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A chemoproteomics method for direct and comprehensive characterization of cysteine reversible oxidation

ABSTRACT: Cysteine oxidation is a series of protein modifications caused by reactive oxygen species (ROS) in cells and can regulate protein functions and cellular activities. Comprehensive and site-specific characterization of the cysteine oxidation is critical for understanding the role of ROS regulating cellular events. Here we developed a chemoproteomics method to directly and systematically detect the reversible cysteine oxidation sites. Different from the commonly used methods for indirect detection of cysteine oxidation, this method allows for direct targeting of oxidized cysteine for MS analysis. The method was validated using small molecules containing oxidized cysteines and the whole cell lysate treated with the probe, demonstrating that it specifically targets reversible oxidized cysteines. In total, 3000-4000 proteins containing cysteine oxidation sites were identified from Jurkat, MCF7 and HEP G2 cells, respectively. Combining with multiplexed proteomics, we applied the method to quantify cysteine oxidation changes in the livers from mice fed with the high or low fat diet (HFD/LFD). It was found that the overall cysteine oxidation in mouse liver was dramatically upregulated with HFD, and the upregulation is correlated with protein distribution, functions, and the flanking residues of the oxidation sites. Many biological processes critical in the pathology of fatty liver diseases are also significantly enriched among the oxidized proteins with higher abundance in the HFD samples, suggesting the potential regulatory role of cysteine oxidation in the diseases. Additionally, the method was applied to study the cysteine oxidation change in THP-1 cells with the lipopolysaccharide treatment, and among >15000 cysteine oxidation sites quantified in THP-1 cells, many of them were upregulated under the treatment. For the oxidized proteins with upregulated oxidation sites, they are associated with inflammation and immune response, indicating the potential role of cysteine oxidation in regulating cellular response to bacterial infection. This chemoproteomics method can be widely applied to comprehensively study cysteine oxidation events in various samples.

INSTRUMENT(S): Orbitrap Exploris 480, LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Ronghu Wu

PROVIDER: MSV000093703 | MassIVE | Wed Dec 20 19:35:00 GMT 2023

REPOSITORIES: MassIVE

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Project description:Most of the redox proteomics strategies are focused on the identification and relative quantification of cysteine oxidation changes without considering the variation in the total levels of the proteins. However, the regulation of protein synthesis and protein degradation are also associated with many of the regulatory mechanisms of the cells, being therefore important to consider the changes in total protein levels in the Post Translational Modifications (PTMs) focused analyses, such as cysteine redox characterization. This aspect was only address in the cysTMTRAQ method, which combines two types of isobaric tags in the same experiment leading to a considerable increase in the costs of the analysis. Therefore, a novel integrative approach combining the SWATH-MS method with differential alkylation using non-isotopically labeled alkylating reagents (oxSWATH) is presented, thus integrating the information regarding relative cysteine oxidation with the comparative analysis of the total protein levels in a cost effective highthrouput approach. The proposed method was tested using a redox regulated protein, and further applied to a comparative analysis of secretomes obtained under control or oxidative stress conditions to strengthen the importance of considering the changes in protein total levels. OxSWATH allowed to determine the relative proportion of reduced and reversible oxidized oxoforms of the proteins, and by considering total protein levels, to determine the total levels of each fraction, which are then used for comparative analysis. This approach can be an important tool in redox centered approaches, being able to not only analyze the overall reduce/reversible oxidized state of proteins but able to be further applied in the characterization of the different oxoforms of the individual cysteines. Moreover, since samples are acquired in SWATH-MS mode, besides the redox centered analysis, a generic differential protein expression analysis can be also performed, allowing a truly comprehensive evaluation of proteomics changes upon oxidative stimulus.

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A chemoproteomics method for direct and comprehensive characterization of cysteine reversible oxidation

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