the result of the reaction of lipid peroxidation products with cysteine, histidine, and lysine [2]. Factors such as amino acid sequence,
tridimensional conformation, cellular location, protein function,
and activity influence the vulnerability of the protein to get carbonylated [3]. Protein carbonylation is, then, a highly selective process of protein oxidation. In fact, only a subset (the so-called
carbonylome) of the total proteins is carbonylated in vivo under
the conditions of oxidative stress.
Carbonylation of amino acid residues frequently involves functional losses and the formation of toxic protein aggregates which
precipitate and accumulate in the cell, breaking the normal cellular
homeostasis. Elevated levels of protein carbonyl groups have been
detected in aging and several other oxidative stress-related diseases
such as obesity, diabetes, Alzheimer, and Parkinson [4]. Therefore,
in the study of these diseases, the development of suitable strategies
becomes crucial to identify and quantify the changes induced on
the carbonylome. However, the low abundance of carbonylated
proteins in vivo, the huge diversity of reactive species and products,
and their relative lability make the analysis of carbonylated proteins
a challenging task for redox proteomic technology.
The most popular methods for studying the carbonylome are
based on dinitrophenyl hydrazine (DNPH). They are the spectrometric and immunological DNPH-based assays. Although they
have demonstrated to be useful, their precision and sensitivity are
limited because the free probe causes interference and the antibody
anti-DNP displays nonspecific interactions with proteins. Fluorescein 5-thiosemicarbazide (FTSC), a fluorescent probe, has been
postulated as an alternative to DNPH for measurement protein
carbonylation [5] because only detect protein-bound carbonyls,
the free probe does not interfere in the quantification and can be
easily removed during sample preparation. Moreover, FTSC, as a
fluorescent probe, shows high sensitivity. FTSC is a hydrazide
derivative that can spontaneously react with aldehyde- or ketonecontaining molecules to form a covalent, hydrazone linkage
(Fig. 1) [6].
In this chapter, we describe a proteomic approach for the study
of the subset of carbonylated proteins among a complex mixture of
proteins. The protocol is based on the FTSC labeling of proteinbound carbonyls regardless of the nature of carbonyl adduct. The
fluorescent probe allows the detection and relative quantification of
each carbonylated protein present in the sample when FTSClabeled proteins are resolved on 2D gels and exposed to UV light.
Finally, the protocol identifies the carbonylated proteins by LC–
MS/MS analysis of the tryptic peptides obtained after in-gel digestion of carbonylated spots (Fig. 2).
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
228
Lucı ´a Me ´ ndez et al.
tridimensional conformation, cellular location, protein function,
and activity influence the vulnerability of the protein to get carbonylated [3]. Protein carbonylation is, then, a highly selective process of protein oxidation. In fact, only a subset (the so-called
carbonylome) of the total proteins is carbonylated in vivo under
the conditions of oxidative stress.
Carbonylation of amino acid residues frequently involves functional losses and the formation of toxic protein aggregates which
precipitate and accumulate in the cell, breaking the normal cellular
homeostasis. Elevated levels of protein carbonyl groups have been
detected in aging and several other oxidative stress-related diseases
such as obesity, diabetes, Alzheimer, and Parkinson [4]. Therefore,
in the study of these diseases, the development of suitable strategies
becomes crucial to identify and quantify the changes induced on
the carbonylome. However, the low abundance of carbonylated
proteins in vivo, the huge diversity of reactive species and products,
and their relative lability make the analysis of carbonylated proteins
a challenging task for redox proteomic technology.
The most popular methods for studying the carbonylome are
based on dinitrophenyl hydrazine (DNPH). They are the spectrometric and immunological DNPH-based assays. Although they
have demonstrated to be useful, their precision and sensitivity are
limited because the free probe causes interference and the antibody
anti-DNP displays nonspecific interactions with proteins. Fluorescein 5-thiosemicarbazide (FTSC), a fluorescent probe, has been
postulated as an alternative to DNPH for measurement protein
carbonylation [5] because only detect protein-bound carbonyls,
the free probe does not interfere in the quantification and can be
easily removed during sample preparation. Moreover, FTSC, as a
fluorescent probe, shows high sensitivity. FTSC is a hydrazide
derivative that can spontaneously react with aldehyde- or ketonecontaining molecules to form a covalent, hydrazone linkage
(Fig. 1) [6].
In this chapter, we describe a proteomic approach for the study
of the subset of carbonylated proteins among a complex mixture of
proteins. The protocol is based on the FTSC labeling of proteinbound carbonyls regardless of the nature of carbonyl adduct. The
fluorescent probe allows the detection and relative quantification of
each carbonylated protein present in the sample when FTSClabeled proteins are resolved on 2D gels and exposed to UV light.
Finally, the protocol identifies the carbonylated proteins by LC–
MS/MS analysis of the tryptic peptides obtained after in-gel digestion of carbonylated spots (Fig. 2).
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
228
Lucı ´a Me ´ ndez et al.