Shotgun Proteomics and Protein-Based Bioinformatics for the Characterization of Food-Derived Bioactive Peptides.

A workflow for the characterization of food-derived bioactive peptides is described in this chapter. The workflow integrates two consecutive steps: a discovery phase and a protein-based bioinformatic phase. In the first step (discovery phase), a shotgun bottom-up proteomics approach is used to create a reference data set for a selected food proteome. Afterward, in a second step (bioinformatic phase), the reference proteome is subjected to several in silico protein-based bioinformatic analyses to predict and characterize potential bioactive peptides after an in silico human gastrointestinal digestion. Using this workflow, bioactive collagen peptides, antihypertensive, antimicrobial, and antitumor peptides were predicted as potential valuable bioactive peptides from seafood and marine by-products. It is concluded that the combination of the global shotgun proteomic analysis and the analysis by protein-based bioinformatics can provide a rapid strategy for the characterization of new potential food-derived bioactive peptides.

Serum proteomics of active tuberculosis patients and contacts reveals unique processes activated during Mycobacterium tuberculosis infection.

Tuberculosis (TB) is the most lethal infection among infectious diseases. The specific aim of this study was to establish panels of serum protein biomarkers representative of active TB patients and their household contacts who were either infected (LTBI) or uninfected (EMI-TB Discovery Cohort, Pontevedra Region, Spain). A TMT (Tamdem mass tags) 10plex-based quantitative proteomics study was performed in quintuplicate containing a total of 15 individual serum samples per group. Peptides were analyzed in an LC-Orbitrap Elite platform, and raw data were processed using Proteome Discoverer 2.1. A total of 418 proteins were quantified. The specific protein signature of active TB patients was characterized by an accumulation of proteins related to complement activation, inflammation and modulation of immune response and also by a decrease of a small subset of proteins, including apolipoprotein A and serotransferrin, indicating the importance of lipid transport and iron assimilation in the progression of the disease. This signature was verified by the targeted measurement of selected candidates in a second cohort (EMI-TB Verification Cohort, Maputo Region, Mozambique) by ELISA and nephelometry techniques. These findings will aid our understanding of the complex metabolic processes associated with TB progression from LTBI to active disease.

Molecular characterization of B-cell epitopes for the major fish allergen, parvalbumin, by shotgun proteomics, protein-based bioinformatics and IgE-reactive approaches.

Parvalbumins beta (ß-PRVBs) are the main fish allergens. The only proven and effective treatment for this type of hypersensitivity is to consume a diet free of fish. We present the molecular characterization of B-cell epitopes by shotgun proteomics of different ß-PRVBs combined with protein-based bioinformatics and IgE-reactive approaches. The final goal of this work is to identify potential peptide vaccine candidates for fish allergy. Purified ß-PRVBs from the main fifteen different fish species that cause allergy were analyzed by shotgun proteomics. Identified ß-PRVBs peptide sequences and ninety-eight ß-PRVB protein sequences from UniProtKB were combined, aligned and analyzed to determine B-cell epitopes using the Kolaskar and Tongaonkar algorithm. The highest rated predicted B-cell peptide epitopes were evaluated by ELISA using the corresponding synthetic peptides and sera from healthy and fish allergic patients. A total of 35 peptides were identified as B-cell epitopes. The top B-cell peptide epitopes (LKLFLQV, ACAHLCK, FAVLVKQ and LFLQNFV) that may induce protective immune responses were selected as potential peptide vaccine candidates. The 3D model of these peptides were located in the surface of the protein. This study provides the global characterization of B-cell epitopes for all ß-PRVBs sequences that will facilitate the design of new potential immunotherapies. SIGNIFICANCE: This work provides the global characterization of B-cell epitopes for all ß-PRVBs sequences by Shotgun Proteomics combined with Protein-based Bioinformatics and IgE-reactive approaches. This study will increase our understanding of the molecular mechanisms whereby fish allergens elicit allergic reactions and will facilitate the design of new potential peptide vaccine candidates.

Proteome profiling of L3 and L4 Anisakis simplex development stages by TMT-based quantitative proteomics.

