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FASEB J. 2019 Nov;33(11):12336-12347. doi: 10.1096/fj.201900693R. Epub 2019 Aug 26.

Activated Thiol Sepharose-based proteomic approach to quantify reversible protein oxidation.

Author information

1
Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
2
Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, Ontario, Canada.
3
Laura and Isaac Perlmutter Cancer Center, New York University Langone Medical Center, New York, New York, USA.
4
Proteomics Laboratory, Division of Advanced Research Technologies, New York University (NYU) Langone Health, New York, New York, USA.
5
Department of Biochemistry and Molecular Pharmacology, New York University (NYU) Langone Health, New York, New York, USA.

Abstract

Reactive oxygen species (ROS) can act as second messengers in various signaling pathways, and abnormal oxidation contributes to multiple diseases, including cancer. Detecting and quantifying protein oxidation is crucial for a detailed understanding of reduction-oxidation reaction (redox) signaling. We developed an Activated Thiol Sepharose-based proteomic (ATSP) approach to quantify reversible protein oxidation. ATSP can enrich H2O2-sensitive thiol peptides, which are more likely to contain reactive cysteines involved in redox signaling. We applied our approach to analyze hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a type of kidney cancer that harbors fumarate hydratase (FH)-inactivating mutations and has elevated ROS levels. Multiple proteins were oxidized in FH-deficient cells, including many metabolic proteins such as the pyruvate kinase M2 isoform (PKM2). Treatment of HLRCC cells with dimethyl fumarate or PKM2 activators altered PKM2 oxidation levels. Finally, we found that ATSP could detect Src homology region 2 domain-containing phosphatase-2 and PKM2 oxidation in cells stimulated with platelet-derived growth factor. This newly developed redox proteomics workflow can detect reversible oxidation of reactive cysteines and can be employed to analyze multiple physiologic and pathologic conditions.-Xu, Y., Andrade, J., Ueberheide, B., Neel, B. G. Activated Thiol Sepharose-based proteomic approach to quantify reversible protein oxidation.

KEYWORDS:

ATSP; HLRCC; PKM2; redox regulation

PMID:
31451050
DOI:
10.1096/fj.201900693R

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