Effects of benzo(a)pyrene on protein expression in Jurkat T-cells

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants of air, water and soil, and are produced by the incomplete combustion of organic materials. The International Agency for Research on Cancer has characterized PAHs as carcinogens. In this study, we investigated the effects of benzo(a)pyrene (B(a)P), which is the most carcinogenic member of the PAHs, on Jurkat cell protein by proteomic analysis. Jurkat cells were treated with various concentrations of B(a)P (0, 2.5, 5, 10, 20 or 40 microM) for 24 or 48 h and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and lactate dehydrogenase assays were carried out to determine cytotoxicity and a Comet assay was used to determinate genotoxicity. The cytotoxicity assays showed that 2.5 microM of B(a)P was the maximal concentration that did not cause any toxicity, but nevertheless, at this level B(a)P produced significant DNA damage in Jurkat cells at 48 h. Proteomic analysis using three different pI ranges and large two-dimensional gel electrophoresis showed 3427 protein spots. A total of 46 (13 up- and 33 down-regulated) proteins were identified as biomarkers of B(a)P and showed dose-dependent expressions in Jurkat T-cell line exposed to B(a)P. Of these, 27 protein spots were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Two functionally differentiated protein groups were found. The protein group involving apoptosis and tumor suppression were found to be up-regulated, and B(a)P down-regulated enzyme was involved in energy metabolism, DNA synthesis and in cell structure and motility.     

Evaluation of toxicological monitoring markers using proteomic analysis

In our study, we chose three different concentrations of FA (0, 5, and 10 ppm), and cytotoxic (lipid peroxidation and protein oxidation) and genotoxic assays (DNA damage) were carried out on plasma, blood, and liver cells of rats subjected to FA-inhalation treatment. The profiles of plasma protein changes determined using 2-DE analysis were also evaluated to identify potential toxicological monitoring markers in FA-exposed rats. Concern was raised that our genotoxic analyses did not follow previously published research data and that the results of our rat plasma proteomic studies were difficult to interpret because we did not directly determine the plasma concentration of FA. However, we had already determined the concentration of FA using HPLC in an exposure chamber to monitor FA inhalation concentrations. We suggest that our experimental design was suitable to determine the FA effects on rat using an inhalation chamber system. For the similarity of genotoxic effects in lymphocytes and liver cells, we chose to present our data on the general cytological toxic effects on lipid peroxidation and protein oxidation which revealed a similarity between plasma and liver cells of FA-exposed rats. We have shown strong correlations between genotoxicity and lipid peroxidation, and lipid peroxidation is known to mediate DNA damage in many in vitro, and in vivo studies. We are well aware of the 'implausibility' of leukemia induction by FA, but for precisely this reason, we feel the need for further study to prove the systemic genotoxic effects of FA.     

Toxicological biomarkers of 2,3,4,7,8-pentachlorodibenzofuran in proteins secreted by HepG2 cells

Using a proteomic approach, a study was conducted for determination of the effects of 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PCDF) on proteins secreted by HepG2 cells. Briefly, HepG2 cells were exposed to various concentrations of 2,3,4,7,8-PCDF for 24 or 48 h. MTT and comet assays were then conducted for determination of cytotoxicity and genotoxicity, respectively. Results of an MTT assay showed that 1 nM of 2,3,4,7,8-PCDF was the maximum concentration that did not cause cell death. In addition, a dose- and time dependent increase of DNA damage was observed in HepG2 cells exposed to 2,3,4,7,8-PCDF. Therefore, two different concentrations of 2,3,4,7,8-PCDF, 1 and 5 nM, were selected for further analysis of proteomic biomarkers using two different pI ranges (4-7 and 6-9) and large two dimensional gel electrophoresis. Results showed identification of 32 proteins ( 29 up- and 3 down-regulated) by nano-LC-ESI-MS/MS and nano-ESI on a Q-TOF2 MS. Among these, the identities of pyridoxine-5'-phosphate oxidase, UDP-glucose 6-dehydrogenase, plasminogen activator inhibitor I precursor, plasminogen activator inhibitor-3, proteasome activator complex subunit 1, isoform 1 of 14-3-3 protein sigma, peptidyl-prolyl cis-trans isomerase A, 14-3-3 protein gamma, protein DJ-1, and nucleoside diphosphate kinase A were confirmed by western blot analysis. The differential expression of protein DJ-1, proteasome activator complex subunit 1 and plasminogen activator inhibitor-3 was further validated in plasma proteins from rats exposed to 2,3,4,7,8-PCDF. These proteins could be used as potential toxicological biomarkers of 2,3,4,7,8-PCDF.     