Anisakis simplex is a parasitic nematode that can cause anisakiosis and/or allergic reactions in humans. The presence of invasive third-stage larvae (L3) in many different consumed fish species and the fourth-stage larvae (L4) in marine mammals, where L3 can accidentally affect to humans and develop as far as stage L4. World Health Organization and food safety authorities aim to control and prevent this emerging health problem. In the present work, using Tandem Mass Tag (TMT)-based quantitative proteomics we analyzed for the first time the global proteome of two A. simplex development stages, L3 and L4. The strategy was divided into four steps: (a) protein extraction of L3 and L4 development stages, (b) high intensity focused ultrasound (HIFU)-assisted trypsin digestion, (c) TMT-isobaric mass tag labeling following by high-pH reversed-phase fractionation, and (d) LC-MS/MS analysis in a LTQ-Orbitrap Elite mass spectrometer. A total of 2443 different proteins of A. simplex were identified. Analysis of the modulated proteins provided the specific proteomic signature of L3 (i.e. pseudocoelomic globin, endochitinase 1, paramyosin) and L4 (i.e. neprilysin-2, glutamate dehydrogenase, aminopeptidase N). To our knowledge, this is the most comprehensive dataset of proteins of A. simplex for two development stages (L3 and L4) identified to date. SIGNIFICANCE: A. simplex is a fish-borne parasite responsible for the human anisakiosis and allergic reactions around the world. The work describes for the first-time the comparison of the proteome of two A. simplex stages (L3 and L4). The strategy is based on four steps: (i) protein extraction, (ii) ultra-fast trypsin digestion under High-Intensity Focused Ultrasound (HIFU), (iii) TMT-isobaric mass tag labeling followed by high-pH reversed-phase fractionation and (iv) peptide analysis using a LTQ-Orbitrap Elite mass spectrometer. The workflow allows to select the most modulated proteins as proteomic signature of those specific development stages (L3 and L4) of A. simplex. Obtained stage-specific proteins, could be used as targets to control/eliminate this parasite and in future eradicate the anisakiosis disease.

High-resolution quantitative proteomics applied to the study of the specific protein signature in the sputum and saliva of active tuberculosis patients and their infected and uninfected contacts.

Our goal was to establish panels of protein biomarkers that are characteristic of patients with microbiologically confirmed pulmonary tuberculosis (TB) and their contacts, including latent TB-infected (LTBI) and uninfected patients. Since the first pathogen-host contact occurs in the oral and nasal passages the saliva and sputum were chosen as the biological fluids to be studied. Quantitative shotgun proteomics was performed using a LTQ-Orbitrap-Elite platform. For active TB patients, both fluids exhibited a specific accumulation of proteins that were related to complement activation, inflammation and modulation of immune response. In the saliva of TB patients, a decrease of in proteins related to glucose and lipid metabolism was detected. In contrast, the sputum of uninfected contacts presented a specific proteomic signature that was composed of proteins involved in the perception of bitter taste, defense against pathogens and innate immune response, suggesting that those are key events during the initial entry of the pathogen in the host. SIGNIFICANCE: This is the first study to compare the saliva and sputum from active TB patients and their contacts. Our findings strongly suggest that TB patients show not only an activation of processes that are related to complement activation and modulation of inflammation but also an imbalance in carbohydrate and lipid metabolism. In addition, those individuals who do not get infected after direct exposure to the pathogen display a typical proteomic signature in the sputum, which is a reflection of the secretion from the nasal and oral mucosa, the first immunological barriers that M. tuberculosis encounters in the host. Thus, this result indicates the importance of the processes related to the innate immune response in fighting the initial events of the infection.

A lipidomic study on the regulation of inflammation and oxidative stress targeted by marine ω-3 PUFA and polyphenols in high-fat high-sucrose diets.

The ability of polyphenols to ameliorate potential oxidative damage of ω-3 PUFAs when they are consumed together and then, to enhance their potentially individual effects on metabolic health is discussed through the modulation of fatty acids profiling and the production of lipid mediators. For that, the effects of the combined consumption of fish oils and grape seed procyanidins on the inflammatory response and redox unbalance triggered by high-fat high-sucrose (HFHS) diets were studied in an animal model of Wistar rats. A standard diet was used as control. Results suggested that fish oils produced a replacement of ω-6 by ω-3 PUFAs in membranes and tissues, and consequently they improved inflammatory and oxidative stress parameters: favored the activity of 12/15-lipoxygenases on ω-3 PUFAs, enhanced glutathione peroxidases activity, modulated proinflammatory lipid mediators synthesis through the cyclooxygenase (COX) pathways and down-regulated the synthesis de novo of ARA leaded by Δ5 desaturase. Although polyphenols exerted an antioxidative and antiinflammatory effect in the standard diet, they were less effective to reduce inflammation in the HFHS dietary model. Contrary to the effect observed in the standard diet, polyphenols up-regulated COX pathways toward ω-6 proinflammatory eicosanoids as PGE2 and 11-HETE and decreased the detoxification of ω-3 hydroperoxides in the HFHS diet. As a result, additive effects between fish oils and polyphenols were found in the standard diet in terms of reducing inflammation and oxidative stress. However, in the HFHS diets, fish oils seem to be the one responsible for the positive effects found in the combined group.

