Application of proteomics in environmental science | SpringerLink

Front. Environ. Sci. Engin. China 2009, 3(4): 393–403 DOI 10.1007/s11783-009-0145-x

REVIEW ARTICLE

Application of proteomics in environmental science Xiaona CHU, Jiangyong HU (✉), Say Leong ONG Center for Water Research Division of Environmental Science and Engineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore

© Higher Education Press and Springer-Verlag 2009

Abstract Proteomics involves the separation of proteins, identification of the amino acid sequence of the interested or target proteins, study of the function of the proteins, modification, structure and ultimate assignments to functional pathways in the cell. The proteomic investigations have contributed greatly to human diseases studies, new drugs discovery researches, and environmental science in recent years. This article provides a review on the development of the main proteomic technologies, including both the gel based and non-gel based technologies, and their applications in environmental science. Proteomic technologies have been utilized in the environmental stresses studies to analyze the induction or reduction of proteins at expression level and identify the target proteins to investigate their function in response to environmental stresses, such as high or low pH, oxidation stress, and toxic chemicals. Such protein responses are also helpful to understand the mechanisms of some cellular activities and the functions of some proteins. Keywords proteomics, environmental stress, two dimensional (2D) gel electrophoresis, mass spectrometry

1

Introduction

Although the first biggest study of biology, the human genome project (HGP), had been completed in 2001, we still face many unsolved questions and unclear secrets. We still have difficulty in diagnosing or treating many human diseases, especially epidemics and lethal diseases such as AIDS, cancer, and tuberculosis, which indicates that our understanding of disease biology is still insufficient [1,2]. The main reason is that researchers were previously not able to investigate the complicated relationships between proteins and genes. Systematic analysis of gene functions Received December 30, 2008; accepted July 16, 2009 E-mail: [email protected]

is preferably at the protein level rather than at the genetic level, since it is the proteins that perform most of the reactions necessary for the cell. With the completion of the HGP, it is possible for researchers to study protein expressions, modifications, structures, and interactions in complex systems, and to understand the biological functions of the proteins, which will pave the way for a more detailed and complete understanding of human diseases [3,4]. The completion of HGP suggests that we are entering the post genome era, the era of proteome. Proteomics is a developing field that is poised to have a significant impact on future researches into human diseases and other related biological fields. Proteomics is concerned with the global analysis of complex protein mixtures for the purpose of qualitative, quantitative, structural, functional, and interactional analysis of all the proteins present in a sample. Therefore, it is necessary to identify a protein in the context of its cellular environment to understand its function and regulation [3]. Proteomic analyses have been applied in a wide range of research. Advances in two-dimensional (2D) electrophoresis, chromatography, mass spectrometry, and other proteomic related technologies have provided tremendous opportunities in biological researches. In the investigation of diseases, the changes in protein levels of some diseases have been identified as markers in the diagnosis of diseases [5–9]. The proteomic analyses could also be applied in the study of protein changes in pathogen bacteria to come up with new antibacterial drugs and better understand the effects of antibacterial drugs [10,11]. Proteomic analyses have also been utilized to fathom the complex mechanisms involved in toxicology, a branch of knowledge that concerns itself with the assessment of the toxic effects of carcinogens and other exogenous chemicals, toxins, pesticides, and pharmaceutical drugs [12–15]. The proteomic approach to the analysis and identification of proteins has been regarded as a new method of studying the cellular effects of environmental presses [16,17]. Proteomic analysis can be used to isolate chemical-specific protein expression signatures (PES),

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Front. Environ. Sci. Engin. China 2009, 3(4): 393–403

which is a sophisticated technique to study the intracellular response and mechanisms of cells when exposed to environmental toxicants or exterior stress. Protein expression changes with the state of development, the tissue, and the internal and external environmental conditions of an organism. There is growing evidence that sets of proteins up and down regulated are specific to a stressor [18]. The understanding of functional proteomics and global characterization of functional features of the proteins is necessary to better understand these events, which constitute the metabolic and structural signals that control growth, development, replication, and stress response of cells [3]. In this review, the technological developments in proteomics are introduced, and the proteomic approaches to environmental exposures are also discussed.