Toxicological biomarkers of 2,3,4,7,8-pentachlorodibenzofuran in proteins secreted by HepG2 cells

Using a proteomic approach, a study was conducted for determination of the effects of 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PCDF) on proteins secreted by HepG2 cells. Briefly, HepG2 cells were exposed to various concentrations of 2,3,4,7,8-PCDF for 24 or 48h. MTT and comet assays were then conducted for determination of cytotoxicity and genotoxicity, respectively. Results of an MTT assay showed that 1nM of 2,3,4,7,8-PCDF was the maximum concentration that did not cause cell death. In addition, a dose- and time dependent increase of DNA damage was observed in HepG2 cells exposed to 2,3,4,7,8-PCDF. Therefore, two different concentrations of 2,3,4,7,8-PCDF, 1 and 5nM, were selected for further analysis of proteomic biomarkers using two different pI ranges (4-7 and 6-9) and large two dimensional gel electrophoresis. Results showed identification of 32 proteins ( 29 up- and 3 down-regulated) by nano-LC-ESI-MS/MS and nano-ESI on a Q-TOF2 MS. Among these, the identities of pyridoxine-5'-phosphate oxidase, UDP-glucose 6-dehydrogenase, plasminogen activator inhibitor I precursor, plasminogen activator inhibitor-3, proteasome activator complex subunit 1, isoform 1 of 14-3-3 protein sigma, peptidyl-prolyl cis-trans isomerase A, 14-3-3 protein gamma, protein DJ-1, and nucleoside diphosphate kinase A were confirmed by western blot analysis. The differential expression of protein DJ-1, proteasome activator complex subunit 1 and plasminogen activator inhibitor-3 was further validated in plasma proteins from rats exposed to 2,3,4,7,8-PCDF. These proteins could be used as potential toxicological biomarkers of 2,3,4,7,8-PCDF.     

Proteome analysis of rat pancreas induced by pancreatectomy

The previous study demonstrated that the streptozotocin (STZ)-induced diabetic mice can be cured by injecting the regenerating pancreatic extract (RPE) of the partially pancreatectomized Wistar-Kyoto rats. In this study, to characterize the complex pattern of protein expression in RPE, the proteins of altered expression level after the pancreatectomy were identified by 2-dimensional electrophoresis (2-DE) and mass spectrometry. Of 76 significantly up- or down-regulated protein spots, 61 were identified by MALDI-TOF/MS. Moreover, the whole RPE was fractionated into 4 groups using an anion-exchange chromatography and each fraction's cell proliferating activity was measured by MTT assay. Compared to the normal pancreatic extract, fraction 3 and 4 of RPE showed the maximal cell proliferating activity. On 2-DE of 3 and 4 fractions, a total of 10 spots, which are differentially expressed after the pancreatectomy, were identified by MS/MS. Of these identified proteins, Reg III which might be functionally associated with well known regenerating factor (Reg I) was found. Taken together, our results demonstrated that the differential protein expression associated with pancreas regeneration could be sought by 2-DE and mass spectroscopy and suggested that the pre-fractionation method combined with in vitro cell proliferation assay is effectively used to pinpoint the active components for pancreas regeneration.     

