Reverse Thiol Trapping Approach to Assess the Thiol Status of Metal-Binding Mitochondrial Proteins.
AMS
Cysteine disulfide
Cysteine oxidation
IAA
Mitochondria
Redox biology
Thiol trapping
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2024
2024
Historique:
medline:
15
7
2024
pubmed:
15
7
2024
entrez:
15
7
2024
Statut:
ppublish
Résumé
Thiol-disulfide interconversions are pivotal in the intricate chemistry of biological systems. They play a vital role in governing cellular redox potential and shielding against oxidative harm. These interconversions can also act as molecular switches within an expanding array of redox-regulated proteins, facilitating dynamic and responsive processes. Furthermore, metal-binding proteins often use thiols for coordination. Reverse thiol trapping is a valuable analytical tool to study the redox state of cysteines in biological systems. By selectively capturing and stabilizing free thiol species with an alkylating agent, reverse thiol trapping allows for their subsequent identification and quantification. Various methods can be employed to analyze the trapped thiol adducts, including electrophoresis-based methods, mass spectrometry, nuclear magnetic resonance spectroscopy, and chromatographic techniques. In this chapter, we will focus on describing a simple and sensitive method to sequentially block thiols in their cellular state with a cell-permeant agent (iodoacetamide), and following reduction and denaturation of the samples, trap the native disulfides with a second blocker that shifts the apparent molecular weight of the protein. The oxidation status of proteins for which suitable antibodies are available can then be analyzed by immunoblotting. We present examples of mitochondrial proteins that use cysteine thiols to coordinate metal factors such as iron-sulfur clusters, zinc, and copper.
Identifiants
pubmed: 39008259
doi: 10.1007/978-1-0716-4043-2_15
doi:
Substances chimiques
Sulfhydryl Compounds
0
Mitochondrial Proteins
0
Iodoacetamide
ZRH8M27S79
Disulfides
0
Metals
0
Cysteine
K848JZ4886
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
249-259Informations de copyright
© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
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