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Development of proteomics

Proteomics involves the separation of proteins, identification of the amino acid sequence of the interested or target proteins, function of the proteins, modifications, structure, and ultimate assignments to functional pathways in the cell [9,19]. The improvements in the protein separation and identification technologies in recent years have remarkably accelerated the developments of proteomic science [20]. In the study of proteomics, the first step is to separate the proteins. 2D gel electrophoresis is the traditional technique used to separate proteins, and it can provide high throughout separation for thousands of proteins in one gel. The principle of the 2D gel is that proteins are separated by their different pI values and molecular masses. In the first dimension of the electrophoresis, proteins are separated based on net charges by isoelectric focusing (IEF). In the second dimension, proteins are further separated based on their size by sodium dodecyl sulfate porous polyacrylamide gel electrophoresis (SDSPAGE) [21]. Silver or Coomassie blue staining is then applied to mark the protein spots in the gels for further observation. The development of immobilized pH gradients (IPG strips) of different ranges contributed greatly to the improvements in the classical process of 2D gel electrophoresis. Previously, different labs used different methods to process the first dimension of the electrophoresis, therefore the gels from different labs could not be compared together. The immobilized pH gradients (IPG strips) of different ranges thus allowed different laboratory comparisons of results to be possible, and the development also increased the loading capacity of proteins [22–24]. High protein loads, especially in the presence of more sensitive detection methods, enhanced the detection of less abundant proteins and subsequently provided sufficient amounts for protein sequencing [5]. On the other hand, with the wider pH range offered by wide pH strips from more acidic to more basic pH ranges proteins could now be separated with excellent resolution [25,26].

After the separation of proteins has been completed, the next step of analyzing the protein spots in the gels requires the aid of powerful software to locate the absent or induced proteins or determine the quantitatively changed proteins after comparing the different gels. To increase the sensitivity and accuracy of the 2D gel electrophoresis, the development of a more powerful image analysis platform is necessary. The PDQUEST system, MELANIE system, Klepler system (Large Scale Proteomics, Rockville, Maryland, USA), RosettaTM bioinformatics tools developed by Oxford GlycoSciences (OGC) and LifeExpress (OGS and InCyte Genomics, Palo Alto, California, USA) [5,27,28] are some examples. There are still some shortcomings of the 2D gel electrophoresis technology, such as the tedious procedures, poor repetition, and gel bending. A newly developed technology, the fluorescence 2D differential gel electrophoresis (2D-DIGE) [29,30], has been introduced into the proteomics field. This technology was developed by Amersham Biosciences (Piscataway, NJ, USA), and it relies on covalent labeling of the proteins with Cy2, Cy3, and Cy5 dye. The labeled protein samples were then mixed and separated in a single 2D gel electrophoresis. The protein samples were separated and compared based on the fluorescence signals of the labeled dyes, and up to three dye signals could be detected in one gel because the dyes have distinct excision and emission wavelengths [5,9,10]. There are also some other technological advancements in the separation procedure in proteomic researches regarding throughput and automation besides the immobilized IPG strips and 2D-DIGE technology. One is the newly launched ZOOM IPGRunner system from Invitrogen (Carlsbad, CA) which allows 2D-PAGE separation in 24 h from rehydration of the sample into the IPG strip for the first dimension to protein staining. Furthermore, NextGen Sciences Ltd. (Cambridgeshire, UK) introduced a fully automated robot that is capable of analyzing three 2D gels at a time under highly reproducible conditions. Finally, image analysis software packages have evolved to facilitate the quantification of all protein spots within large sets of 2D gels, which requires different levels of user interaction to ensure data quality [9]. Besides the 2D gel electrophoresis, liquid chromatographic (LC) strategies are utilized to separate hydrophobic, extremely small, or extremely large proteins that cannot be separated by the classic 2D gel method. The chromatographic separations are based on different properties of proteins. The size exclusion chromatography can separate proteins based on their size, ion exchange chromatography based on their charge, and reverse phase chromatography based on their different hydrophobicities [3,31]. After proteins have been separated successfully by 2D gel or liquid chromatography, the next step in the analysis of proteins is to identify the separated proteins. Previously,

