PARTIAL PURIFICATION OF GLUTATHIONE PEROXIDASE ENZYME FROM WOMEN WITH BREAST CANCER.

The study included the purification of glutathione peroxidase enzyme (GPX) in the serum of women with breast cancer, which involved 60 samples of serum from women with breast cancer, and 30 samples from healthy individuals. The results of the study showed a significant decrease at a probability level of p<0.0001 for the activity of the GPX enzyme in the serum of women with breast cancer. Additionally, the GPX enzyme was purified from the serum of women with breast cancer through precipitation with ammonium sulfate and dialysis, and the use of DEAE-Cellulose ion exchange chromatography and gel filtration chromatography using Sephadex G-100, where a main protein band was separated, which was relied upon in determining the optimal conditions for the partially purified enzyme. The optimal conditions for the partially purified enzyme from the serum of women with breast cancer were determined and the highest activity was for the substrate concentration of 0.1 mM H2O2. The maximum speed Vmax was 3.125IU/L and the Michaelis-Menten constant Km was 0.0179 M using Lineweaver-Burk plot, the optimal pH was at 8.5, temperature at 37°C, and the highest activity time was at 5 minutes.

Molecular mechanism investigation on the interactions of copper (II) ions with glutathione peroxidase 6 from Arabidopsis thaliana.

Accumulation of copper (II) ions in plant leads to the excessive reactive oxygen species (ROS) which attributes to the depletion of the antioxidants in the cell and destruction to antioxidant enzymes. The antioxidant enzyme glutathione peroxidase has been used as biomarkers to reflect metal-induced oxidative stress. However, the underlying toxic mechanisms of the copper ions(II)-induced oxidative damage to plants remain unknown. In the work, a detailed molecular interaction of copper (II) ions with Arabidopsis thaliana glutathione peroxidase 6 (AtGPX6) in relation with poisonous effects of exposure to heavy metal was investigated by multiple spectroscopic techniques. The intrinsic fluorescence of AtGPX6 was quenched upon the addition of copper (II) ions by the combination of static and dynamic quenching mechanisms accompanied by complex formation and conformational changes. A single binding site was revealed for AtGPX6 towards copper ions. The binding process was hydrophobic effect accompanied by positive entropy change and enthalpy change. The secondary structure of AtGPX6 was changed by the addition of copper ions investigated by synchronous fluorescence spectroscopy, ultraviolet-visible absorption spectroscopy and circular dichroism spectroscopy, resulting in loosened and deconstructed protein skeleton and increased hydrophobicity around the Trp residues. This study helps to illuminate the detailed interactions between copper ions and plant glutathione peroxidase and elucidate the destructive mechanism to antioxidative defense system caused by heavy metal exposure at molecular level.

Glutathione peroxidase 4-catalyzed reduction of lipid hydroperoxides in membranes: The polar head of membrane phospholipids binds the enzyme and addresses the fatty acid hydroperoxide group toward the redox center.

GPx4 is a monomeric glutathione peroxidase, unique in reducing the hydroperoxide group (-OOH) of fatty acids esterified in membrane phospholipids. This reaction inhibits lipid peroxidation and accounts for enzyme's vital role. Here we investigated the interaction of GPx4 with membrane phospholipids. A cationic surface near the GPx4 catalytic center interacts with phospholipid polar heads. Accordingly, SPR analysis indicates cardiolipin as the phospholipid with maximal affinity to GPx4. Consistent with the electrostatic nature of the interaction, KCl increases the KD. Molecular dynamic (MD) simulation shows that a -OOH posed in the core of the membrane as 13 - or 9 -OOH of tetra-linoleoyl cardiolipin or 15 -OOH stearoyl-arachidonoyl-phosphaphatidylcholine moves to the lipid-water interface. Thereby, the -OOH groups are addressed toward the GPx4 redox center. In this pose, however, the catalytic site facing the membrane would be inaccessible to GSH, but the consecutive redox processes facilitate access of GSH, which further primes undocking of the enzyme, because GSH competes for the binding residues implicated in docking. During the final phase of the catalytic cycle, while GSSG is produced, GPx4 is disconnected from the membrane. The observation that GSH depletion in cells induces GPx4 translocation to the membrane, is in agreement with this concept.