Determination of the Geographical Origin of All Commercial Hake Species by Stable Isotope Ratio (SIR) Analysis.

The determination of the geographical origin of food products is relevant to comply with the legal regulations of traceability, to avoid food fraud, and to guarantee food quality and safety to the consumers. For these reasons, stable isotope ratio (SIR) analysis using an isotope ratio mass spectrometry (IRMS) instrument is one of the most useful techniques for evaluating food traceability and authenticity. The present study was aimed to determine, for the first time, the geographical origin for all commercial fish species belonging to the Merlucciidae family using SIR analysis of carbon (δ13C) and nitrogen (δ15N). The specific results enabled their clear classification according to the FAO (Food and Agriculture Organization of the United Nations) fishing areas, latitude, and geographical origin in the following six different clusters: European, North African, South African, North American, South American, and Australian hake species.

Changes in liver proteins of rats fed standard and high-fat and sucrose diets induced by fish omega-3 PUFAs and their combination with grape polyphenols according to quantitative proteomics.

This study considered the physiological modulation of liver proteins due to the supplementation with fish oils under two dietary backgrounds: standard or high in fat and sucrose (HFHS), and their combination with grape polyphenols. By using a quantitative proteomics approach, we showed that the capacity of the supplements for regulating proteins depended on the diet; namely, 10 different proteins changed into standard diets, while 45 changed into the HFHS diets and only scarcely proteins were found altered in common. However, in both contexts, fish oils were the main regulatory force, although the addition of polyphenols was able to modulate some fish oils' effects. Moreover, we demonstrated the ability of fish oils and their combination with grape polyphenols in improving biochemical parameters and reducing lipogenesis and glycolysis enzymes, enhancing fatty acid beta-oxidation and insulin signaling and ameliorating endoplasmic reticulum stress and protein oxidation when they are included in an unhealthy diet.

Characterization of Foodborne Strains of Staphylococcus aureus by Shotgun Proteomics: Functional Networks, Virulence Factors and Species-Specific Peptide Biomarkers.

In the present work, we applied a shotgun proteomics approach for the fast and easy characterization of 20 different foodborne strains of Staphylococcus aureus (S. aureus), one of the most recognized foodborne pathogenic bacteria. A total of 644 non-redundant proteins were identified and analyzed via an easy and rapid protein sample preparation procedure. The results allowed the differentiation of several proteome datasets from the different strains (common, accessory, and unique datasets), which were used to determine relevant functional pathways and differentiate the strains into different Euclidean hierarchical clusters. Moreover, a predicted protein-protein interaction network of the foodborne S. aureus strains was created. The whole confidence network contains 77 nodes and 769 interactions. Most of the identified proteins were surface-associated proteins that were related to pathways and networks of energy, lipid metabolism and virulence. Twenty-seven virulence factors were identified, and most of them corresponded to autolysins, N-acetylmuramoyl-L-alanine amidases, phenol-soluble modulins, extracellular fibrinogen-binding proteins and virulence factor EsxA. Potential species-specific peptide biomarkers were screened. Twenty-one species-specific peptide biomarkers, belonging to eight different proteins (nickel-ABC transporter, N-acetylmuramoyl-L-alanine amidase, autolysin, clumping factor A, gram-positive signal peptide YSIRK, cysteine protease/staphopain, transcriptional regulator MarR, and transcriptional regulator Sar-A), were proposed to identify S. aureus. These results constitute the first major dataset of peptides and proteins of foodborne S. aureus strains. This repository may be useful for further studies, for the development of new therapeutic treatments for S. aureus food intoxications and for microbial source-tracking in foodstuffs.

Protein biomarker discovery and fast monitoring for the identification and detection of Anisakids by parallel reaction monitoring (PRM) mass spectrometry.