Xiaona CHU et al. Application of proteomics in environmental science

the identification depended on the cycles of Edman degradation to sequence the proteins [32], which is very tedious and expensive. With the rapid progress in mass spectrometry in the 1990s, the classical sequencing method was replaced mostly by mass spectrometry (MS) identification [9]. In brief, the procedure of analyzing proteins by MS includes three steps: first, the protein samples are ionized in gas phase; second, the ions are separated according to their mass to charge (m/z) ratio; finally, the ions are detected. So far, protein analysis (primary sequence, post translational modifications (PTMs), or protein-protein interactions) by MS has been most successful when applied to small sets of proteins isolated in specific functional contexts [33,34]. The mass spectrometry has been developed in several areas of design in the last two decades. Development of the “soft” desorption ionization techniques, namely the matrix assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) in the late 1980s, revolutionized proteomic research by rendering the study of macromolecules feasible [10]. ESI and MALDI are the two techniques most commonly used to volatize and ionize the proteins or peptides for mass spectrometric analysis. ESI ionizes the analytes out of a solution and is therefore readily coupled to liquid based (for example, chromatographic and electrophoretic) separation tools. MALDI sublimates and ionizes the samples out of a dry, crystalline matrix via laser pulses. MALDI-MS is normally used to analyse relatively simple peptide mixtures, while liquid chromatography ESI-MS system (LC-MS) is usually applied for the analysis of complex samples [33]. Besides the development of the ESI and MALDI methodologies, there are other important achievements in the improvements of MS. The time of flight (TOF), ion trap, quadrupole, and Fourier transform ion cyclotron (FTIC) are the four major types of mass analyzers currently in use. Each of them shows distinct advantages and weaknesses with regards to the sensitivity, mass accuracy, and mass resolving power they provide. These analysers can be operated alone, or amalgamated in tandem to take advantage of the strengths of each one. MALDI is usually coupled with TOF analysers that measure the mass of intact peptides, while ESI has mostly been coupled with ion traps or triple quadrupole instruments to generate fragmented ion spectra (collision induced (CID) spectra) of selected precursor ions [33,35,36]. In addition, the automated applications in MS have increased the efficiency and throughput of protein identification. Robots can excise protein spots from 2D gels and transfer the gel plugs into microtiter plates to digest, or some digest robots perform the in gel tryptic digests directly in the microtiter plate, then a spotting robot applies peptide samples to the MALDI targets. MALDITOF mass spectrometers acquire the data, and software packages are available to automatically extract peptide masses from the derived spectra, which are submitted to

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the database search [5,9,37]. Following this procedure, the only thing left is to check the quality to ensure that the hits from the database match the predominant peaks on the spectra. Experimentally obtained peptide masses of a digested protein spot were compared to a database that contained all theoretical peptide masses derived from an in silico digestion of the proteins predicted from the genome sequence [38]. There are several 2D gel, protein, and genomic databases available from the Internet, such as World 2D-PAGE (http://www.expasy.ch/ch2d/2d-index. html) at the ExPASy server in Geneva. Even the software by which the user could match his local gel with any gel image from any of the www-based 2D gel databases [39] and the program is available on the website (http://www. lecb.ncifcrf.gov/flicker). The web-based gel matching limits and achievements were both described in previous reports [40,41]. With the rapid development of proteomics, a very large scale of data were produced and need handling and analysis, therefore a new technology of bioinformatics was developed in recent years to solve the problems. Bioinformatics uses computers to handle information problems in the life sciences by creating extensive electronic databases on genomes and protein sequences [42,43]. Besides the gel related technologies 2D gel electrophoresis, and the non-gel based proteomics chromatography and MS, the other high throughput and multiplexed protein technologies have been developed with the update of computer technology in recent decades. The progress in protein biochips, protein, and antibody microarrays provided a chip- or array-based proteomics profiling platform for researchers to study wider areas, such as protein and gene interaction, proteins interaction, disease researches, etc. [44–46].

