Within the past decade, the role of cysteine oxidation in organismal stress response has been established as a fundamental regulatory mechanism [1, 2, 3, 4, 5, 6]. With a redox-sensitive thiol side chain, cysteines can act as reversible oxidative switch and initiate signaling pathways in response to stress. They are commonly oxidized by reactive oxygen species (ROS) and reactive nitrogen species (RNS) such as hydrogen peroxide (H2O2) and peroxynitrite (ONOO−), respectively, which accumulate in biological systems in stress states and can post translationally modify cysteines to affect and change protein and cellular function.

Cysteine oxidation can be characterized into two main categories: reversible oxidation and irreversible oxidation [7]. Reduced cysteines can be oxidized by ROS to form a relatively unstable and reactive sulfenic acid (R-SOH) and by RNS to form a nitrosothiol (R-SNO), which can then be hydrolyzed to form sulfenic acid. In the presence of proximal thiols, sulfenic acid can be stabilized by other cysteine containing molecules (e.g., proteins, glutathione) to form disulfide bonds (R1-S-S-R2). In addition, sulfenic acid can react with amines and amides to form sulfenamides (R1-S-N-R2), with cyclic sulfenamides forming to stabilize sulfenic acids through adjacent amides along the protein backbone. In the absence of proximal thiols or amides to form disulfide bonds/sulfenamides or in the presence of excess oxidant, sulfenic acid can be oxidized to sulfinic acid (R-SO2H) and further oxidized to sulfonic acid (R-SO3H). Although sulfinic acid can be reduced back to sulfenic acid in the presence of sulfiredoxin, these two increased oxidative states are often indicative of oxidative damage and can lead to protein dysfunctionality, degradation, and possibly cell death [8, 9, 10].

Interestingly, cysteines are one of the most uncommon amino acid residues within sequenced proteomes. In the reviewed Uniprot/Swissprot proteomes (accessed 20230601), nearly 83% of all proteins have a cysteine residue. However, cysteine residues only comprise 1.4% of all amino acid residues in these proteomes, making them difficult to study via traditional proteomic methods. Recent advances in mass spectrometry have furthered the ability to study low abundant species, such as cysteine-containing peptides. Traditional data dependent acquisition (DDA) methods operate by selecting the top n most abundant species within a sample for fragmentation. DDA methods are biased by abundance and can lead to data loss for low abundant species. In comparison, data independent acquisition (DIA) can be used to improve coverage of low abundant species [11]. DIA methods operate by fragmenting all coeluting species simultaneously within a specified mass-to-charge range. This leads to complex fragmentation spectra and until now required generation of detailed spectral libraries via DDA methods to ease downstream data analysis. Advancements in spectral library generation [12], development of library-free DIA methods [13], and creation of DIA-specific software [14, 15, 16] are helping to mitigate the complexity of data analysis, but these nascent software/methods still require development and optimization for utility in the most complex proteomes. DIA-based proteomics has allowed for increased coverage of sub-stoichiometric post translational modifications and has been used to accurately examine cysteine oxidation [17]. In addition to advancements in DIA, the development of isobaric tags applied to study cysteine oxidation, such as tandem mass tags (TMT), allows samples across biological replicates and conditions to be multiplexed to increase the abundance of precursor peptide ions within a survey scan [18]. These isobaric tags can then be distinguished by distinct reporter ions in the fragmentation spectra such that peptide abundances across biological replicates and sample conditions can be accurately quantified.

Although cysteines are uncommon, the functional diversity of post translational modifications that occur on cysteine residues point for a range of biological functions and pathways in which cysteines are involved. Yet the role of cysteine and how its different modification types play distinct roles in stress response is still being understood, especially in non-model and understudied organisms. As such, this review summarizes traditional methods that use mass spectrometry to interrogate the reversible redoxome and unveils new advances in investigating the cellular response to oxidative stress.