Recent advances in mass spectrometry-based methods to investigate reversible cysteine oxidation

Protein Oxidative Modifications in Neurodegenerative Diseases: From Advances in Detection and Modelling to Their Use as Disease Biomarkers

“…Given the importance of Cys oxidation in a range of biological functions and pathways, methods specifically targeting cysteines and their redox-PTMs have been developed [ 2 , 16 , 23 , 106 ]. These methods take advantage of the ability to irreversibly label the highly reactive thiolate ion present in Cys residues using specific compounds known as Cys alkylating agents; these agents can be modified to be used as isotope tags or as baits for enrichment techniques ( Figure 2 ) [ 23 , 106 ].…”

“…Thus, most methods focus on the reversible oxidized forms of Cys, which can then be chemically reduced to their thiolate form for further alkylation—an approach commonly called a tag-based reductive switch-labelling method [ 107 ]. Briefly, the ‘switch assay’ is comprised of 4 main steps ( Figure 2 ): (1) alkylation of free reduced Cys residues immediately after protein extraction, (2) reduction of reversibly oxidized Cys using either a general reducing agent such as dithiothreitol (DTT) or a modification-specific reductant such as ascorbate (to reduce sulfenylated Cys or nitrosothiols), or glutaredoxin (to reduce S-glutathionylated Cys), and (3) alkylation of nascent thiols with a distinct alkylating reagent, and finally, (4) detection using an appropriate method, typically an MS-based approach [ 2 , 23 , 108 ]. Variants of this protocol can be applied to focus on specific redox-PTMs; however, they all share the advantage of replacing labile oxidation-modified Cys with highly stable alkylated forms for subsequent analysis, while preserving the thiol oxidation state observed under physiological conditions [ 23 , 108 ].…”

“…Other examples are the use of antibodies on both Cys sulfinic and sulfonic acids or the use of reductive ligation of triarylphosphine derivatives and nitrosylated thiols. In addition, a combination of chemoproteomics probes with proximity-labelling methods has been developed to provide insights into subcellular Cys oxidation [ 2 , 121 ]. These new methods will help shed light on the functional role of specific redox-PTMs of Cys, such as how Cys oxidation affects the structure and interactome of proteins, as well as how redox-PTMs are spatially and temporally regulated in cells.…”

“…As discussed above, the study of redox-PTMs is relevant to understanding both health and disease. On the one hand, in physiological conditions, many redox-PTMs targeting Cys residues are reversible and act as a redox molecular switch through participation in intra- and inter-cellular signalling pathways [ 10 ]; on the other hand, Cys modified by irreversible redox-PTMs are often indicative of oxidative damage and can lead to protein misfolding, aggregation and dysfunction [ 2 , 136 ].…”

“…These redox-PTMs play essential roles in the cellular response to stress conditions and maintenance of redox homeostasis and, more generally, in cellular signalling pathways. Redox-PTMs are thus fundamental both in physiological conditions and under chronic oxidative stress, as observed in neurodegenerative diseases (NDDs), such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Dementia (FTD), Dementia with Lewy Bodies (DLB), and neuroinflammatory disease Multiple Sclerosis (MS) [ 2 , 3 , 4 , 5 , 6 , 7 , 8 ]. Indeed, redox-PTMs are essential for the integration and transformation of oxidant signals into biological responses through redox-dependent intracellular signalling pathways, a topic that has been reviewed elsewhere [ 9 , 10 , 11 ].…”