3 Application of proteomics in environmental research With the development of the global industry and growth of human population, thousands of man-made chemicals are released to the environment by agriculture, transport, industries, and other activities. Proteomic approaches have been applied in environmental science as they are capable of detecting subtle changes in the level and structure of individual proteins in response to the environmental stresses. The proteomic approaches also provide an insight into underlying mechanisms of toxicity [47]. The application of proteomic approaches in environmental research could be divided into two areas: 1) mechanism study of proteomic response to stresses, which helps us understand how the cells respond to environmental stresses and why some organisms could resist extreme or toxic environmental stresses; 2) screening environmental samples with proteomic approaches, which can monitor concerned pollutants. The proteomic approaches have been applied

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in environmental research from microorganisms and plants to invertebrates and vertebrates. 3.1

Studies on prokaryote microorganisms

For prokaryotes, Escherichia coli (E. coli) and Bacillus subtilis have been used as the gram negative and positive model strain separately to study the normal environmental stress, such as pH, oxidation, etc. To study the cellular response of E. coli to various pH values, mediums at pH 4.9, 6.0, 8.0, and 9.1 were used to culture E. coli strains. 22 proteins were found to be regulated by pH. These proteins included periplasmic proteins, membrane proteins, and three other proteins whose functions were not clear. When exposed to external acid (pH was from 4.9 to 6.0), several acetate induced proteins were increased (e.g., LuxS and YfiD). Otherwise, protein RibB, the homolog protein of LuxS, was not induced by acetate although it was also induced at low pH, showing a different pH induced mechanism. When exposed to alkaline environment, tryptophanase (TnaA) becomes one of the most highly expressed proteins in the cells [48], which is a versatile enzyme, deaminates serine and cysteine. Besides TnaA, three more additional enzymes were reported to be induced under high pH environment (namely AstD, GabT, and CysK) with different functions [49]. Streptococcus mutans (S. mutan) is a well known pathogen, which is associated with the initiation of dental caries because the byproducts of its acid fermentation are harmful to the teeth’s’ enamel. S. mutans was cultured at pH 7.0 and pH 5.0 separately to study its acid tolerance. Among the identified proteins, 106 protein spots were found to be associated with metabolism, and the remaining 61 protein spots were related to regulatory and/or stress, including DNA replication, transcription, translation, protein folding, and proteolysis. The 61 protein spots were changed from 30 different proteins, and 25 of them were induced or up regulated at pH 5.0 and were assessed as the acid stress related proteins. The functions of the proteins were DNA binding (Ssb), transcription elongation factor (GreA), RNA exonuclease (PnpA), and two proteinases PepB and PepD [50]. Oxidative stress, a common environmental stress, has been studied in many organisms [51]. In E. coli, two aconitases AcnA and AcnB were reported to be induced by oxidative stress [52,53]. The mutant E. coli strains which lesion the gene acnA or acnB or both were cultured, and their proteome were compared with the wild strain after exposure to peroxide. At least 11 polypeptides were changed in the acnB mutant, and 4 of them have been identified by sequence analysis, i.e., ODH, YedA, dihydrofolate reductase, and SodA. The results affirmed that the acn lesions affected the synthesis proteins which were related to the oxidative stress response [54]. Bacillus subtilis has been studied under the oxidative stresses, and the oxidative stress was achieved by the addition of 58 μmol/L of H2O2 or 100 μmol/L of paraquat. The

