Quantitative proteomic characterization of redox-dependent post-translational modifications on protein cysteines

Abstract

Protein thiols play a crucial role in redox signaling, in the regulation of enzymatic activity and protein function, and in maintaining redox homeostasis in living systems. The unique chemical reactivity of the thiol group makes protein cysteines susceptible to reactions with reactive oxygen and nitrogen species that form various reversible and irreversible post-translational modifications (PTMs). The reversible PTMs in particular are major components of redox signaling and are involved in the regulation of various cellular processes under physiological and pathological conditions. The biological significance of these redox PTMs in both healthy and disease states has been increasingly recognized. Herein, we review recent advances in quantitative proteomic approaches for investigating redox PTMs in complex biological systems, including general considerations of sample processing, chemical or affinity enrichment strategies, and quantitative approaches. We also highlight a number of redox proteomic approaches that enable effective profiling of redox PTMs for specific biological applications. Although technical limitations remain, redox proteomics is paving the way to a better understanding of redox signaling and regulation in both healthy and disease states.

Fig. 1

Redox-dependent modifications on protein cysteines.

Fig. 2

Direct approaches for reversible Cys PTMs. (a) Dimedone-based chemical probes for SOH. (b) Organomercury and phosphine-based probes for SNO. (c) Tag-switch method and Biotin-Thiol-Assay for SSH.

Fig. 3

Indirect strategies for reversible Cys modifications.

Fig. 4

Stable isotope labeling approaches for redox PTMs. (a) Relative quantification; (b) Stiochiometric measurement for redox PTMs with differential alkylation approaches.

Fig. 5

Isobaric labeling approaches for Cys PTMs.

Fig. 6

Targeted quantification of Cys PTMs.