Influence of Selenium Content in the Culture Medium on Protein Profile of Yeast Cells Candida utilis ATCC 9950.

Selenium is an essential trace element for human health and it has been recognized as a component of several selenoproteins with crucial biological functions. It has been identified as a component of active centers of many enzymes, as well as integral part of biologically active complexes. The aim of the study was to evaluate the protein content and amino acid profile of the protein of fodder yeast Candida utilis ATCC 9950 cultured in media control and experimental enriched selenium. Protein analysis was performed using SDS-PAGE method consisting of polyacrylamide gel electrophoresis in the presence of SDS. The highest contents of soluble protein (49,5 mg/g) were found in yeast cells after 24-hour culture conducted in control (YPD) medium. In the presence of selenium there were determined small amounts of protein content. With increasing time of yeast culture (to 72 hours) the control and experimental media were reported to reduce soluble protein content. In electropherogram proteins from control cultures was observed the presence of 10 protein fractions, but in all the experimental cultures (containing 20, 30, and 40 mg/L selenium) of 14 protein fractions. On the basis of the molecular weights of proteins, it can be concluded that they were among others: selenoprotein 15 kDa and selenoprotein 18 kDa.

Identification and characterization of two phospholipid hydroperoxide glutathione peroxidase genes from the Mediterranean species of the whitefly Bemisia tabaci complex.

Phospholipid hydroperoxide glutathione peroxidases (PHGPXs) are essential enzymes of the cellular antioxidant defense system during insect-plant interactions. However, little attention has been devoted to the functional characterization of PHGXPs in the whitefly Bemisia tabaci. Here, we report the identification and characterization of two PHGPX genes, designated as BtQ-PHGPX1 and BtQ-PHGPX2 from the Mediterranean species of the B. tabaci complex. Sequence analysis indicated that the length of BtQ-PHGPX1 is of 942 bp with a 729 bp open-reading frame (ORF) encoding 242 amino acids, and BtQ-PHGPX2 is of 699 bp with a 567 bp ORF encoding 188 amino acids. Sequence alignment analysis showed that BtQ-PHGPX1 and BtQ-PHGPX2 shared high similarity with other known PHGPXs. The NVASXCGXT, FPCNQFXXQEPG, and IKWNFXKFLV surrounded the reactive cysteine, glutamine, and tryptophan residues, respectively. Recombinant BtQ-PHGPX1 and BtQ-PHGPX2 were overexpressed in Escherichia coli and purified. quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis with whiteflies of different development stages showed that the mRNA levels of BtQ-PHGPX2 were significantly higher in larvae than in other stages. The mRNA levels of BtQ-PHGPX2 were significantly higher than BtQ-PHGPX1 during all the developmental stages. The mRNA levels of BtQ-PHGPX1 and BtQ-PHGPX2 in female adults were relatively higher than in male adults. The expression of BtQ-PHGPX1 and BtQ-PHGPX2 was induced by the insecticide imidacloprid. These results suggest that BtQ-PHGPX1 and BtQ-PHGPX2 may participate in detoxification of oxidative hazards in B. tabaci.

Characterization and structural analysis of human selenium-dependent glutathione peroxidase 4 mutant expressed in Escherichia coli.