proteomic analysis revealed that under peroxide stress, 55 proteins were up regulated and 150 proteins were down regulated; while under the paraquat stress, 65 protein expressions were increased and 200 protein expressions were decreased. After comparison, there were around 20 up regulated proteins and 140 down regulated proteins for both peroxide and paraquat treatment. The transcriptome analysis revealed that the PerR regulated genes, fur gene, and CtsR regulated operon were significantly induced after exposure to both the oxidative stresses. And that σB regulon and SOS regulon increased after H2O2 treatment. Sulfur limitation regulated genes were induced after paraquat [55]. The oxidative stress responses were also studied in other bacteria: hydrogen peroxide stress in Francisella tularensis LVS [56] and Staphylococcus aureus [57], and disulfide stress which is a subcategory oxidative stress in B. subtilis [58]. 2,4,6-trinitrotoluene (TNT) is a nitroaromatic explosive. After exploding, it would be released into the soil and water ecosystems, and the released components were toxic to microorganisms, green algae, fish, and animals. Several bacteria had been reported to metabolize TNT in the aerobic environment [59–61]. Stenotrophomonas sp. OK 5, a TNT degrading bacteria, was utilized to study the TNT induced stress shock proteins (SSPs) using proteomic techniques. Among the 300 protein spots that were detected by 2D gel, 10 protein spots were observed to be induced by 0.6 mmol/L of TNT treatment for 6 h. Four of the 10 proteins were then identified by ESI Q TOF mass spectrometry. After comparing with other reported protein sequences, the 4 proteins were identified as DnaK protein, outer membrane protein (OmpW), osmotically inducible protein (OsmC), and a putative membrane protein [62]. Chlorophenoxy herbicides are toxic agents that may disturb the energy conservation system of bacteria located in the cytoplasmic membrane [63]. Delftia acidovorans MC1 is able to grow on chlorophenoxy herbicides such as 2,4-dichlorophenoxypropionic acid (2,4-DCPP) and 2,4dichlorophenoxyacetic acid as sole sources of carbon and energy, and the responses of Delftia acidovorans MC1 to high concentration of chlorophenoxy herbicides was studied at the protein level by Benndorf et al. In the identified proteins, many were involved in the metabolism of 2,4-DCPP, such as chlorocatechol 1,2-dioxygenases (TfdC and TfdCII), 2,4-DCP hydroxylase (TfdB), and so on. And apart from DnaK, only a weak or no induction of the classical stress proteins was observed. The other identified proteins were AspG, TufA, OdhB, and YceI, whereas their contribution to response to 2,4-DCPP were not elucidated [64]. Benzoic acid as a toxic chemical was used to induce E. coli, and 2D-PAGE as a high throughput analysis technique was applied to study the global proteome of E. coli after benzoic acid treatment. After 2D gel electrophoresis, the gels were analyzed by the 2D Master imager and Typhoon scanner. After treatment with benzoic acid,


the largest protein expression changes were discovered to be the protein ompF (including 3 proteins) and protein znuA. The function of protein ompF was to allow the nonspecific absorption of small hydrophilic compounds into the cells, and the reduction of the expression level was supposed to decrease the absorption of benzoic acid. The down regulation of znuA protein was supposed to be due to the homologues function to fur, which was required for acid tolerance, as a result induced fur could cause znuA reduction [65]. 3.2

Studies on eukaryotes microorganisms

Fission yeast Schizosaccharomyces pombe and budding yeast S. cerevisiae have been used as the eukaryotic cell models to study the cellular reactions to the toxic environmental stress because of their easy to handle genetic manipulation and their complete genome information. The proteomic response of S. cerevisiae to oxidative stress was investigated, and 0.4 mmol/L of H2O2 was used to treat the cells. The global proteomic study showed that at least 115 proteins were stimulated, whereas 52 proteins were repressed by this treatment. 71 proteins of the induced 115 proteins were identified, and besides unknown function proteins they were sorted into seven different functional classes: antioxidant defense, heat shock proteins, proteases and proteasome subunits, translation apparatus components, carbohydrate metabolism enzymes, and enzymes involved in amino acid metabolism. Most of the repressed proteins were translational apparatus components and metabolic enzymes [66]. S. cerevisiae was exposed to 0.9 mmol/L of sorbic acid at pH 4.5, and the changes in protein presented that 10 proteins were up regulated and 3 proteins were down regulated after exposing to sorbic acid. Among those up regulated proteins, most were stress proteins and chaperones, and were induced by H2O2, heat shock, low pH, and so on. In addition to the stress proteins, the up-regulation of energy related proteins was also observed. Exposure to sorbic acid also induced proteins that are involved in carbohydrate metabolism and glycolytic repression [67]. Most metals are sufficient for cell survival at trace amounts, while at high concentrations they are fatal to most cells. In the report of Hu et al., 15 different metals were used to treat S. cerevisiae, 1000 to 2000 different proteins in each gel were detectable, and around 20% of which changed significantly after being treated with different metals. The proteins whose expression levels changed upon treatments of most metals were focused on in the study as their functions were supposed to be related with the overall cellular survival mechanism. More than 100 protein spots were chosen, and more than 50 proteins were identified. The identified proteins were grouped into several categories based on their known biological functions, antioxidant, heat shock proteins, proteases,

