct oxidative pathways^,–, each involving two molecules of NO for each nitrosothiol formed. Superoxide radical = O₂^−•. Oxygen = O₂.

Figure 2

Biotin-switch technique for ascorbate-dependent reduction of nitrosothiols. Enriched proteins are either analyzed by SDS-PAGE or annotated by mass spectrometry.

Figure 3

Chemoselective nitrosothiol labeling methods. In both approaches, free thiols are first blocked by addition of MMTS. (A) Triaryl-phosphine ligation methods. Three reactions are shown that describe recent reports of nitrosothiol-selective phosphine reactions. The reductive ligation approach was demonstrated on fixed cells, but led to over-reduction to the free thiol. The one-step disulfide formation reaction was demonstrated on cell lysates after nitric oxide donor treatment. (B) Phenyl-mercury enrichment of nitrosothiols for proteomic annotation. Sepharose beads or biotin are linked to phenyl-mercury for nitrosothiol enrichment, followed by trypsin digestion. Nitrosylated peptides are released from the resin by perfomic acid oxidation to the sulfonic acid for mass spectrometry annotation.