Glutathione peroxidase 4 (GPx4) is a monomeric selenium-dependent glutathione peroxidase highly expressed in mammalian cells, which can reduce phospholipid hydroperoxides. However, it has been difficult to express recombinant mammalian GPx4 in Escherichia coli because of the differences in the selenocysteine (Sec) incorporation machinery between eukaryotes and prokaryotes. In this study, an E. coli BL21(DE3)cys auxotrophic strain was used to express GPx4 mutants. We found that untargeted substitution of Cys-2, Cys-37, Cys-75, Cys-107, and Cys-148 with Sec led to loss of activity, suggesting that mutation of any of these Cys residues in GPx4 could result in a structural change. Additionally, we found that the catalytic activity of GPx4 mutants increased as the number of noncatalytic Sec residues decreased, indicating that the negative effects could be mitigated by replacing these Cys residues with Ser residues. A GPx4 mutant with all Cys residues converted to Ser exhibited a "Ping-Pong" mechanism and structure similar to that of native GPx4, indicating that it could act as a substitute for GPx4, when heterologously expressing the protein in E. coli. This research provides an important foundation for biosynthesis of selenium-dependent GPx4 mutants in E. coli.

Oxidative profiles of endurance horses racing different distances / Perfil oxidativo em cavalos de enduro competindo em diferentes distâncias

Increased oxidative stress during prolonged endurance exercises may result in muscle damage, fatigue and decreased performance. An adequate stress response during training is critical to obtain improved results and high animal welfare standards. The aim of this study was to evaluate the red blood cell haemolysate concentrations of superoxide dismutase (SOD), glutathione peroxidase (GPx), reduced glutathione (GSH) and catalase (CAT) and the plasma concentrations of malondialdehyde (MDA) from endurance horses in different distances at high speed in a tropical climate. Fifteen horses were tested; five at 160km (18.54 - 17.16km/h race speed), five at 120km (21.53 - 17km/h race speed) and five at 80km (20.06 - 18.01km/h race speed). Blood samples were collected at rest, immediately after and three hours after the horses left the final vet check and three, seven and fourteen days after the race. No significant increases (P > 0.05) in the levels of SOD, GPx, GSH, CAT or MDA were observed for any of the times or distances examined. Based on these observations, we conclude that reactive oxygen species (ROS) formation during exercise evokes specific adaptations, such as increased antioxidant/oxidative damage-repairing enzyme activity, increased resistance to oxidative stress and lower levels of oxidative damage...

Aumento do estresse oxidativo durante o exercício prolongado pode resultar em fadiga muscular, lesões e diminuição do desempenho. Uma adequada resposta a esse estresse durante o treinamento é fundamental para a obtenção de melhores resultados e bem-estar dos animais. O objetivo deste estudo foi avaliar a concentração de superóxido dismutase (SOD), glutationa peroxidase (GPx), glutationa reduzida (GSH) e catalase (CAT) no hemolisado sanguíneo e malondialdeído (MDA) plasmático em cavalos de enduro correndo em diferentes distâncias, com alta média de velocidade, em clima tropical. Quinze cavalos foram testados, cinco em 160km (18.54-17.16km/h), cinco em 120km (21.53-17km/h) e cinco em 80km (20.06-18.01km/h). Amostras de sangue foram coletadas em repouso, imediatamente e três horas depois que os cavalos passaram pela inspeção veterinária final e três, sete e 14 dias após a corrida. Não houve aumentos significativos (P>0,05) dos níveis de SOD, GPx, GSH, CAT ou MDA em nenhum tempo nem distâncias analisadas. Com base nessas observações, pode-se concluir que as espécies reativas de oxigênio (ROS) formadas durante o exercício provocam adaptações específicas, tais como atividade antioxidante aumentada da enzima, maior resistência ao estresse oxidativo e menores níveis de danos oxidativos...

GPX3 from Arabidopsis thaliana: cloning, expression, purification, crystallization and preliminary X-ray analysis.

The Arabidopsis thaliana glutathione peroxidase 3 (GPX3) gene encodes a glutathione peroxidase with roles in H2O2 homeostasis and signalling. The GPX3 gene sequence was cloned into pGEX-6P1 and overexpressed in Escherichia coli. The GPX3 protein was purified to homogeneity in two chromatographic steps. Various lengths of the GPX3 sequence were used to obtain proteins that yielded crystals using vapour-diffusion techniques, but only GPX3ΔN36 (lacking 36 amino acids from the N-terminus) showed a good diffraction pattern. Its crystals diffracted to 2.8 Šresolution and belonged to space group P65, with unit-cell parameters a = b = 98.241, c = 42.057 Å.