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carbohydrate, DNA/protein synthesis, transcription regulation, and other unknown function proteins [68]. Cadmium is very toxic at low concentrations for organisms, and is probably carcinogenic too. In the report of Vido et al., the proteomic responses of S. cerevisiae to cadmium stress were analyzed after S. cerevisiae were cultured with 0 μmol/L, 25 μmol/L, 50 μmol/L, 75 μmol/L, 100 μmol/L, and 200 μmol/L of cadmium sulfate for 2–5 d. 54 induced and 43 repressed proteins were identified. The proteomic response to cadmium revealed that eight enzymes of the sulfur amino acid and GSH biosynthesis pathway were strongly induced, and several proteins with antioxidant properties were also induced. All the results indicated that the two cellular thiol redox systems, glutathione and thioredoxin, are essential for cellular defense against cadmium [69]. The cellular responses of the S. pombe to cadmium were also studied using an integrated proteomic strategy. Amino acid coded mass tagging (AACT) was integrated with LC-MS/MS to study the cellular response to cadmium. A total of 1133 proteins were identified, and among the 319 quantitated proteins 106 were up regulated and 55 were down regulated. Most of the up-regulated and down-regulated proteins have protein biosynthesis function. The second prevalent class of the up-regulated proteins included heat shock proteins, oxygen and radical detoxification, and stress response proteins, whereas the proteins involved in nucleotide metabolism belonged to the second most prevalent class of down-regulated proteins [70]. 2,4-dichlorophenoxyacetic acid (2,4-D) is one of the most widely used herbicides, and the intensive use of it may give rise to a lot of toxicological problems in nontargeted organisms. S. cerevisiae was incubated with 0.3 mmol/L of 2,4-D, and the global mechanisms underlying adaptation to 2,4-D were studied. A total of 26 protein spots whose expression increased more than 2 times under the 2,4-D stress were identified, corresponding to 22 different proteins. On the other hand, the decreased protein under 2,4-D stress was identified as a single protein Ado1p. The functions of the identified proteins included mRNA and protein degradation, carbohydrate and energy metabolism, vacuolar, amino acid and nucleotide metabolism, and stress response. Three proteins that increased during exposure to 2,4-D were involved in cell protection against environmental stress. There are (i) antioxidant enzyme Ahp1p, with a specific role in the reduction of alkyl hydroperoxides; (ii) Ssb2p, belonging to the heat shock proteins of the Hsp70 family, whose function is promoting proper protein folding; and (iii) Hsp12p, a small heat shock molecular chaperone over expressed under a wide range of environmental stresses, while the function of it was not clear now [71]. Three commercial herbicides Pointer (P), Silglif (S), and Proper Energy (PE) were used to treat wild-type wine S. cerevisiae strain, and the comparative proteomic analysis showed the effects of these herbicides on yeast. In the

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presence of P and PE, the proteins belonging to cell rescue and defense group were either induced or repressed. The treatment with S evoked a stronger and more uniform induction of nearly all the identified proteins in this group. The proteins belonging to carbohydrate metabolism group were found to be repressed after the treatment with herbicides. The herbicides treatment was found to alter the yeast proteome producing responses that share homologies with those observed with the herbicide 2,4-D or with wellknown oxidizing agents H2O2 [72]. 3.3