采用蛋白组学技术分析质粒介导siRNA的“Off-target”效应

siRNA的"脱靶效应"(off-target effects)是RNA干扰应用研究领域的热点问题.采用蛋白组学技术对质粒介导的siRNA稳定沉默原癌基因c-myc可能存在的"off-target"效应进行初步研究,为siRNA靶向特异性的系统评价奠定理论与实验基础.构建靶向c-myc的siRNA真核表达质粒p-Mat01-1及其错配质粒p-Mis09-1,空质粒pEGFP-C1为对照,并稳定转染MCF-7人乳腺癌细胞.通过RT-PCR和Western印迹分析结果显示p-Mat01-1稳定转染克隆中c-myc/c-MYC的表达降低.采用2-DE及LC-ESI-MS/MS等方法,研究了p-Mat01-1与pEGFP-C1稳定转染克隆的蛋白组表达差异.结果显示,p-Mat01-1稳定转染克隆中有47个c-myc非调控蛋白点表达升高或降低,约占423个随机检测蛋白点的11.1%.这些蛋白涉及细胞骨架、代谢、增殖、信号传导、分子伴侣、氧化还原等多条途径.实验结果表明,质粒介导靶向c-myc的siRNA在MCF-7细胞中存在明显的"off-target"效应,提示在设计siRNA实验及应用研究时应系统考察其靶向特异性.     

Proteomic identification of differentially expressed proteins in mature and germinated maize pollen

The identification of proteins involved in pollen germination and tube growth is important for fundamental studies of fertility and reproduction in flowering plants. We used 2-DE and MALDI-TOF-MS to identify differentially expressed proteins in mature (P0) and 1-h germinated (P1) maize pollen. Among about 470 proteins separated in 2D gels, the abundances of 26 protein spots changed (up- or down-regulation) between P0 and P1. The 13 up-regulated protein spots were mainly involved in tube wall modification, actin cytoskeleton organization, energy metabolism, signaling, protein folding and degradation. In particular, pectin methylesterase, inorganic pyrophosphatase, glucose-1-phosphate uridylyltransferase and rab GDP dissociation inhibitor α are highly up-regulated, suggesting their potential role in pollen tube growth. The down-regulated 13 protein spots mainly include defense-related proteins, pollen allergens and some metabolic enzymes. This study would contribute to the understanding of the changes in protein expression associated with pollen tube development.     

A modified alkaline Comet assay for in vivo detection of oxidative DNA damage in Drosophila melanogaster

Modifications to the alkaline Comet assay by using lesion-specific endonucleases, such as formamidopyrimidine-DNA glycosylase (FPG) and endonuclease III (ENDOIII, also known as Nth), can detect DNA bases with oxidative damage. This modified assay can be used to assess the genotoxic/carcinogenic potential of environmental chemicals. The goal of this study was to validate the ability of this modified assay to detect oxidative stress-induced genotoxicity in Drosophila melanogaster (Oregon R(+)). In this study, we used three well known chemical oxidative stress inducers: hydrogen peroxide (H(2)O(2)), cadmium chloride (CdCl(2)) and copper sulfate (CuSO(4)). Third instar larvae of D. melanogaster were fed various concentrations of the test chemicals (50-200μM) mixed with a standard Drosophila food for 24h. Alkaline Comet assays with and without the FPG and ENDOIII enzymes were performed with midgut cells that were isolated from the control and treated larvae. Our results show a concentration-dependent increase (p<0.05-0.001) in the migration of DNA from the treated larvae. ENDOIII treatment detected more oxidative DNA damage (specifically pyrimidine damage) in the H(2)O(2) exposed larvae compared to FPG or no enzyme treatment (buffer only). In contrast, FPG treatment detected more oxidative DNA damage (specifically purine damage) in CuSO(4) exposed larvae compared to ENDOIII. Although previously reported to be a potent genotoxic agent, CdCl(2) did not induce more oxidative DNA damage than the other test chemicals. Our results show that the modified alkaline Comet assay can be used to detect oxidative stress-induced DNA damage in D. melanogaster and thus may be applicable for in vivo genotoxic assessments of environmental chemicals.     

The effect of oxidation or alkylation on the separation of wool keratin proteins by two-dimensional gel electrophoresis