Relationship between Paratuberculosis and the microelements Copper, Zinc, Iron, Selenium and Molybdenum in Beef Cattle

To study the deficiency of minerals and its relationship with Paratuberculosis, blood, serum, and fecal samples were obtained from 75 adult bovines without clinical symptoms of the disease and from two bovines with clinical symptoms of the disease, from two beef herds with a previous history of Paratuberculosis in the Province of Buenos Aires, Argentina. Serum samples were processed by ELISA and feces were cultured in Herrolds medium. Copper, zinc and iron in serum were quantified by spectrophotometry and selenium was measured by the activity of glutathione peroxidase. We also determined copper, zinc, iron and molybdenum concentrations in pastures and the concentration of sulfate in water. Mycobacterium avium subsp paratuberculosis (Map) was isolated from 17.3% of fecal samples of asymptomatic animals and from the fecal samples from the two animals with clinical symptoms. All the Map-positive animals were also ELISA-positive or suspect, and among them, 84.6% presented low or marginal values of selenium and 69.2% presented low or marginal values of copper. The two animals with clinical symptoms, and isolation of Map from feces and organs were selenium-deficient and had the lowest activity of glutathione peroxidase of all the animals from both herds. All the animals negative to Map in feces and negative to ELISA had normal values of Se, while 13.8% of animals with positive ELISA or suspect and culture negative presented low levels of Se. Half of the animals that were negative both for ELISA and culture in feces were deficient in copper but none of them presented low values of selenium. The content of molybdenum and iron in pasture was high, 2.5 ppm and 1.13 ppm in one herd and 2.5 ppm and 2.02 ppm in the other, respectively, whereas the copper:molybdenum ratio was 1.5 and 5.2, respectively. These results do not confirm an interaction between imbalances of the micronutrients and clinical Paratuberculosis, but show evidence of the relationship between selenium...

Identification and cloning of a selenium-dependent glutathione peroxidase from tiger shrimp, Penaeus monodon, and its transcription following pathogen infection and related to the molt stages.

Complementary (c)DNA encoding glutathione peroxidase (GPx) messenger (m)RNA of the tiger shrimp Penaeus monodon was obtained from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) method. The 1321-bp cDNA contained an open reading frame (ORF) of 564bp, a 69-bp 5'-untranslated region (UTR), and a 688-bp 3'-UTR containing a poly A tail and a conserved selenocysteine insertion sequence (SECIS) element. The molecular mass of the deduced amino acid (aa) sequence (188 aa) was 21.05kDa long with an estimated pI of 7.68. It contains a putative selenocysteine residue which is encoded by the unusual stop codon, (190)TGA(192), and forms the active site with residues Glu(75) and Trp(143). Comparison of amino acid sequences showed that tiger shrimp GPx is more closely related to vertebrate GPx1, in accordance with those in Litopenaeus vannamei and Macrobrachium rosenbergii. GPx cDNA was synthesised in lymphoid organ, gills, heart, haemocytes, the hepatopancreas, muscles, and intestines. After injected with either Photobacterium damsela or white spot syndrome virus (WSSV), the respiratory bursts of shrimp significantly increased in order to kill the pathogen, and induced increases in the activities of superoxide dismutase and GPx, and regulation in the expression of cloned GPx mRNA to protect cells against damage from oxidation. The GPx expression significantly increased at stage D(0/1), and then gradually decreased until stage C suggesting that the cloned GPx might play a role in the molt regulation of shrimp.

SOD and CAT cDNA cloning, and expression pattern of detoxification genes in the freshwater bivalve Unio tumidus transplanted into the Moselle river.