Studies on plants

The environmental researches on plants have been performed with proteomic approaches also. Arabidopsis thaliana and rice were the first plants whose genomes were completed. Therefore, these two plants have been considered as dicotyledons and monocotyledons models, respectively, in molecular biology, genomics, and proteomics study [73]. The effects of Cd exposure were evaluated in poplar leaves by proteomic methods during 14 d [74] and 56 d [75]. In the 14-d Cd exposure study, 125 spots were identified from 717 spots. Among them, 73 proteins were down-regulated and 52 spots were up-regulated. The identified proteins indicated that Cd had a negative effect on the light phase of photosynthesis via down-regulated expression of proteins of the electron transport chain, oxygen evolving enhancer proteins. Furthermore, the proteins involved in carbon metabolism were downregulated, while the protein involved in remobilizing carbon from other energy sources were up-regulated [74]. In the 56-d Cd exposure study, 185 proteins were determined from approximately 1000 spots with an absolute variation, among which 125 proteins were identified. 31% of the identified proteins belonged to the primary carbon metabolism. Contrary to the 14-d study, only one protein from the light dependent reaction was found to be down-regulated. Cd had a negative effect on the carbon fixation, as the related enzymes, such as ribulose bisphosphage carboxylase/oxygenase (RuBisCO) activases, and RiBisCO subunit binding proteins were down-regulated in both the short term (14 d) and long term (56 d) Cd exposure studies [75]. Ozone is one of the most important secondary atmospheric pollutants, which is formed by the interaction of NO2 and UV radiation [73]. The proteomic responses of poplar leaves to 120 ppb of ozone for up to 35 d were studied. Among the matched 529 spots, 147 proteins showed absolute variation and 71 of them were identified. The proteins associated with the Calvin cycle (25 proteins) and electron transport (17 proteins) in the chloroplast were found to decrease in abundance, and the proteins associated with glucose catabolism were increased during ozone exposure. The other identified proteins with changes in abundance included nitrogen metabolism enzymes,

enzymes with oxidoreductase activity, and protein isoforms involved in protein folding [76]. 3.4

Studies on invertebrates

Mussels have been widely used as the indicator of marine pollution due to its capacity of bioaccumulating and concentrating organic and metallic pollutants [77]. Mytilus edulis, a blue mussel that is isolated from the Baltic Sea is the widely used mussel indicator of marine pollution. The proteomic profiles of M. edulis when exposed to several marine pollutants, diallyl phthalate (DAP), 2,2’,4,4’tetrabromodiphenl ether (PBDE-47), and bisphenol-A (BPA) were identified. 170 spots showed a significant increase or decrease in protein abundance in the 2-D electrophoresis maps from the groups exposed to pollutants. These proteomic profiles could be used as a valuable tool to monitor the presence of pollutants [77]. The PESs of M. edulis, which is composed of 13 proteins including oxidation, amino acid metabolism, detoxification, protein degradation, organelle biogenesis, and protein folding were used to distinguish the clean sample from the polluted marine water samples in the pilot study. The pollutants in the pilot study included anthropogenic compounds such as organic chlorines, polychlorobiphenyls, petroleum hydrocarbons, polyaromatic hydrocarbons, furans, dioxins, and heavy metals [78]. The PESs of M. edulis to three different stresses were studied after it was exposed to copper (70 ppb), Aroclor 1248 (1 ppb), and lowered salinity (3 ppb) for 7 d. The three stress related gels were compared with the control gels separately, and around 500 to 600 protein spots were compared. In the PCBs intimated M. edulis cells, 5 protein spots were induced and 18 protein spots repressed. In the copper exposed cells, 13 proteins were induced and 10 proteins repressed, and in the cells with salinity stress, 17 proteins were induced and 9 proteins repressed [18]. 3.5

Studies on vertebrates

Because the liver and kidney are both common sites for toxicity within the body, several proteomic studies have been performed to define changes in the liver cells or kidney cells in response to damage [13]. By using 2D gel electrophoresis technologies, a prototype Molecular Effects Database describing xenobiotic effects in rodent liver was developed by Anderson and colleagues. This database can detect, classify, and characterize a broad range of liver toxicity mechanisms. It contained approximately 107 protein measurements, including data on the liver effects of 43 compounds [79]. In recent years, many more researches by using proteomics technologies on liver cells or kidney cells to study different toxicoids and their mechanisms were reported [80–84]. Besides kidney and liver cells, other vertebrate cells also have been used to study environmental stresses. Human