Comparative two-dimensional electrophoretic (2-DE) studies were performed over a time-course to examine the effect of oxidation or alkylation on the separation of wool keratin proteins. The effect of oxidation was followed by treating scoured wool fibres with increasing levels of hydrogen peroxide, ranging from 0-12 g/L, using conditions mimicking the industrial wool bleaching process. Peroxide treatment was found to have only a minor effect on the 2-DE separation of the intermediate filament protein (IFP) class. Conversely, peroxide treatment of the 24-28 kDa high sulphur protein (HSP) class, which contains up to 40 cysteine residues per protein, resulted in the gradual disappearance of the major HSP spots correlated with the appearance of a few discrete spots at lower isoelectric point (pI). This suggested that only a few specific cysteine residues were being oxidized to cysteic acid by treatment with hydrogen peroxide. Peroxide treatment also appeared to have affected a discrete number of cysteine residues among proteins in the high glycine-tyrosine protein (HGTP) class, reducing the intensity of the high pI spots, while correspondingly increasing the intensity of those at lower pI. In a separate study, wool proteins were alkylated with iodoacetamide (1 M, pH 8) for periods ranging from 10 min to 48 h. In contrast to treatment with peroxide, the pI values of the HSP spots were unaffected by alkylation, irrespective of the length of this treatment. Alkylation resulted in a shift to lower pI and a loss of resolution of individual spots in the Type I and II IFP trains, to the extent that after 24 h alkylation individual spots in these trains merged. In addition after 1 h the intensity of the high pI Type II IFPs decreased until they were no longer visible on the 2-DE map after 24 h. Similarly as alkylation time increased, the major, high pI HGTP spots decreased in intensity. In unison with their decrease, some of the lower pI spots increased in intensity, while new spots appeared at more acidic pIs. Mass spectral studies indicated that cysteine alkylation was relatively fast, with 70-95% of the cysteines in the keratin proteins being alkylated within the first 10 min, while in the case of the HGTPs there was evidence for noncysteine alkylation occurring within this period. Alkylation of proteins for periods of up to 6 h prior to electrofocusing is being promoted as a better alternative to the current 2-DE protocol of the inclusion of a reductant in the immobilized pH gradient rehydration solution. This study has clearly demonstrated that long alkylation times do not suit all protein types or classes.     

Analysis of the cytosolic proteome of Halobacterium salinarum and its implication for genome annotation

The halophilic archaeon Halobacterium salinarum (strain R1, DSM 671) contains 2784 protein-coding genes as derived from the genome sequence. The cytosolic proteome containing 2042 proteins was separated by two-dimensional gel electrophoresis (2-DE) and systematically analyzed by a semi-automatic procedure. A reference map was established taking into account the narrow isoelectric point (pI) distribution of halophilic proteins between 3.5 and 5.5. Proteins were separated on overlapping gels covering the essential areas of pI and molecular weight. Every silver-stained spot was analyzed resulting in 661 identified proteins out of about 1800 different protein spots using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting (PMF). There were 94 proteins that were found in multiple spots, indicating post-translational modification. An additional 141 soluble proteins were identified on 2-D gels not corresponding to the reference map. Thus about 40% of the cytosolic proteome was identified. In addition to the 2784 protein-coding genes, the H. salinarum genome contains more than 6000 spurious open reading frames longer than 100 codons. Proteomic information permitted an improvement in genome annotation by validating and correcting gene assignments. The correlation between theoretical pI and gel position is exceedingly good and was used as a tool to improve start codon assignments. The fraction of identified chromosomal proteins was much higher than that of those encoded on the plasmids. In combination with analysis of the GC content this observation permitted an unambiguous identification of an episomal insert of 60 kbp ("AT-rich island") in the chromosome, as well as a 70 kbp region from the chromosome that has integrated into one of the megaplasmids and carries a series of essential genes. About 63% of the chromosomally encoded proteins larger than 25 kDa were identified, proving the efficacy of 2-DE MALDI-TOF MS PMF technology. The analysis of the integral membrane proteome by tandem mass spectrometric techniques added another 141 identified proteins not identified by the 2-DE approach (see following paper).     