The cDNA sequences encoding manganese superoxide dismutase (Mn-SOD) and catalase (CAT) were isolated in the freshwater bivalve Unio tumidus by reverse-transcription polymerase chain reaction (RT-PCR) using degenerate primers. Quantitative real-time PCR approach was used to evaluate the mRNA expression patterns of SOD, CAT, selenium-dependent glutathione peroxidase (Se-GPx), pi class glutathione S-transferase (pi-GST) and metallothionein (MT), in the digestive gland of Unio tumidus transplanted from a control site to four stations in the Moselle River (M1-M4), for periods of 8 and 21 days. These sites were chosen upstream and downstream of populated areas. Chemical analysis performed on sediments from the Moselle river sites did not show high levels of pollutants. Decrease of SOD, CAT, Se-GPx and MT mRNA levels were observed at M3 site after a 21-day exposure compared to control site. These results suggest inefficiency of antioxidant systems affected by cytotoxic mechanisms and confirm an environmental perturbation. Organisms transplanted at M4 site showed a strong increase of biomarkers transcription levels after 21 days of exposure. These inductions could correspond to an adaptive response to an altered environment. Our results showed that biological approaches using multibiomarkers appear as essential tools complementary to measurement of contaminants, to detect environmental degradations.

Association of mitochondrial antioxidant enzymes with mitochondrial DNA as integral nucleoid constituents.

Mitochondrial DNA (mtDNA) is organized in protein-DNA macrocomplexes called nucleoids. Average nucleoids contain 2-8 mtDNA molecules, which are organized by the histone-like mitochondrial transcription factor A. Besides well-characterized constituents, such as single-stranded binding protein or polymerase gamma (Pol gamma), various other proteins with ill-defined functions have been identified. We report for the first time that mammalian nucleoids contain essential enzymes of an integral antioxidant system. Intact nucleoids were isolated with sucrose density gradients from rat and bovine heart as well as human Jurkat cells. Manganese superoxide dismutase (SOD2) was detected by Western blot in the nucleoid fractions. DNA, mitochondrial glutathione peroxidase (GPx1), and Pol gamma were coimmunoprecipitated with SOD2 from nucleoid fractions, which suggests that an antioxidant system composed of SOD2 and GPx1 are integral constituents of nucleoids. Interestingly, in cultured bovine endothelial cells the association of SOD2 with mtDNA was absent. Using a sandwich filter-binding assay, direct association of SOD2 by salt-sensitive ionic forces with a chemically synthesized mtDNA fragment was demonstrated. Increasing salt concentrations during nucleoid isolation on sucrose density gradients disrupted the association of SOD2 with mitochondrial nucleoids. Our biochemical data reveal that nucleoids contain an integral antioxidant system that may protect mtDNA from superoxide-induced oxidative damage.

New strategies for the isolation and activity determination of naturally occurring type-4 glutathione peroxidase.

Type 4 glutathione peroxidase (GPx4) is a widely expressed mammalian selenoenzyme known to play a vital role in cytoprotection against lipid hydroperoxide (LOOH)-mediated oxidative stress and regulation of oxidative signaling cascades. Since prokaryotes are not equipped to express mammalian selenoproteins, preparation of recombinant GPx4 via commonly used bacterial transformation is not feasible. A published procedure for isolating the enzyme from rat testis employs affinity chromatography on bromosulfophthalein-glutathione-linked agarose as the penultimate step in purification. Since this resin is no longer commercially available and preparing it in satisfactory operational form is tedious, we have developed an alternative purification approach based on sequential anion exchange, size exclusion, and cation exchange chromatography. Final preparations were found to be essentially homogeneous in GPx4 (M(r) approximately 20 kDa), as demonstrated by SDS-PAGE with protein staining and immunoblotting. Specific enzymatic activity was determined using a novel thin-layer chromatographic approach in which the kinetics of phosphatidylcholine hydroperoxide loss or cholesterol-7alpha-hydroperoxide loss was monitored. A >400-fold purification of active enzyme has been attained. The relatively straightforward isolation procedure described should prove valuable for further functional studies on GPx4, e.g. how its ability to catalyze LOOH reduction compares with that of other LOOH detoxifying enzymes.

