Chemical Modification Of Proteins Research Articles

Environmental-stress-induced Chromatin Regulation and its Heritability.

Chromatin is subject to proofreading and repair mechanisms during the process of DNA replication, as well as repair to maintain genetic and epigenetic information and genome stability. The dynamic structure of chromatin modulates various nuclear processes, including transcription and replication, by altering the accessibility of the DNA to regulatory factors. Structural changes in chromatin are affected by the chemical modification of histone proteins and DNA, remodeling of nucleosomes, incorporation of variant histones, noncoding RNAs, and nonhistone DNA-binding proteins. Phenotypic diversity and fidelity can be balanced by controlling stochastic switching of chromatin structure and dynamics in response to the environmental disruptors and endogenous stresses. The dynamic chromatin remodeling can, therefore, serve as a sensor, through which environmental and/or metabolic agents can alter gene expression, leading to global cellular changes involving multiple interactive networks. Furthermore its recent evidence also suggests that the epigenetic changes are heritable during the development. This review will discuss the environmental sensing system for chromatin regulation and genetic and epigenetic controls from developmental perspectives.

Grafting synthetic transmembrane units to the engineered low-toxicity α-hemolysin to restore its hemolytic activity.

The chemical modification of proteins to provide desirable functions and/or structures broadens their possibilities for use in various applications. Usually, proteins can acquire new functions and characteristics, in addition to their original ones, via the introduction of synthetic functional moieties. Here, we adopted a more radical approach to protein modification, i.e., the replacement of a functional domain of proteins with alternative chemical compounds to build "cyborg proteins." As a proof of concept model, we chose staphylococcal α-hemolysin (Hla), which is a well-studied, pore-forming toxin. The hemolytic activity of Hla mutants was dramatically decreased by truncation of the stem domain, which forms a β-barrel pore in the membrane. However, the impaired hemolytic activity was significantly restored by attaching a pyrenyl-maleimide unit to the cysteine residue that was introduced in the remaining stem domain. In contrast, negatively charged fluorescein-maleimide completely abolished the remaining activity of the mutants.

Aldehyde Stress-Mediated Novel Modification of Proteins: Epimerization of the N-Terminal Amino Acid

Various kinds of aldehyde-mediated chemical modifications of proteins have been identified as being exclusively covalent. We report a unique noncovalent modification: the aldehyde-mediated epimerization of the N-terminal amino acid. Epimerization of amino acids is thought to cause conformational changes that alter their biological activity. However, few mechanistic studies have been performed, because epimerization of an amino acid is a miniscule change in a whole protein. Furthermore, it does not produce a mass shift, making mass spectrometric analysis difficult. Here, we have demonstrated epimerization mediated by endogenous aldehydes. A model peptide, with an N-terminal l- or d-FMRFamide, was incubated with an endogenous or synthetic aldehyde [acetaldehyde, methylglyoxal, pyridoxal 5'-phosphate (PLP), 4-oxo-2(E)-nonenal, 4-hydroxy-2(E)-nonenal, d-glucose (Glc), 4- or 2-pyridinecarboxaldehyde] under physiological conditions. Each reaction mixture was analyzed by liquid chromatography with ultraviolet detection and/or electrospray ionization mass spectrometry. Considerable epimerization occurred after incubation with some endogenous aldehydes (PLP, 40.6% after 1 day; Glc with copper ions, 6.5% after 7 days). Moreover, the epimerization also occurred in whole proteins (human serum albumin and PLP, 26.3% after 1 day). Tandem mass spectrometric studies, including deuterium labeling and sodium borohydride reduction, suggested that the epimerization results from initial Schiff base formation followed by tautomerization to ketimine that causes the chirality to be lost. This suggests that the epimerization of the N-terminal amino acid can also occur in vivo as a post-translational modification under a high level of aldehyde stress.