Proteome Changes in Thai Indigenous Chicken Muscle during Growth Period

Proteomic profiling of the pectoralis muscle of Thai indigenous chickens during growth period was analyzed using two-dimensional gel electrophoresis (2-DE) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/MS). A total of 259, 161, 120 and 107 protein spots were found to be expressed in the chicken pectoralis muscles at 0, 3, 6 and 18 weeks of age, respectively. From these expressed proteins, five distinct protein spots were significantly associated with chicken age. These protein spots were characterized and showed homology with phosphoglycerate mutase 1 (PGAM1), apolipoprotein A1 (APOA1), triosephosphate isomerase 1 (TPI1), heat shock protein 25 kDa (HSP25) and fatty acid binding protein 3 (FABP3). These five protein spots were categorized as follows: (i) the expression levels of PGAM1 and TPI1 proteins were positively correlated with chicken aging (p<0.05), (ii) the expression levels of APOA1 and FABP3 proteins were negatively correlated with chicken aging (p<0.05) and (iii) the expression levels of the HSP25 protein were up- and down-regulated during growth period. Moreover, the mRNA expression levels of the FABP3 and HSP25 genes were significantly decreased in muscle during the growth period (p<0.05), whereas no significant changes of the PGAM1, TPI1 and APOA1 gene expression from the chicken muscle was observed. The identified proteins were classified as metabolic and stress proteins. This demonstrates a difference in energy metabolism and stress proteins between age groups and shows that proteomics is a useful tool to uncover the molecular basis of physiological differences in muscle during the growth period.     

Genotoxic and mutagenic effects of diesel oil water soluble fraction on a neotropical fish species

Numerous spills and leakages involving petroleum and its derivatives have recently occurred in Brazilian rivers. Considering the lack of information regarding the genotoxic response of neotropical fish to these events and the predominance of information regarding saltwater fish, which offers no genuine comparisons, the present work aimed to evaluate the genotoxicity and mutagenicity of the diesel water soluble fraction (DWSF) on the neotropical fish Prochilodus lineatus under acute (6, 24 and 96h) and subchronic (15 days) exposures, using the comet (SCGE) and micronucleus assays. The results indicated genotoxic and mutagenic damage in erythrocytes of P. lineatus exposed to DWSF. Comet scores for fish exposed to DWSF in all experimental periods were significantly higher than the respective negative control groups (fish exposed to clean water for the same period). The relative frequencies of micronucleated erythrocytes for P. lineatus exposed to DWSF under acute and subchronic treatment were also significantly higher than their respective negative controls. Taken together these results showed that acute and subchronic exposures to DWSF produce mutagenic and genotoxic effects on the blood cells of P. lineatus and that the combination of comet and micronucleus assays proved to be both suitable and useful in the evaluation of the genotoxicity of diesel oil due to their complementary action.     

Comparative proteome analysis of the embryo proper and yolk sac membrane of day 11.5 cultured rat embryos

BACKGROUND: Proteomic analysis of cultured postimplantation rat embryos is expected to be useful for investigation into embryonic development. Here we analyzed protein expression in cultured postimplantation rat embryos by two-dimensional electrophoresis (2-DE) and mass-spectrometric protein identification. METHODS: Rat embryos were cultured from day 9.5 for 48 h or from day 10.5 for 24 h. Proteins of the embryo proper and yolk sac membrane were isolated by 2-DE and differentially analyzed with a 2-D analysis software. Selected protein spots in the 2-DE gels were identified by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometric analysis and protein database search. RESULTS: About 800 and 1,000 protein spots were matched through the replicate 2-DE gels each from one embryo in the embryo proper and yolk sac membrane, respectively, and virtually the same protein spots were observed irrespective to the length of culture period. From protein spots specific to the embryo proper (126 spots) and yolk sac membrane (304 spots), proteins involved in tissue-characteristic functions, such as morphogenesis and nutritional transfer, were identified: calponin, cellular retinoic acid binding protein, cofilin, myosin, and stathmin in the embryo proper, and Ash-m, dimerization cofactor of hepatocyte nuclear factor, ERM-binding phosphoprotein, cathepsin, and legumain in the yolk sac membrane. CONCLUSION: Proteomic analysis of cultured postimplantation rat embryos will be a new approach in developmental biology and toxicology at the protein level.     

Proteomic profiling of cholangiocarcinoma cell line treated with pomiferin from Derris malaccensis

Pomiferin, a prenylated isoflavonoid from Derris malaccensis with strong anti-fungal and anti-oxidant activities, showed cytotoxic activity towards human cholangiocarcinoma cells (HuCCA-1), with IC(50) of 0.9 microg/mL. Pomiferin caused apoptosis, detectable by DNA fragmentation. Two-dimensional PAGE showed increased expression of 12 proteins, namely glucose-regulated protein 75 (grp 75), calcyclin (S100A6), degraded cytokeratin 19, ATP synthase D, ribosomal protein P0, degraded cytokeratin 18 (two spots pI/MW 6.03/29.9 and pI/MW 4.66/21.5), cofilin, annexin A1, triose phosphate isomerase, peroxiredoxin-1, calgizzarin, and profilin. In contrast, cytokeratins (CK) 7, 18 and 19 were down-regulated, and were shown by 1-DE immunodetection to be degraded.     

