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.

Mass spectrometry-based proteomics for system-level characterization of biological responses to engineered nanomaterials

“…The ability to measure a variety of PTMs is a unique aspect of advanced proteomics technologies. Currently, more than 200 biological relevant PTMs have been reported [47] and several types of them, such as phosphorylation, acetylation, glycosylation, and thiol-based redox modifications, have been studied extensively [24,32,48–50]. …”

“…In addition to ROS, reactive nitrogen species (RNS) have also been implicated in ENM-induced toxicity [12]. ROS/RNS are known mediators of thiol-based redox modifications [32], which include several types of reversible modifications such as S-nitrosylation (SNO), S-sulfenylation (SOH), S-glutathionylation (SSG), and redox-sensitive disulfide formation, and irreversible modifications such as sulfinic acid (SO 2 H) and sulfonic acid (SO 3 H). These modifications could either play a role in the signaling and regulation, or represent pathological signatures of cellular toxicity (Fig.…”

“…Although these studies provide valuable information on how ENMs alter gene expression, the changes at the mRNA level do not necessarily correlate with changes at the protein level. Furthermore, ENM exposure can generate ROS by both direct and indirect mechanisms, which may induce protein post-translational modifications (PTMs) that affect cellular functions [24,31,32]. MS-based proteomics is an ideal tool for not only large-scale quantitative profiling of thousands of proteins, but also for extensive profiling of different types of PTMs, which play a critical role in cellular signaling and regulation.…”

Detecting post-translational modification signatures as potential biomarkers in clinical mass spectrometry

“…Although the functional relevance of Cys oxidative PTMs in human diseases, such as cancer [189], cardiovascular [188,190], inflammatory [191], and neurodegenerative diseases [191,192] is clear, the molecular mechanisms of redox signaling still remain unclear. A number of proteomics techniques have been developed to study the targets and modulations of redox signaling [193]. Because redox proteomics is still an emerging field with continuing method development, research has mainly focused on basic method development and the application of the newly-developed methods to cell culture and animal models [193,194]; only a few redox proteomics studies have involved human subjects [195,196].…”

Modified cysteine S-phosphopeptide standards for mass spectrometry-based proteomics

“…Overnight incubation of the thiol-deprived analyte at a low pH results in a total loss of acid-labile phosphorylations, including those that are S-linked. Therefore, any reappearance of thiol groups may be attributed to hydrolytically released Cys-peptides, which can be effectively extracted on thiol-affinity materials (Gu et al 2015;Duan et al 2017).…”