caveat in the analysis of carbonylated proteins is their inherent instability towards further covalent modification such as Schiff base-formation (Mirzaei and Regnier, 2005). The latter would not be detected via any of the carbonyl-specific methods involving derivatization with hydrazines. Another problem with most of the proteomic data so far presented on protein carbonyl formation is the general lack of tandem mass spectrometry data which would (1) confirm the carbonyl content by a complementary technique, and (2) locate the modified amino acids. A potential source of error in the immunochemical identification of carbonyl-containing proteins only by Western blot could be the contamination of a nonmodified protein in a 2D gel spot by a trace amount of a second, carbonylated, protein. Mirzaei and Regnier (2005) demonstrated that tandem mass spectrometric information on the carbonyl-containing peptide sequences can be obtained after biotin hydrazide derivatization of protein carbonyls, followed by immunoenrichment on immobilized monomeric avidin columns.
Analysis of 3-Nitrotyrosine Increasing evidence is accumulating that the formation of 3-nitrotyrosine in vivo is associated with changes in protein structure and function and represents a valid biomarker for conditions of oxidative stress (Ischiropoulos and Beckman, 2003). To date, most proteomic strategies for the detection of 3-nitrotyrosine utilized 2DE followed by Western blot detection of 3-nitrotyrosine (Aulak et al., 2001; Miyagi et al., 2002; Turko et al., 2003; Casoni et al., 2005). Analogous to the proteomic analysis of protein-associated carbonyls, the age-dependent accumulation of 3-nitrotyrosine is rather selective to a subset of proteins (Kanski et al., 2003; 2005a; 2005b). Interestingly, predominantly cyto-solic proteins displayed an age-dependent increase in 3-nitrotyrosine in skeletal muscle (Kanski et al., 2003; 2005b) while cardiac aging caused 3-nitrotyrosine accumulation to a significant extent also on mitochondrial proteins (Kanski et al., 2005a). It remains to be shown whether complementary analytical strategies can confirm this rather unexpected selectivity.
Most of the 2DE analysis of 3-nitrotyrosine-containing proteins in vivo suffer from the absence of tandem mass spectrometric sequence information, confirming the presence of 3-nitrotyrosine on the identified proteins. Exceptions are noted for the 2DE analysis of human pituitary (Zhan and Desiderio, 2004) and rat cardiac proteins (Kanski et al., 2005a). This general lack of tandem mass spectrometric information is likely due to the actual yield and recovery of 3-nitrotyrosine-containing peptides/proteins from biological tissue. However, when skeletal muscle proteins were resolved by solution isoelectric focusing/SDS-PAGE instead of 2DE, permitting significantly higher sample loads, eleven 3-nitro-tyrosine-containing proteins were identified and confirmed by tandem mass spectrometric data (Kanski et al., 2005b).
Information on the actual location of 3-nitrotyrosine in the identified proteins led to some interesting observations. In the cytosolic creatine kinase, age-dependent tyrosine nitration occurred predominantly on residues at positions Tyr14 and Tyr20. Instead, the exposure of creatine kinase to peroxynitrite (ONOO~) in vitro led to nitration of predominantly Tyr82, a residue not affected in vivo. Peroxynitrite forms through the reaction of nitric oxide (NO) with superoxide (O. _) and represents one important tyrosine-nitrating species in vivo. However, additional nitrating systems are known such as the peroxidase-nitrite system, affording nitrogen dioxide (*NO2). Moreover, while identifying nitrated proteins in vivo we must always be aware of the fact that any observed selectivity will be the result not only of the nitration chemistry, but also of protein turnover and repair. Nitrated proteins are subject to accelerated turnover, and Murad and coworkers have identified a potential repair enzyme for nitrated proteins, a putative ''denitrase,'' which targets selected nitrated proteins such as histone H1.2 (Irie et al., 2003). Proteomic studies in isolated mitochondria have indicated that nitrated proteins can be repaired quite rapidly when the oxygen tension decreases (Aulak et al., 2004; Koeck et al., 2004).
Analysis of Cys oxidation and S-glutathiolation Cys oxidation is one of the most prevalent protein modifications under conditions of oxidative stress. Proteomic evidence for Cys oxidation has been obtained through tandem MS characterization of cysteic acid-containing peroxiredoxins in Jurkat T-cell lymphoma cells exposed to an organic peroxide or glucose oxidase (Rabilloud et al., 2002). Lin et al. (2002) utilized a lipophilic cation, (4-iodobutyl)triphenylphosphonium (IBTP), which enriches inside the mitchondria, and an antibody directed against the triphenylphosphonium moiety, for the pro-teomic characterization of mitochondrial protein thiols, which are sensitive towards oxidation. Intracellular proteins encounter large concentrations of the endogenous antioxidant glutathione (GSH). Therefore, S-glutathiolation is a common endogenous process, and proteomic methods for the detection of S-glutathiolated proteins have been developed, using either biotinylated GSH ester for the selective immuno-affinity purification of glutathiolated proteins (Sullivan et al., 2000), or [35S]-labeled GSH for the specific detection of [35S] incorporation into proteins resolved on 2D gels (Fratelli et al., 2002). Obviously, these methods are applicable only to cell cultures or tissue homogenates but cannot be performed in vivo. S-glutathiolation in vivo would need to be characterized by direct tandem MS analysis of S-glutathiolated peptide sequences isolated from tissue. A subset of proteins, which are susceptible to S-glutathiolation by S-nitrosoglutathione, was identified utilizing S-nitrosoglutathione-sepharose (Klatt et al., 2000).
Analysis of protein glycation The accumulation of advanced glycation endproducts (AGEs) is a hallmark of age-dependent protein modification. AGEs represent a class of chemical structures generated via breakdown or cross-linking of initial sugar-protein adducts. Poggioli et al. (2002) could demonstrate that age-dependent glycation does not occur randomly but appears to selectively target a few proteins, though no mass spectrometric studies to characterize these proteins were presented. In contrast, Crabb et al. (2002) performed both HPLC-tandem MS and Western blot experiments to identify proteins, including AGE-modified proteins, present in debris-like material, referred to as "drusen," accumulating below the retinal pigment epithelium on Bruch's membrane during age-related macular degeneration (AMD).
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