Redox proteomics: from protein modifications to cellular dysfunction and disease

1. Oxidatively Modified Proteins and Proteomic Technologies. 1.1 Chemical Modification of Proteins by Reactive Oxygen Species (E. Stadtman & R. Levine). 1.2 The Chemistry of Protein Modifications Elicited By Nitric Oxide and Related Nitrogen Oxides (D. Thomas, et al.). 1.3 Mass Spectrometry Approaches for the Molecular Characterization of Oxidatively/Nitrosatively Modified Proteins (A. Scaloni). 1.4 Thiol-disulfide Oxidoreduction of Protein Cysteines: Old Methods Revisted for Proteomics (V. Bonetto & P. Ghezzi). 1.5 Carbonylated Proteins and their Implication in Physiology and Pathology (R. Levine & E. Stadtman). 1.6 S-Nitrosation of Cysteine thiols as a Redox Signal (Y. Zhang & N. Hogg). 1.7 Detection of Glycated and GlycoOxidated Proteins (A. Lapolla, et al.). 1.8 MudPIT (Multidimensaional Protein Identification Technology) for Identification of Post-translational Protein Modifications in Complex Biological Mixtures (S. Thomas, et al.). 1.9 Use of a Proteomic Technique to Identify Oxidant-Sensitive Thiol Proteins in Cultured Cells (M. Hampton, et al.). 1.10 ICAT (Isotope-Code Affinity Tag) Approach to Redox Proteomics: Identification and Quantification of Oxidant-Sensitive Protein Thiols (M. Sethuraman, et al.). 1.11 Quantitative Determination of Free and Protein-Associated 3-nitrotyrosine and S-nitrosothiols in the Circulation by Mass Spectrometry and Other Methodologies: A Critical Review and Discussion from the Analytical and Review Point of View (D. Tsikas). 2. Cellular Aspects of Protein Oxidation. 2.1 The Covalent Advantage: A New Paradigm for Cell Signaling Mediated by Thiol Reactive Lipid Oxidation Products (D. Dickinson, et al.). 2.2 Early Molecular Events During Response to Oxidative Stress in Human Cells by Differential Proteomics (G. Tell). 2.3 Oxidative Damage to Proteins: Structural Modifications and Consequences in Cell Function (E. Cabiscol & J. Ros). 2.4 Oxidative Damage and Cellular Senescence: Lessons from Bacteria and Yeast (T. Nystrom). 3. Redox Proteomic Analysis in Human Diseases. 3.1 Proteins as Sensitive Biomarkers of Human Conditions Associated with Oxidative Stress (I. Dalle-Donne, et al.). 3.2 Degradation and Accumulation of Oxidized Proteins in Age-Related Diseases (P. Voss & T. Grune). 3.3 Redox Proteomics: A New Approach to Investgate Oxidative Stress in Alzheimer's Diseases (D. Butterfield, et al.). 3.4 Oxidized Proteins in Cardiac Ischemia-Reperfusion (J. Brennan & P. Eaton). 3.5 Proteome Anaylsis of Oxidative Stress: Glutathionyl Hemoglobin in Diabetic and Uremic Patients (T. Niwa). 3.6 Glyco-Oxidative Biochemistry in Diabetic Renal Injury (T. Miyata). 3.7 Quantitative Screnning of Protein Glycation, Oxidation, and Nitration Adducts by LC-MS/MS: Protein Damage in Diabetes, Uremia, Cirrhosis, and Alzheimer's Disease (P. Thornalley). 3.8 Protein Targets and Functional Consequences of Tyrosine Nitration in Vascular Disease (L. Baker, et al.). 3.9 Oxidation of Artery Wall Proteins by Myeloperoxidase: A proteomics Approach (T. Vaisar & J. Heinecke). 3.10 Oxidative Stress and Protein Oxidation in Pre-Eclampsia (M. Raijmakers, et al.). 3.11 The Involvement of Oxidants in the Etiology of Chronic Airway Diseases: Proteomic Approaches to Identify Redox Processes in Epithelial Cell Signal and Inflammation (A. van der Vliet, et al.). 3.12 Sequestering Agents of Intermediate Reactive Aldehydes as Inhibitors of Advanced Lipoxidation End-Products (ALEs) (M. Carini, et al.).

Structural basis for the site-specific chemical modification of proteins

Structural basis for the site-specific chemical modification of proteins

Leonidas Zervas award lecture: Abiotic ligands for new quaternary architectures of peptides and proteins

This review is the result of a kind invitation to provide a report on the basis of the Leonidas Zervas award lecture at the 32nd European Peptide Society in Athens, Greece, September 2012. The lecture covered several topics including contributions toward new methods for the assembly of peptides and chemical modification of proteins. The present review will focus on another topic from the Zervas lecture, namely, our recent efforts at creating new, artificial architectures for the organization of the quaternary structure of proteins. Crucially, this is achieved with abiotic ligands and not protein surface engineering. This concept was demonstrated for the important biopharmaceutical peptide insulin, by using both abiotic metal ion binding ligands and perfluoroalkyl chains.