[Isolation and purification of glutathione peroxidase].

Electrophoretically homogeneous glutathione peroxidase (EC 1.11.1.9) preparation from rat liver with a specific activity of 1.46 U/mg of protein and a yield of 7.2% was obtained using the purification procedure developed. The K(M) values for reduced glutathione and hydrogen peroxide were 0.033 and 0.208 mM, respectively. The enzymatic reaction had the following characteristics: the temperature optimum, 32 degrees C; the pH optimum, 7.4; and the activation energy, 29.1 kJ/mol. The molecular weight of the enzyme was 88 kDa.

Sperm nuclei glutathione peroxidases and their occurrence in animal species with cysteine-containing protamines.

The selenoenzyme sperm nuclei glutathione peroxidase (snGPx), also called the nuclear form of phospholipid hydroperoxide glutathione peroxidase (n-PHGPx), was found to be involved in the stabilization of condensed sperm chromatin, most likely by thiol to disulfide oxidation of the cysteine residues of the mammalian protamines, small nuclear basic proteins in the nuclei of sperm cells. By applying Acidic Urea-PAGE in combination with SDS-PAGE, snGPx with an apparent molecular mass of 34 kDa and a 24-kDa protein were purified from rat sperm nuclei. The 24-kDa protein was identified by means of mass spectrometry as a truncated form of snGPx produced by cleavage at the N-terminal end. After defined processing of spermatozoa and detergent treatment of the sperm nuclei fraction, snGPx and its truncated form were shown to be the only selenoproteins present in mature mammalian sperm nuclei. Both forms were found in mature rat and horse sperm nuclei but in man only snGPx was detected. In trout and chicken, species with sperm cells which likewise undergo chromatin condensation but do not contain cysteine in their protamines, the snGPx proteins were missing. This can be taken as an indirect proof of the function of snGPx to act as protamine cysteine thiol peroxidase in the mammalian species with cysteine-containing protamines.

Cytosolic glutathione peroxidase from liver of pacu (Piaractus mesopotamicus), a hypoxia-tolerant fish of the Pantanal.

Pacu (Piaractus mesopotamicus Holmberg, 1887, Characiformes) dwells in waters of Pantanal, in which it has adapted for alternate concentrations of dissolved oxygen. Intracellular antioxidant protection should be vital for such an adaptation. Accordingly, we found that cytosol from liver of pacu has the highest antioxidant glutathione peroxidase activity so far reported for fish and murine species. To clarify whether this activity was due to a selenium independent glutathione S-transferase or to a glutathione peroxidase, we purified it and studied its kinetics. The substrates cumene hydroperoxide and hydrogen peroxide were promptly reduced by the enzyme, but peroxidized phosphatidylcholine had to undergo previous fatty acid removal with phospholipase A(2). Augmenting concentrations (from 2 to 6 mM) of reduced glutathione activated the pure enzyme. Curves of velocity versus different micromolar concentrations of hydrogen peroxide in the presence of 2, 4 or 8 mM reduced glutathione indicated that at least 2.5 mM reduced glutathione should be available in vivo for an efficient continuous destruction of micromolar concentrations of hydrogen peroxide by this peroxidase. Molecular exclusion HPLC and SDS-polyacrylamide gel electrophoresis indicated that the purified peroxidase is a homotetramer. Data from internal sequences showed selenocysteine in its primary structure and that the enzyme was a homologue of the type-1 glutathione peroxidase found in rat, bull, trout, flounder and zebra fish. Altogether, our data establish that in liver cells of pacu, a hypoxia-tolerant fish from South America, there are high levels of a cytosolic GPX-1 capable of quenching hydrogen peroxide and fatty acid peroxides, providing an effective antioxidant action.