UNLABELLED: Anisakids are fish-borne parasites that are responsible for a large number of human infections and allergic reactions around the world. World health organizations and food safety authorities aim to control and prevent this emerging health problem. In the present work, a new method for the fast monitoring of these parasites is described. The strategy is divided in three steps: (i) purification of thermostable proteins from fish-borne parasites (Anisakids), (ii) in-solution HIFU trypsin digestion and (iii) monitoring of several peptide markers by parallel reaction monitoring (PRM) mass spectrometry. This methodology allows the fast detection of Anisakids in <2h. An affordable assay utilizing this methodology will facilitate testing for regulatory and safety applications. SIGNIFICANCE: The work describes for the first time, the Protein Biomarker Discovery and the Fast Monitoring for the identification and detection of Anisakids in fishery products. The strategy is based on the purification of thermostable proteins, the use of accelerated in-solution trypsin digestions under an ultrasonic field provided by High-Intensity Focused Ultrasound (HIFU) and the monitoring of several peptide biomarkers by Parallel Reaction Monitoring (PRM) Mass Spectrometry in a linear ion trap mass spectrometer. The workflow allows the unequivocal detection of Anisakids, in <2h. The present strategy constitutes the fastest method for Anisakids detection, whose application in the food quality control area, could provide to the authorities an effective and rapid method to guarantee the safety to the consumers.

Quantification of proteome changes in bovine muscle from two-dimensional electrophoresis data.

Proteome changes in the longissimus thoracis bovine muscle in response to pre-slaughter stress were assessed on the basis of two-dimensional electrophoresis (2-DE) data. In this study, the bootstrap resampling statistical technique and a new measure of relative change of the volume of 2-DE protein spots are shown to be more efficient than commonly used statistics to reliably quantify changes in protein abundance in stress response. The data are supplied in this article and are related to "Tackling proteome changes in the longissimus thoracis bovine muscle in response to pre-slaughter stress" by Franco et al. [1].

Tackling proteome changes in the longissimus thoracis bovine muscle in response to pre-slaughter stress.

Pre-slaughter stress has adverse effects on meat quality that can lead to the occurrence of Dark Firm Dry (DFD) meat in cattle. This study explores the previously uncharacterized proteome changes linked to pre-slaughter stress in the longissimus thoracis (LT) bovine muscle. Differential proteome profiles of DFD and normal (non-DFD) LT meat samples from male calves of the Rubia Gallega breed were assessed by 2-DE coupled to MS analysis (LC-MS/MS and MALDI TOF/TOF MS). A total of seven structural-contractile proteins (three different myosin light chain isoforms, two fast skeletal myosin light chain 2 isoforms, troponin C type 2 and cofilin-2) and three metabolism enzymes (triosephosphate isomerase, ATP synthase and beta-galactoside alpha-2,6-sialyltransferase) were found to have statistically significant differential abundance in sample groups. In addition, 2-DE in combination with the phosphoprotein-specific fluorescent dye Pro-Q DPS revealed that highly phosphorylated fast skeletal myosin regulatory light chain 2 isoforms underwent the most intense relative change in muscle conversion to DFD meat. Therefore, they appear to be the most sensitive biomarkers of stress just prior to slaughter in Rubia Gallega. Overall, these findings will facilitate a more integrative understanding of the biochemical processes associated with stress in cattle muscle and their effects in meat quality. BIOLOGICAL SIGNIFICANCE: Pre-slaughter stress is a crucial factor in meat production. Animals destined for slaughter are stressed by a variety of endogenous and exogenous factors that negatively affect the complex post-mortem biochemical events underlying the conversion of muscle into meat. The study of the muscle proteome has a great relevance for understanding the molecular mechanisms associated with stress. However, there is no information available on the molecular changes linked to pre-slaughter stress in cattle on the proteome scale. Our study led to the identification of a number of candidate proteins associated with the response to pre-slaughter stress in the LT bovine muscle of the Rubia Gallega breed. The functions of those significantly changed proteins have a clear biological relationship with stress response. These findings contribute to a deeper insight into the molecular pathways that respond to stress in cattle.

Investigation of production method, geographical origin and species authentication in commercially relevant shrimps using stable isotope ratio and/or multi-element analyses combined with chemometrics: an exploratory analysis.