Steric Effects in Peptide and Protein Exchange with Activated Disulfides

Disulfide exchange is an important bioconjugation tool, enabling chemical modification of peptides and proteins containing free cysteines. We previously reported the synthesis of a macromer bearing an activated disulfide and its incorporation into hydrogels. Despite their ability to diffuse freely into hydrogels, larger proteins were unable to undergo in-gel disulfide exchange. In order to understand this phenomenon, we synthesized four different activated disulfide-bearing model compounds (Mn = 300 Da to 10 kDa) and quantified their rate of disulfide exchange with a small peptide (glutathione), a moderate-sized protein (β-lactoglobulin), and a large protein (bovine serum albumin) in four different pH solutions (6.0, 7.0, 7.4, and 8.0) to mimic biological systems. Rate constants of exchange depend significantly on the size and accessibility of the thiolate. pH also significantly affects the rate of reaction, with the faster reactions occurring at higher pH. Surprisingly, little difference in exchange rates is seen between macromolecular disulfides of varying size (Mn = 2 kDa - 10 kDa), although all undergo exchange more slowly than their small molecule analogue (MW = 300 g/mol). The maximum exchange efficiencies (% disulfides exchanged after 24 h) are not siginificantly affected by thiol size or pH, but somewhat affected by disulfide size. Therefore, while all three factors investigated (pH, disulfide size, and thiolate size) can influence the exchange kinetics and extent of reaction, the size of the thiolate and its accessibility plays the most significant role.

Epigenomics comes of age with expanding roles in biological understanding and clinical application

Epigenetics is a mechanism that regulates gene expression not depending on the underlying DNA sequence, but on the chemical modification of DNA or histone proteins. Thanks to current advanced technologies, the understanding of epigenetic modifications or ‘epigenome’ in the human genome has been growing rapidly.

Spatially selective surface platforms for binding fibrinogen prepared by particle lithography with organosilanes.

We introduce an approach based on particle lithography to prepare spatially selective surface platforms of organosilanes that are suitable for nanoscale studies of protein binding. Particle lithography was applied for patterning fibrinogen, a plasma protein that has a major role in the clotting cascade for blood coagulation and wound healing. Surface nanopatterns of mercaptosilanes were designed as sites for the attachment of fibrinogen within a protein-resistant matrix of 2-[methoxy(polyethyleneoxy)propyl] trichlorosilane (PEG-silane). Preparing site-selective surfaces was problematic in our studies, because of the self-reactive properties of PEG-organosilanes. Certain organosilanes presenting hydroxyl head groups will cross react to form mixed surface multi-layers. We developed a clever strategy with particle lithography using masks of silica mesospheres to protect small, discrete regions of the surface from cross reactions. Images acquired with atomic force microscopy (AFM) disclose that fibrinogen attached primarily to the surface areas presenting thiol head groups, which were surrounded by PEG-silane. The activity for binding anti-fibrinogen was further evaluated using ex situ AFM studies, confirming that after immobilization the fibrinogen nanopatterns retained capacity for binding immunoglobulin G. Studies with AFM provide advantages of achieving nanoscale resolution for detecting surface changes during steps of biochemical surface reactions, without requiring chemical modification of proteins or fluorescent labels.

A mild synthesis of N-functionalised bromomaleimides, thiomaleimides and bromopyridazinediones

Bromomaleimides are useful building blocks in synthesis and powerful reagents for the selective chemical modification of proteins. A mild new synthesis of these reagents is described, along with the convenient transferability of the approach to dithiomaleimides and bromopyridazinediones.

Structure–activity studies on the upstream splice junction of a semisynthetic intein

Protein trans-splicing by split inteins holds great potential for the chemical modification and semisynthesis of proteins. However, the structural requirements of the extein sequences immediately flanking the intein are only poorly understood. This knowledge is of particular importance for protein labeling, when synthetic moieties are to be attached to the protein of interest as seamlessly as possible. Using the semisynthetic Ssp DnaB intein both in form of its wild-type sequence and its evolved M86 mutant, we systematically varied the sequence upstream of the short synthetic IntN fragment using both proteinogenic amino acids and unnatural building blocks. We could show for the wild-type variant that the native N-extein sequence could be reduced to the glycine residue at the (−1) position directly flanking the intein without significant loss of activity. The glycine at this position is strongly preferred over building blocks containing a phenyl group or extended alkyl chain adjacent to the scissile amide bond of the N-terminal splice junction. Despite their negative effects on the splicing yields, these unnatural substrates were well processed in the N–S acyl shift to form the respective thioesters and did not result in an increased decoupling of the asparagine cyclization step at the C-terminal splicing junction. Therefore, the transesterification step appeared to be the bottleneck of the protein splicing pathway. The fluorophore 7-hydroxycoumarinyl-4-acetic acid as a minimal N-extein was efficiently ligated to the model protein, in particular with the M86 mutant, probably because of its higher resemblance to glycine with an aliphatic c-α carbon atom at the (−1) position. This finding indicates a way for the virtually traceless labeling of proteins without inserting extra flanking residues. Due to its overall higher activity, the M86 mutant appears most promising for many protein labeling and chemical modification schemes using the split intein approach.

