Redox proteomics in melanoma cells: An optimized protocol

Cancer development and progression are intimately related with post-translational protein modifications, e.g., highly reactive thiol moiety of cysteines enables structural rearrangements resulting in redox biological switches. In this context, redox proteomics techniques, such as 2D redox DIGE, biot...

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Bibliographic Details
Published in:Analytical biochemistry 2024-08, Vol.691, p.115543-115543, Article 115543
Main Authors: Cunha, E.S., Mazepa, E., Batista, M., Marchini, F.K., Martinez, G.R.
Format: Article
Language:English
Subjects:
biotin
Biotin-switch assay
carcinogenesis
Iodoacetamide
Melanoma
moieties
N-ethylmaleimide
oxidation
peptides
Proteomics
Redox
Thiol oxidation
thiols
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Summary:Cancer development and progression are intimately related with post-translational protein modifications, e.g., highly reactive thiol moiety of cysteines enables structural rearrangements resulting in redox biological switches. In this context, redox proteomics techniques, such as 2D redox DIGE, biotin switch assay and OxIcat are fundamental tools to identify and quantify redox-sensitive proteins and to understand redox mechanisms behind thiol modifications. Given the great variability in redox proteomics protocols, problems including decreased resolution of peptides and low protein amounts even after enrichment steps may occur. Considering the biological importance of thiol's oxidation in melanoma, we adapted the biotin-switch assay technique for melanoma cells in order to overcome the limitations and improve coverage of detected proteins. [Display omitted] •Redox proteomics protocols are limited by low peptide amount and resolution.•A biotin-switch redox proteomics was improved for B16–F10 melanoma cells.•We demonstrated a gain of identification and enrichment of oxidized thiols.•This optimized protocol will help to address important cellular alterations in melanoma.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2024.115543