Three factors defining the traceability of a food product are production method (wild or farmed), geographical origin and biological species, which have to be checked and guaranteed, not only in order to avoid mislabelling and commercial fraud, but also to address food safety issues and to comply with legal regulations. The aim of this study was to determine whether these three factors could be differentiated in shrimps using stable isotope ratio analysis of carbon and nitrogen and/or multi-element composition. Different multivariate statistics methods were applied to different data subsets in order to evaluate their performance in terms of classification or predictive ability. Although the success rates varied depending on the dataset used, the combination of both techniques allowed the correct classification of 100% of the samples according to their actual origin and method of production, and 93.5% according to biological species. Even though further studies including a larger number of samples in each group are needed in order to validate these findings, we can conclude that these methodologies should be considered for studies regarding seafood product authenticity.

Protein carbonylation associated to high-fat, high-sucrose diet and its metabolic effects.

The present research draws a map of the characteristic carbonylation of proteins in rats fed high-caloric diets with the aim of providing a new insight of the pathogenesis of metabolic diseases derived from the high consumption of fat and refined carbohydrates. Protein carbonylation was analyzed in plasma, liver and skeletal muscle of Sprague-Dawley rats fed a high-fat, high-sucrose (HFHS) diet by a proteomics approach based on carbonyl-specific fluorescence-labeling, gel electrophoresis and mass spectrometry. Oxidized proteins along with specific sites of oxidative damage were identified and discussed to illustrate the consequences of protein oxidation. The results indicated that long-term HFHS consumption increased protein oxidation in plasma and liver; meanwhile, protein carbonyls from skeletal muscle did not change. The increment of carbonylation by HFHS diet was singularly selective on specific target proteins: albumin from plasma and liver, and hepatic proteins such as mitochondrial carbamoyl-phosphate synthase (ammonia), mitochondrial aldehyde dehydrogenase, argininosuccinate synthetase, regucalcin, mitochondrial adenosine triphosphate synthase subunit beta, actin cytoplasmic 1 and mitochondrial glutamate dehydrogenase 1. The possible consequences that these specific protein carbonylations have on the excessive weight gain, insulin resistance and nonalcoholic fatty liver disease resulting from HFHS diet consumption are discussed.

Lipidomic analysis of polyunsaturated fatty acids and their oxygenated metabolites in plasma by solid-phase extraction followed by LC-MS.

The present work describes the development of a robust and sensitive targeted analysis platform for the simultaneous quantification in blood plasma of lipid oxygenated mediators and fatty acids using solid-phase extraction (SPE) and high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). The concurrent analysis of these lipid mediators is challenging because of their instability, differences in solubility, and the frequent occurrence of isobaric forms with similar fragmentation patterns. Results demonstrated that the reduction of SPE temperature to 4 °C is a critical parameter for preserving the hydroperoxy derivatives. Polymeric HLB cartridges increased 40-50 % ARA, EPA, and DHA sensitivity compared to C18 sorbent and also provided higher global performance for most hydroxides and other oxidation products. The proposed method for the two tested mass analyzers yields high sensitivity, good linearity, and reproducibility, with detection limits ranging 0.002-7 ng/mL and global recoveries as high as 85-112 %. However, the additional advantage of the linear ion trap (LIT) mass analyzer working in full scan product ion mode, compared to the triple quadrupole (QqQ) operating in multiple reaction monitoring (MRM), should be noted: the full scan product ion mode provides the full fragmentation spectra of compounds that allowed the discrimination of coeluting isomers and false positive identifications without additional chromatography development. The proposed lipidomic procedure demonstrates a confident, simple, and sensitive method to profile in plasma a wide range of lipid eicosanoid and docosanoid mediators, including innovatively the analysis of hydroperoxy congeners and nonoxidized PUFA precursors.

Protective effect of the omega-3 polyunsaturated fatty acids: Eicosapentaenoic acid/Docosahexaenoic acid 1:1 ratio on cardiovascular disease risk markers in rats.

