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Quantitative display of the redox status of proteins with maleimide‐polyethylene glycol tagging

Lee, Yu‐Jung, Chang, Geen‐Dong
Electrophoresis 2019 v.40 no.4 pp. 491-498
cysteine, electrophoresis, human cell lines, hydrogen peroxide, insulin, oxidation, oxidative stress, post-translational modification, protein-tyrosine-phosphatase, proteins, quantitative analysis, thiols
Cysteine oxidation, either biologically reversible or irreversible, is the main posttranslational modification associated with redox signaling and oxidative stress. Maleimide‐polyethylene glycol (m‐PEG) has been used to detect reversibly oxidized proteins by reacting to the reduced cysteine residues leading to mobility shift in immunoblots; a method called PEG‐switch. With PEG‐switch, both reduced and oxidized proteins can be observed on the same immunoblot simultaneously, providing a simple quantitative measurement for protein thiol modifications. In this report, we optimized the assay conditions and exploited the applications of PEG‐switch in quantitation of the extent of protein thiol oxidation in cells in response to H₂O₂ and insulin. In addition, we have proposed a redox scoring system for measuring the redox status of any given protein from the m‐PEG immunoblot. Our results provided quantitative data showing that two cysteine residues of protein tyrosine phosphatase 1B are prone to oxidation following insulin treatment in cultured HeLa cells.