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FTSC-Labeling Coupled with 2DE-LC–MS/MS Analysis of Complex Protein Mixtures for Identification and Relative Quantification of Tissue Carbonylome

AuthorsMéndez, Lucía CSIC ORCID; Barros, Lorena CSIC; Muñoz, Silvia; Medina, Isabel CSIC ORCID
KeywordsIn vivo protein carbonylation
Fluorescent tag
Oxidative stress
Redox proteomics
Mass spectrometry
Issue Date2021
CitationShotgun Proteomics: 227-246 (2021)
SeriesMethods in Molecular Biology, v. 2259
AbstractCarbonylation is a nonenzymatic irreversible posttranslational protein modification and the main hallmark of protein oxidative damage. Elevated levels of protein carbonyl groups have been detected in age-related and metabolic diseases such as obesity, diabetes, Alzheimer, Parkinson, and several other oxidative stress-related maladies. Interestingly, many studies have shown that only a subset of proteins is carbonylated under the conditions of oxidative stress, demonstrating that carbonylation is a highly selective process. As a consequence, identifying and quantifying the disease-induced changes on a certain carbonylome are crucial to understanding the etiology and progression of numerous diseases and then designing adequate prevention/palliation strategies. However, the low abundance of carbonylated proteins in vivo, the enormous diversity of reactive species, and their relative lability make the analysis of carbonylated proteins a challenging task for redox proteomic technology. Therefore, we present a proteomic approach based on the labeling of carbonyls formed in vivo on proteins using the fluorescein 5-thiosemicarbazide (FTSC) tag to detect the subset of carbonylated proteins among a complex mixture of proteins regardless of the nature of carbonyl adduct, isolation and relative quantification of carbonylated proteins in 2D gel electrophoresis, and protein identification by LC–MS/MS analysis. This method has been successfully used for the evaluation of in vivo protein carbonylation in very diverse animal tissues (plasma, liver, kidney, skeletal muscle, and adipose tissue) and species (from fish to mammalian) and has also been applied in different research fields (from food technology to nutrition), demonstrating its robustness and reliability
Description20 pages, 1 table, 2 figures
Publisher version (URL)https://doi.org/10.1007/978-1-0716-1178-4_15
Appears in Collections:(IIM) Libros y partes de libros
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