P32: Potential role for 8-Nitro-cGMP in nitrite-mediated hypoxic signaling

Background Nitrite has been shown to elicit both acute and chronic nitric oxide (NO)-dependent effects, especially at lower oxygen tensions.In this paradigm, nitrite reduction by deoxyhemoglobin and deoxymyoglobin activates NO-signaling to mediate several functions including hypoxic vasodilation.The mechanism underlying this however remains unclear, with a major challenge being how to reconcile nitrite-derived NO-signaling in an NO-scavenging environment, considering the high heme concentrations (∼10 mM) and rapid reaction rates between heme and NO ( k ∼ 10 7 M −1 s −1 ).Recent studies also implicate the derivative of cGMP, 8-nitro-cGMP, as a novel second messenger in NO-dependent signaling.Both cGMP and 8-nitro-cGMP activates PKG, however, unlike cGMP, 8-nitro-cGMP is resistant to PDE5 degradation and has the potential to elicit NO-induced signal transduction via a new chemical modification of proteins referred to as S -guanylation.To date, nitrite-dependent NO formation and 8-nitro-cGMP-dependent activation of NO-signaling have been considered independent of each other.In this study, we proposed a link between these two signaling paradigms.We hypothesized that 8-nitro-cGMP is a critical mediator of nitrite-dependent activation of hypoxic NO-signaling and tested if nitrite promotes hypoxic NO-signaling via 8-nitro-cGMP. Methods First, 8-nitro-cGMP formation in rat aorta exposed to nitrite at high O 2 (21%) and low O 2 (1%) tensions was assessed by immunostaining.The role of 8-nitro-cGMP in nitrite-mediated vasodilation was assessed using ex vivo aortic vessel dilation as an index of nitrite-dependent NO-signaling at high O 2 (21%) and low O 2 (1%) tensions, with and without the PDE5 inhibitors, sildenafil or zaprinast. Results The highest intensity of staining for 8-nitro-cGMP was observed in the vessels treated with nitrite at low O 2 (1%) tension and in the presence of RBC, indicating a nitrite-induced formation of 8-nitro-cGMP under hypoxic conditions.Nitrite stimulated vasodilation at 1% O 2 tension in a dose-dependent manner and this response was significantly improved in the presence of RBCs.Treatment of the aortic vessel segments with the PDE5 inhibitors, sildenafil or zaprinast improved RBC-dependent nitrite-mediated vasodilation by ∼2-fold at 21% O 2 .However, PDE5 inhibition had no effect on nitrite-dependent vasodilation at 1% O 2. Moreover, NO-dependent vasodilation using MAHMA NONOate was sensitive to PDE5 inhibition at both 21% and 1% O 2 .These data suggest a hypoxia-dependent shift from a PDE5-sensitive to PDE5-insensitive mechanism for nitrite-dependent vasodilation. Conclusion Collectively, these data indicate the possible role for 8-nitro-cGMP in nitrite-mediated hypoxic signaling paradigms. Disclosure Nothing to disclose.

Effects of chemical modifications in the partition behavior of proteins in aqueous two-phase systems: A case study with RNase A

Chemical modification of proteins is gaining importance due to the improvement in properties and the broader range of applications that these protein conjugates have. Once modified, several purification strategies need to be applied to isolate the conjugates of interest. Aqueous two-phase systems (ATPS) are an attractive alternative for the primary recovery of proteins and their conjugates. However, to better understand which biochemical parameters affect in greater degree the partition behavior of these modified proteins in ATPS, it becomes necessary to characterize the partition behavior of different species. In this work, ribonuclease A (RNase A) was selected as a model protein to address the partition behavior of chemically modified proteins in ATPS. Native, mono-PEGylated, Uniblue A, Dabsyl Chloride, and Direct Red 83 chemically modified RNase A's were partitioned in 16 different polyethylene glycol (PEG)-potassium phosphate ATPS. Results suggest that while the effects of system design parameters govern the partition of native RNase A, the behavior of the chemically modified species is more influenced by the physicochemical characteristics of the modifying molecules, that in most cases promote partition toward the top polymer-rich phase with recovery percentages as high as 86%. It has been found that both, the hydrophobicity and molecular weight of the modifying species play a preponderant role in conjugate partition behavior since as hydrophobicity increases partition is promoted towards the PEG-rich phase balancing the effect of the molecular weight of the modifying molecules that tends to shift partition towards the salt rich phase.