Differentially expressed proteins in the interaction of Xanthomonas axonopodis pv. citri with leaf extract of the host plant

The present study reports the expression of proteins of Xanthomonas axonopodis pv. citri in response to different growth conditions. The bacterium was cultured in the basal medium MM1 and in the presence of leaf extracts from a susceptible host plant (sweet orange) as well as a resistant (ponkan) and a nonhost plant (passion fruit). The protein profiles were analyzed by two-dimensional gel electrophoresis (2-DE). Twelve differential spots (induced, up- and down-regulated and repressed) were observed in the protein profiles of the bacterium cultivated in citrus extract (susceptible host) when compared to that of MM1. The 2-DE profile of the bacterium cultured in the complex medium nutrient yeast glycerol was also obtained and the comparison with that of MM1 revealed 36 differential spots. Five proteins from the different treatments were successfully N-terminally sequenced and the putative functions were assigned by homology searches in databases. Two constitutively expressed proteins, B4 and B5, were identified as pseudouridine synthase and elongation factor P, respectively. The large subunit of ribulose 1,5-biphosphate carboxylase/oxygenase and a sulfate binding protein were found as specifically up-regulated in the presence of citrus extracts. Finally, the heat shock protein G was found exclusively in the complex medium and repressed in all other media.     

Proteomic Screening of Antigenic Proteins from the Hard Tick,Haemaphysalis longicornis (Acari: Ixodidae)

Proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. For detection of antigens from Haemaphysalis longicornis, 1-dimensional electrophoresis (1-DE) quantitative immunoblotting technique combined with 2-dimensional electrophoresis (2-DE) immunoblotting was used for whole body proteins from unfed and partially fed female ticks. Reactivity bands and 2-DE immunoblotting were performed following 2-DE electrophoresis to identify protein spots. The proteome of the partially fed female had a larger number of lower molecular weight proteins than that of the unfed female tick. The total number of detected spots was 818 for unfed and 670 for partially fed female ticks. The 2-DE immunoblotting identified 10 antigenic spots from unfed females and 8 antigenic spots from partially fed females. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF) of relevant spots identified calreticulin, putative secreted WC salivary protein, and a conserved hypothetical protein from the National Center for Biotechnology Information and Swiss Prot protein sequence databases. These findings indicate that most of the whole body components of these ticks are non-immunogenic. The data reported here will provide guidance in the identification of antigenic proteins to prevent infestation and diseases transmitted by H. longicornis.     

Identification of toxicological biomarkers of di(2‐ethylhexyl) phthalate in proteins secreted by HepG2 cells using proteomic analysis

The effects of di(2‐ethylhexyl) phthalate (DEHP) on proteins secreted by HepG2 cells were studied using a proteomic approach. HepG2 cells were exposed to various concentrations of DEHP (0, 2.5, 5, 10, 25, 50, 100, and 250 μM) for 24 or 48 h. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) and comet assays were then conducted to determine the cytotoxicity and genotoxicity of DEHP, respectively. The MTT assay showed that 10 μM DEHP was the maximum concentration that did not cause cell death. In addition, the DNA damage in HepG2 cells exposed to DEHP was found to increase in a dose‐ and time‐dependent fashion. Proteomic analysis using two different pI ranges (4–7 and 6–9) and large size 2‐DE revealed the presence of 2776 protein spots. A total of 35 (19 up‐ and 16 down‐regulated) proteins were identified as biomarkers of DEHP by ESI‐MS/MS. Several differentiated protein groups were also found. Proteins involved in apoptosis, transportation, signaling, energy metabolism, and cell structure and motility were found to be up‐ or down‐regulated. Among these, the identities of cystatin C, Rho GDP inhibitor, retinol binding protein 4, gelsolin, DEK protein, Raf kinase inhibitory protein, triose phosphate isomerase, cofilin‐1, and haptoglobin‐related protein were confirmed by Western blot assay. Therefore, these proteins could be used as potential biomarkers of DEHP and human disease associated with DEHP.