oxSWATH: An integrative method for a comprehensive redox-centered analysis combined with a generic differential

Sandra I Anjo¹, Matilde N Melo², Liliana R Loureiro³

¹Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; Faculty of Sciences and Technology, University of Coimbra, Coimbra, Portugal; Faculty of Medicine, University of Coimbra, Coimbra, Portugal.

|February 10, 2019

Summary

This study introduces oxSWATH, a new method that combines two types of protein analysis: redox changes and total protein levels. Traditional methods often focus only on cysteine oxidation without considering how much of the protein is present overall. The researchers used SWATH-MS and differential alkylation to create a cost-effective, high-throughput approach. They tested the method using a redox-regulated protein and secretomes from cells under stress. The oxSWATH method allows for analysis at both the peptide and protein levels. It also supports a broader proteomics evaluation by including general protein expression data. The results show that oxidative stress affects both redox states and total protein levels. The method provides a more comprehensive view of proteomic changes in response to stress.

Area of Science:

Redox proteomics within molecular biology
Proteomics data integration in bioinformatics

Background:

Many redox proteomics methods focus on cysteine oxidation without considering changes in total protein levels. Protein synthesis and degradation are key regulatory mechanisms. Existing approaches often overlook these broader proteomic shifts. This gap motivated the development of more integrative strategies. Prior research has shown the importance of cysteine redox states in cellular function. However, no prior work had resolved how to combine redox and total protein analyses effectively. The need for high-throughput, cost-effective methods remains unmet. Integrating these levels could improve the understanding of proteomic responses to stress.

Purpose Of The Study:

The aim was to develop an integrative method that combines redox analysis with total protein quantification. This method addresses the need for a comprehensive proteomic evaluation. The study focused on cysteine oxidation and total protein levels simultaneously. The motivation came from the limitations of current redox proteomics strategies. The researchers proposed a new approach using SWATH-MS and differential alkylation. This approach allows for high-throughput and cost-effective analysis. The method was tested on a redox-regulated protein to validate its effectiveness. The goal was to enable comparative analyses at both peptide and protein levels.

Main Methods:

The researchers used SWATH-MS in combination with differential alkylation. They applied a mix of commonly available alkylating reagents. This approach is called oxSWATH. The method integrates cysteine oxidation data with total protein levels. The samples were analyzed under control and oxidative stress conditions. The study used secretomes from cultured cells for validation. The data acquisition was performed in SWATH-MS mode. This allowed for both redox-focused and general proteomics analysis.

Main Results:

The oxSWATH method successfully determined reduced and oxidized cysteine forms. It also measured total protein levels for each oxoform. The method was validated using a redox-regulated protein model. Comparative analyses were performed at both peptide and protein levels. The approach enabled the detection of changes in total protein abundance. The data supported the importance of considering overall proteome shifts. The method provided a cost-effective and high-throughput solution. The results showed that oxidative stress altered both redox and total protein levels.

Conclusions:

The oxSWATH method integrates redox and total protein analysis effectively. It allows for comparative studies at multiple levels. The approach is high-throughput and cost-effective. The method supports both redox-centered and general proteomics analysis. The researchers proposed that this method improves the evaluation of proteomic changes. The results suggest that oxidative stress affects both redox and total protein levels. The method was tested and validated using secretome data. The study supports the need for integrative proteomics strategies.

Keywords:

proteomics analysis oxidative stress SWATH-MS protein oxidation

Frequently Asked Questions

The method allows for the simultaneous analysis of cysteine oxidation and total protein levels.

The method uses a combination of commonly available alkylating reagents.

SWATH-MS enables both redox and general proteomics analysis in the same experiment.

The method provides redox-centered and differential protein expression data.

The researchers tested it using a redox-regulated protein and secretome data.

The study suggests oxidative stress alters both redox and total protein levels.

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