BACKGROUND: High consumption of fish carries a lower risk of cardiovascular disease as a consequence of dietary omega-3 long chain polyunsaturated fatty acid (n-3 PUFA; especially EPA and DHA) content. A controversy exists about the component/s responsible of these beneficial effects and, in consequence, which is the best proportion between both fatty acids. We sought to determine, in healthy Wistar rats, the proportions of EPA and DHA that would induce beneficial effects on biomarkers of oxidative stress, and cardiovascular disease risk. METHODS: Female Wistar rats were fed for 13 weeks with 5 different dietary supplements of oils; 3 derived from fish (EPA/DHA ratios of 1:1, 2:1, 1:2) plus soybean and linseed as controls. The activities of major antioxidant enzymes (SOD, CAT, GPX, and GR) were determined in erythrocytes and liver, and the ORAC test was used to determine the antioxidant capacity in plasma. Also measured were: C reactive protein (CRP), endothelial dysfunction (sVCAM and sICAM), prothrombotic activity (PAI-1), lipid profile (triglycerides, cholesterol, HDLc, LDLc, Apo-A1, and Apo-B100), glycated haemoglobin and lipid peroxidation (LDL-ox and MDA values). RESULTS: After three months of nutritional intervention, we observed statistically significant differences in the ApoB100/ApoA1 ratio, glycated haemoglobin, VCAM-1, SOD and GPx in erythrocytes, ORAC values and LDL-ox. Supplementation with fish oil derived omega-3 PUFA increased VCAM-1, LDL-ox and plasma antioxidant capacity (ORAC). Conversely, the ApoB100/ApoA1 ratio and percentage glycated haemoglobin decreased. CONCLUSIONS: Our results showed that a diet of a 1:1 ratio of EPA/DHA improved many of the oxidative stress parameters (SOD and GPx in erythrocytes), plasma antioxidant capacity (ORAC) and cardiovascular risk factors (glycated haemoglobin) relative to the other diets.

Identification and classification of seafood-borne pathogenic and spoilage bacteria: 16S rRNA sequencing versus MALDI-TOF MS fingerprinting.

The present study aims to compare two molecular technologies, 16S rRNA sequencing and MALDI-TOF MS, for bacterial species identification in seafood. With this aim, 70 reference strains from culture collections, including important seafood-borne pathogenic and spoilage bacterial species, and 50 strains isolated from commercial seafood products, were analysed by both techniques. Genomic analysis only identified the species of 50% of the isolated strains, proving to be particularly poor at identifying members of the Pseudomonas and Bacillus genera. In contrast, MALDI-TOF MS fingerprinting identified 76% of the strains at the species level. The mass spectral data were submitted to the SpectraBank database (http://www.spectrabank.org), making this information available to other researchers. Furthermore, cluster analysis of the peak mass lists was carried out with the web application SPECLUST and the calculated groupings were consistent with results determined by a phylogenetic approach that is based on the 16S rRNA sequences. However, the MALDI-TOF MS analysis demonstrated more discriminating potential that allowed for better classification, especially for the Pseudomonas and Bacillus genera. This is of importance with respect to the varying pathogenic and spoilage character at the intragenus and intraspecies level. In this sense, MALDI-TOF MS demonstrated to be a competent bacterial typing tool that extends phenotypic and genotypic approaches, allowing a more ample classification of bacterial strains.

Proteomic evaluation of myofibrillar carbonylation in chilled fish mince and its inhibition by catechin.

The present study investigates the susceptibility of individual myofibrillar proteins from mackerel (Scomber scombrus) mince to undergo carbonylation reactions during chilled storage, and the antioxidant capacity of (+)-catechin to prevent oxidative processes of proteins. The carbonylation of each particular protein was quantified by combining the labelling of protein carbonyls by fluorescein-5-thiosemicarbazide (FTSC) with 1-D or 2-D gel electrophoresis. Alpha skeletal actin, glycogen phosphorylase, unnamed protein product (UNP) similar to enolase, pyruvate kinase, isoforms of creatine kinase, aldolase A and an isoform of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) showed elevated oxidation in chilled non-supplemented mince. Myosin heavy chain (MHC) was not carbonylated in chilled muscle, but an extensive MHC degradation was observed in those samples. The supplementation of catechin reduced protein oxidation and lipid oxidation in a concentration-dependent manner: control>25>100≈200ppm. Therefore, the highest catechin concentrations (100 and 200ppm) exhibited the strongest antioxidant activity. Catechin (200ppm) reduced significantly carbonylation of protein spots identified as glycogen phosphorylase, pyruvate kinase muscle isozyme, isoforms of creatine kinase. Conversely, catechin was ineffective to inhibit the oxidation of actin and UNP similar to enolase. These results draw attention to the inefficiency of catechin to prevent actin oxidation, in contrast to the extremely high efficiency of catechin in inhibiting oxidation of lipids and other proteins.