Purification and physicochemical characterization of a recombinant phospholipid hydroperoxide glutathione peroxidase from Oryza sativa.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an unique antioxidant enzyme that directly reduces lipid hydroperoxides in biomembranes. In the present work, the entire encoding region for Oryza sativa PHGPx was expressed in Escherichia coli M15, and the purified fusion protein showed a single band with 21.0 kD and pI = 8.5 on SDS- and IFE-PAGE, respectively. Judging from CD and fluorescence spectroscopy, this protein is considered to have a well-ordered structure with 12.2% alpha-helix, 30.7% beta-sheet, 18.5% gamma-turn, and 38.5% random coil. The optimum pH and temperature of the enzyme activity were pH 9.3 and 27 degrees C. The enzyme exhibited the highest affinity and catalytical efficiency to phospholipid hydroperoxide employing GSH or Trx as electron donor. Moreover, the protein displayed higher GSH-dependent activity towards t-Butyl-OOH and H(2)O(2). These results show that OsPHGPx is an enzyme with broad specificity for hydroperoxide substrates and yielded significant insight into the physicochemical properties and the dynamics of OsPHGPx.

Multitechnique mass-spectrometric approach for the detection of bovine glutathione peroxidase selenoprotein: focus on the selenopeptide.

Glutathione peroxidase (isolated from bovine erythrocytes) and its behaviour during alkylation and enzymatic digestion were studied by various hyphenated techniques: gel electrophoresis-laser ablation (LA) inductively coupled plasma (ICP) mass spectrometry (MS), size-exclusion liquid chromatography-ICP MS, capillary high-performance liquid chromatography (capHPLC)-ICP MS, matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) MS, electrospray MS, and nanoHPLC-electrospray ionization (ESI) MS/MS. ESI TOF MS and MALDI TOF MS allowed the determination of the molecular mass but could not confirm the presence of selenium in the protein. The purity of the protein with respect to selenium species could be evaluated by LA ICP MS and size-exclusion chromatography (SEC)-ICP MS under denaturating and nondenaturating conditions, respectively. SEC-ICP MS and capHPLC-ICP MS turned out to be valuable techniques to study the enzymolysis efficiency, miscleavage and artefact formation during derivatization and tryptic digestion. For the first time the parallel ICP MS and ESI MS/MS data are reported for the selenocysteine-containing peptide extracted from the gel; capHPLC-ICP MS allowed the sensitive detection of the selenopeptide regardless of the matrix and nanoHPLC-electrospray made possible its identification.

Proteome analysis of serum from type 2 diabetics with nephropathy.

Diabetic nephropathy (DN) is a renal disease which develops as a consequence of diabetes mellitus. Microalbuminuria is the earliest clinical sign of DN. There are no specific diagnostic biomarkers for type 2 diabetics with nephropathy other than microalbuminuria and macroalbuminuria. However, microalbuminuria does not constitute a sole independent indicator for type 2 diabetics with nephropathy, and thus, another screening method, such as a biomarker assay, is required in order to diagnose it more correctly. Therefore, we have utilized two-dimensional electrophoresis (2-DE) to identify human serum protein markers for the more specific and accurate prediction of progressive nephropathy in type 2 diabetes patients, via comparisons of the serum proteome in three experimental groups: type 2 diabetes patients without microalbuminuria (DM, n = 30), with microalbuminuria (MA, n = 29), and with chronic renal failure (CRF, n = 31). As a result, proteins which were differentially expressed with statistical significance (p < 0.05) in MA and CRF groups as compared to those in DM group were selected and identified by ESI-Q-TOF MS/MS. Among these identified proteins, two proteins which might be useful as diagnostic biomarkers of type 2 diabetics with nephropathy were verified by Western blotting: extracellular glutathione peroxidase (eGPx) and apolipoprotein (ApoE) were found to exhibit a progressive reduction in MA and CRF groups. Notably, eGPx was further verified by ELISA using DM (n = 100) and MA (n = 96) patient samples. Collectively, our results show that the two proteins identified in this study may constitute potential biomarkers for the diagnosis of type 2 diabetics with nephropathy.