Limiting the Hydrolysis and Oxidation of Maleimide–Peptide Adducts Improves Detection of Protein Thiol Oxidation

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...

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Bibliographic Details
Published in:Journal of proteome research 2017-05, Vol.16 (5), p.2004-2015
Main Authors: Boyatzis, Amber E, Bringans, Scott D, Piggott, Matthew J, Duong, Marisa N, Lipscombe, Richard J, Arthur, Peter G
Format: Article
Language:English
Subjects:
Alkylation
Animals
Electrophoresis, Polyacrylamide Gel
Humans
Hydrolysis
Maleimides - metabolism
Molecular Probe Techniques - standards
Oxidation-Reduction
Oxidative Stress
Proteins - metabolism
Proteolysis
Proteomics - methods
Sulfhydryl Compounds - metabolism
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Summary: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.
ISSN:1535-3893
1535-3907
DOI:10.1021/acs.jproteome.6b01060