Chapter 15
FTSC-Labeling Coupled with 2DE-LC–MS/MS Analysis
of Complex Protein Mixtures for Identification and Relative
Quantification of Tissue Carbonylome
Lucı ´a Me ´ ndez, Lorena Barros, Silvia Mun ˜ oz, and Isabel Medina
Abstract
Carbonylation 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 stressrelated 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.
Key words In vivo protein carbonylation, FTSC, Fluorescent tag, Oxidative stress, Redox proteomics,
Mass spectrometry, 2DE
1 Introduction
Protein carbonylation is a nonenzymatic and irreversible posttranslational modification and the major hallmark of oxidative damage
[1]. There are four mechanisms to form carbonyls moieties (aldehyde, ketone, or lactam) in a protein: (a) direct oxidation of tryptophan; (b) metal-catalyzed oxidation of the side chains of arginine,
lysine, proline, and tyrosine; (c) as adducts of advanced glycation
products (AGEs) on lysine and arginine (glycoxidation); and (d) as
Mo ´ nica Carrera and Jesu ´ s Mateos (eds.), Shotgun Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 2259,
https://doi.org/10.1007/978-1-0716-1178-4_15, © Springer Science+Business Media, LLC, part of Springer Nature 2021
227
FTSC-Labeling Coupled with 2DE-LC–MS/MS Analysis
of Complex Protein Mixtures for Identification and Relative
Quantification of Tissue Carbonylome
Lucı ´a Me ´ ndez, Lorena Barros, Silvia Mun ˜ oz, and Isabel Medina
Abstract
Carbonylation 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 stressrelated 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.
Key words In vivo protein carbonylation, FTSC, Fluorescent tag, Oxidative stress, Redox proteomics,
Mass spectrometry, 2DE
1 Introduction
Protein carbonylation is a nonenzymatic and irreversible posttranslational modification and the major hallmark of oxidative damage
[1]. There are four mechanisms to form carbonyls moieties (aldehyde, ketone, or lactam) in a protein: (a) direct oxidation of tryptophan; (b) metal-catalyzed oxidation of the side chains of arginine,
lysine, proline, and tyrosine; (c) as adducts of advanced glycation
products (AGEs) on lysine and arginine (glycoxidation); and (d) as
Mo ´ nica Carrera and Jesu ´ s Mateos (eds.), Shotgun Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 2259,
https://doi.org/10.1007/978-1-0716-1178-4_15, © Springer Science+Business Media, LLC, part of Springer Nature 2021
227