Halogenation Generates Effective Modulators of Amyloid-Beta Aggregation and Neurotoxicity

Halogenation of organic compounds plays diverse roles in biochemistry, including selective chemical modification of proteins and improved oral absorption/blood-brain barrier permeability of drug candidates. Moreover, halogenation of aromatic molecules greatly affects aromatic interaction-mediated self-assembly processes, including amyloid fibril formation. Perturbation of the aromatic interaction caused by halogenation of peptide building blocks is known to affect the morphology and other physical properties of the fibrillar structure. Consequently, in this article, we investigated the ability of halogenated ligands to modulate the self-assembly of amyloidogenic peptide/protein. As a model system, we chose amyloid-beta peptide (Aβ), which is implicated in Alzheimer’s disease, and a novel modulator of Aβ aggregation, erythrosine B (ERB). Considering that four halogen atoms are attached to the xanthene benzoate group in ERB, we hypothesized that halogenation of the xanthene benzoate plays a critical role in modulating Aβ aggregation and cytotoxicity. Therefore, we evaluated the modulating capacities of four ERB analogs containing different types and numbers of halogen atoms as well as fluorescein as a negative control. We found that fluorescein is not an effective modulator of Aβ aggregation and cytotoxicity. However, halogenation of either the xanthenes or benzoate ring of fluorescein substantially enhanced the inhibitory capacity on Aβ aggregation. Such Aβ aggregation inhibition by ERB analogs except rose bengal correlated well to the inhibition of Aβ cytotoxicity. To our knowledge, this is the first report demonstrating that halogenation of aromatic rings substantially enhance inhibitory capacities of small molecules on Aβ-associated neurotoxicity via Aβ aggregation modulation.

Shaking and stirring: Comparison of controlled laboratory stress conditions applied to the human growth hormone

Shaking or stirring in a miniaturized device is often applied in the development of protein pharmaceuticals, serving as a test of stability under physical stress conditions also occurring in the real process. The overall purpose of these “stress tests” is to accelerate protein aggregation that could otherwise take place at a much slower rate, thereby enhancing experimental throughput to speed up determination of critical process parameters of stability. Results are useful to determine critical parameters for formulation development. However, shaking differs from stirring in the forces applied on proteins in solution and therefore, there is the possibility that characteristics of protein stability interrogated with each method may not be the same. We have performed a detailed, time-resolved analysis of aggregation of the human growth hormone (hGH), exposed to stirring and shaking in a well-defined and comparable mini-reactor set-up. We show that aggregation of hGH, which in its native form is a monomer, is not the result of protein deamidation, a chemical protein modification that occurs in hGH under the incubation conditions used. We also show that both under stirring and shaking, hGH appears to precipitate into mainly spherical particles in the presence of only small amounts of soluble protein oligomers. The precipitate appears to contain protein that has a substantial amount of hydrophobic surface exposed. An air–liquid interface, constantly renewed by stirring or shaking, was identified to be mainly responsible for aggregation of hGH under each of the stress conditions applied. In hGH, therefore, controlled shaking and stirring lead to identical conclusions about the overall protein stability and the main critical parameter driving the aggregation, thus informing stabilization strategies and formulation development.

Fluorescent labeling of specific cysteine residues using CyMPL.

The unique reactivity and relative rarity of cysteine among amino acids makes it a convenient target for the site-specific chemical modification of proteins. Commercially available fluorophores and modifiers react with cysteine through a variety of electrophilic functional groups. However, it can be difficult to achieve specific labeling of a particular cysteine residue in a protein containing multiple cysteines, in a mixture of proteins, or in a protein's native environment. This unit describes a procedure termed CyMPL (Cysteine Metal Protection and Labeling), which enables specific labeling by incorporating a cysteine of interest into a minimal binding site for group 12 metal ions (e.g., Cd2+ and Zn2+). These sites can be inserted into any region of known secondary structure in virtually any protein and cause minimal structural perturbation. Bound metal ions protect the cysteine from reaction while background cysteines are covalently blocked with non-fluorescent modifiers. The metal ions are subsequently removed and the deprotected cysteine is labeled specifically.

