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Oxidation sites Despite cysteine is one of the least abundant amino acids, it is highly conserved across many species. A major mode of post-translational metabolic regulation is initiated through production of reactive oxygen species (ROS) and related species that modify protein function by covalent adduction of cysteine residues. Because cysteine oxidation is fast, reversible, and often highly selective, it is employed for adaptive modulation of protein function, for example serving to either induce or abolish enzyme activity, or to facilitate or disrupt protein interactions, in response to acute metabolic perturbations. As such, redox regulatory metabolites have been implicated in a vast array of tissue-specific regulatory processes. Moreover, dysregulation of ROS and oxidative signaling is one of the longest postulated underlying causes of physiological decline with age. In this website, we provide oxidation stoichiometry data on cysteine sites. On the “Sites” page, every heatmap shows the full length of a protein. Oxidation sites are colored according to their oxidation% values in each tissue. The second plot aligns observed phosphorylation sites in Phosphomouse and quantified oxidation sites in Oximouse. The barplot presents oxidation% values of a cysteine oxidation site across 20 young and old tissues.

Quantitation and stoichiometry Despite the fundamental importance of redox regulation in tissue-specific physiology and mammalian aging, there is a surprising dearth of information regarding the protein modifications that explain the molecular basis for these processes in vivo. Comprehensive analyses of this type are lacking in large part because existing methods are non-quantitative and/or cover a very small proportion of the cysteine proteome. To address this problem, we developed a novel protocol, enriching very lowly abundant cysteines from highly complex biological samples using Cysteine-reactive phosphate tags (CPT) and Immobilized metal affinity chromatography (IMAC), and combined it with SPS- MS3- TMT to determine modification stoichiometry on cysteines (oxidation%). Cysteine oxidation stoichiometry is crucial because, for instance, a five-fold increase of cysteine oxidation could mean 0.2%-1% or 20%-100% on the stoichiometry level. While a 0.2%-1% change might be important if this oxidation event activates the protein, it would hardly be of any biological meaning if the modification inhibits protein functions. Determining stoichiometry would provide a more direct avenue to asses biological relevance of cysteine oxidation events.

Network Analysis This analysis seeks answers for two questions: (1) ROS signal through what protein interaction networks to regulate tissue-specific biological processes? and (2) what protein complexes could be forged together by interchain disulfide bonds? By overlaying OxiMouse on Bioplex 2.0, this analysis sheds light on these problems. On the community page, the heatmap presents the enrichment of oxidized proteins in each protein network observed in Bioplex 2.0. Clicking on any network in any tissue, the network is displayed and colored by oxidation% values (red-over 20% oxidized; orange- between 10-20%; skyblue- under 10%; and gray-not quantified).

Downloading OxiMouse Data Protein/sites of interest can be downloaded. The file includes oxidation% ± SE in each young and old tissue, along with peptide sequence information and protein annotations.