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Published on: June 21, 2021
Limiting the Hydrolysis and Oxidation of Maleimide-Peptide Adducts Improves Detection of Protein Thiol Oxidation
Amber E Boyatzis1, Scott D Bringans2, Matthew J Piggott1
1School of Chemistry and Biochemistry, University of Western Australia , Crawley, Western Australia 6009, Australia.
Abstract:
Oxidative stress, caused by reactive oxygen and nitrogen species (RONS), is important in the pathophysiology of many diseases. A key target of RONS is the thiol group of protein cysteine residues. Because thiol oxidation can affect protein function, mechanistic information about how oxidative stress affects tissue function can be ascertained by identifying oxidized proteins. The probes used must be specific and sensitive, such as maleimides for the alkylation of reduced cysteine thiols. However, we find that maleimide-alkylated peptides (MAPs) are oxidized and hydrolyzed under sample preparation conditions common for proteomic studies. This can result in up to 90% of the MAP signal being converted to oxidized or hydrolyzed MAPs, decreasing the sensitivity of the analysis. A substantial portion of these modifications were accounted for by Coomassie "blue silver" staining (∼14%) of gels and proteolytic digestion buffers (∼20%). More than 40% of the MAP signal can be retained with the use of thioglycolic acid during gel electrophoresis, trichloroethanol-UV protein visualization in gels, and proteolytic digestion buffer of pH 7.0 TRIS. This work demonstrates that it is possible to decrease modifications to MAPs through changes to the sample preparation workflow, enhancing the potential usefulness of maleimide in identifying oxidized peptides.
Insights
Identifying oxidized proteins is key to understanding disease. This study shows that modifying sample preparation can prevent signal loss, improving the detection of oxidized peptides using maleimides.
Area of Science:
- Biochemistry
- Proteomics
- Oxidative Stress
Background:
- Oxidative stress from reactive oxygen and nitrogen species (RONS) impacts disease pathophysiology.
- Protein cysteine thiols are primary targets of RONS, affecting protein function.
- Identifying oxidized proteins is crucial for understanding oxidative stress in tissues.
Purpose of the Study:
- To investigate the stability of maleimide-alkylated peptides (MAPs) during proteomic sample preparation.
- To identify factors contributing to MAP oxidation and hydrolysis.
- To optimize sample preparation methods to preserve MAP signals for sensitive analysis.
Main Methods:
- Proteomic sample preparation techniques were analyzed for their impact on MAPs.
- Quantification of MAP oxidation and hydrolysis under various conditions.
- Evaluation of specific reagents and buffer conditions (e.g., thioglycolic acid, pH 7.0 TRIS buffer) for preserving MAPs.
Main Results:
- Maleimide-alkylated peptides (MAPs) are susceptible to oxidation and hydrolysis during standard proteomic sample preparation.
- Significant MAP signal loss (up to 90%) can occur due to factors like Coomassie staining and proteolytic digestion.
- Optimized methods using thioglycolic acid and specific buffer conditions retained over 40% of the MAP signal.
Conclusions:
- Standard proteomic sample preparation protocols can compromise the analysis of oxidized peptides.
- Modifications to sample preparation workflows, including reagent choice and buffer pH, can significantly reduce MAP degradation.
- Optimized methods enhance the sensitivity and utility of maleimide-based approaches for identifying oxidized proteins in disease research.