ChIP–seq and beyond: new and improved methodologies to detect and characterize protein–DNA interactions

Chromatin immunoprecipitation experiments followed by sequencing (ChIP-seq) detect protein-DNA binding events and chemical modifications of histone proteins. Challenges in the standard ChIP-seq protocol have motivated recent enhancements in this approach, such as reducing the number of cells that are required and increasing the resolution. Complementary experimental approaches - for example, DNaseI hypersensitive site mapping and analysis of chromatin interactions that are mediated by particular proteins - provide additional information about DNA-binding proteins and their function. These data are now being used to identify variability in the functions of DNA-binding proteins across genomes and individuals. In this Review, I describe the latest advances in methods to detect and functionally characterize DNA-bound proteins.

Minimizing immunogenicity of biopharmaceuticals by controlling critical quality attributes of proteins

Adverse immune responses severely hamper the success of biopharmaceutical therapies. Possible clinical consequences include anaphylaxis, reduced drug half-life and neutralization of the therapeutic protein as well as the endogenous human homologue. Controlling potential triggers of the immune system helps to minimize the immunogenicity of biopharmaceuticals, a crucial consideration in biopharmaceutical manufacturing. This review summarizes the latest advancements that have been made towards insight into the impact of structural characteristics on the immunogenicity of therapeutic proteins. Examples are given to illustrate the role of critical quality attributes, such as protein conformation, glycosylation, chemical modifications and aggregation, in immunogenicity. During the development of biopharmaceutical products, it is important to not just assess the risk for immunogenicity in clinical trials, but to ensure product quality throughout drug design, cell-line selection, upstream and downstream processing, all the way to to the final product.

Epigenetic aspects in schizophrenia etiology and pathogenesis

Epidemiological evidence suggests that etiology of schizophrenia may involve both the influence of genetic factors specific for the individual and the impact of the environment. It is quite likely that a crucial role in the disease development is played by molecular mechanisms mediating the interaction between genes and environment. Modern research have shown that epigenetic mechanisms or chemical modifications of deoxyribonucleic acids (DNA) and histone proteins remain unstable throughout life and can be changed by environmental factors. Thus the epigenetic mechanisms outline an attractive molecular hypothesis of the environment modelling role and the environmental contribution to schizophrenia progression. We give in the present study a general outline of schizophrenia as a pathological entity and discuss the role and involvement of environment versus genetic determinant (nature versus nurture) in the pathophysiolgical processes. Additionally, we focus on DNA methylation discussing the evidence for the role of that process in schizophrenia. Thirdly, we review the post-translational histone modifications and their role in schizophrenia. These investigations might surely lead further to the development of epigenetic therapy that looks promising in regard to symptom alleviation and the disease-associated cognitive deficit.

Formation of S-(carboxymethyl)-cysteine in rat liver mitochondrial proteins: effects of caloric and methionine restriction

Maillard reaction contributes to the chemical modification and cross-linking of proteins. This process plays a significant role in the aging process and determination of animal longevity. Oxidative conditions promote the Maillard reaction. Mitochondria are the primary site of oxidants due to the reactive molecular species production. Mitochondrial proteome cysteine residues are targets of oxidative attack due to their specific chemistry and localization. Their chemical, non-enzymatic modification leads to dysfunctional proteins, which entail cellular senescence and organismal aging. Previous studies have consistently shown that caloric and methionine restrictions, nutritional interventions that increase longevity, decrease the rate of mitochondrial oxidant production and the physiological steady-state levels of markers of oxidative damage to macromolecules. In this scenario, we have detected S-(carboxymethyl)-cysteine (CMC) as a new irreversible chemical modification in mitochondrial proteins. CMC content in mitochondrial proteins significantly correlated with that of the lysine-derived analog N (ε)-(carboxymethyl)-lysine. The concentration of CMC is, however, one order of magnitude lower compared with CML likely due in part to the lower content of cysteine with respect to lysine of the mitochondrial proteome. CMC concentrations decreases in liver mitochondrial proteins of rats subjected to 8.5 and 25 % caloric restriction, as well as in 40 and 80 % methionine restriction. This is associated with a concomitant and significant increase in the protein content of sulfhydryl groups. Data presented here evidence that CMC, a marker of Cys-AGE formation, could be candidate as a biomarker of mitochondrial damage during aging.