amnion epithelial cells (FL cells) were used to study the cellular responses to Benzo[α] pyrene (B[a]P), which is a prototype of polycyclic hydrocarbons (PAHs) and a potent procarcinogen generated from the combustion of fossil fuel and cigarette smoke [85]. The effects of effluent from a wastewater treatment plant (EWWTP) on intestinal epithelial Caco-2 cells were investigated. Previous studies showed that the wastewater constituent nonylphenol and lipopolysaccharide (LPS) induce the overexpression of specific proteins. From the results of proteomics analysis of human intestinal Caco-2 cells treated with the wastewater effluent, the overexpression of specific proteins, namely elongation factor 1 β and enolase 1 were found, which suggested that specific proteins can be used as biomarkers for the risk assessment of water and wastewater [86]. The proteomic profiles of MCF 7 cells to estrogenic endocrine disrupters (EDs) were studied by treating MCF 7 cell with 3.710–9 mol/L of 17β estradiol (E2) for 48 h and 72 h. It was observed that approximately 40 proteins were down regulated and 5 proteins were up regulated when MCF 7 cells were treated with E2 at 72 h. Likewise, 15 proteins were down regulated and 4 proteins up regulated when treated with EE2 at 48 h. Such down regulated dominant pattern might indicate that the inhibition factors of cell proliferation were reduced by E2. The change of protein expression pattern provides a great potential of predicting the presence of estrogen as it represents estrogen (E2 in this case) inducing protein expression profiles. This study provided a new avenue to investigate the insight mechanism of estrogenic EDs and a new opportunity to screen EDs in the physiologically trustworthy method [87].

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Summary and conclusions

Protein, which is the functional unit for most activities in living organisms, has been recognized from the end of the last century as a key aspect in life sciences. With the completion of the human genome project, the complete human genomic information and other lives genomic data would no doubt provide the necessary reference and foundation for further developments in the field of proteomics. This paper reviewed the development of proteomics and the main technologies applied in the environmental researches. Although the application of proteomic technology in environmental science was studied only from the end of the last century, the development was rapid and remarkable. Under the environmental stresses, some proteins were either up regulated or down regulated, and the changes in expression level could be studied to discover the function of the proteins. On the other hand, if the functions of the proteins were clear already, the expression varieties could contribute to understanding the cellular mechanisms of the

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stress responses or even other activities. For example, the membrane proteins were induced under TNT stress, which indicated that the membrane of Stenotrophomonas sp. OK 5 could be one of the reasons why it can resist TNT [62]. In addition, the genomic information can be applied in tandem with proteomic researches, and inversely, the proteomic study could help us understand the genomic information better [55]. To study some interested genes, the wild strain and mutant strain whose target genes had been mutated or damaged were cultured together with the same outer stress. After the treatment, both of their proteomics were compared. Such researches provide not only valuable information on the function of the target genes and proteins and the interaction of gene and protein, but also the protein interaction at both the genomic and proteomic levels. The proteomic study of mutant E. coli with gene acn lesions under oxidative stress is an example using such strategy [54]. For microorganisms, it was found that once the cells were under the toxic chemical stresses, besides the specific proteins which were toxic chemical metabolism related proteins, the heat-shock proteins and oxidative related proteins were mostly up-regulated by the chemical. The proteomic study results of 2,4-D, sorbic acid, and metals stress to S. cerevisiae [67,68,71] and cadmium stress to S. pombe [70] all indicated this finding. On the other hand, some researches showed that such classic stress related proteins were not regulated by the individual chemical stress, such as the proteomic responses of Delftia acidovorans MC1 to herbicides 2, 4-DCPP [64]. The nucleotide metabolism proteins, protein synthesis proteins, and energy related proteins were also regulated by the chemical stresses, and mostly were down-regulated [66,70]. For almost all organisms, from microorganisms to vertebrate cells, any environmental stress such as extreme pH, temperature, and toxic chemicals, would arise global proteomic responses. However, the responses to different stresses were different. In addition, the individual proteomic profiles to specific stresses as PESs could be used as the signature of the specific stress to monitor the environment. The PESs of M. edulis have been used to monitor polluted marine water [77,78], and the study of MCF 7 also indicated the potential capability of using PESs to monitor estrogen compounds from environmental samples [87]. Besides the proteomic signature method, the specific protein that is induced by individual chemicals could be used as a biomarker to monitor the environment [86]. For example, under the treatment of 2,4-DCPP, Delftia acidovorans MC1 was found to be induced specific proteins which were involved in the metabolism of 2,4DCPP, such as chlorocatechol 1,2-dioxygenases (TfdC, and TfdCII) and 2,4-DCP hydroxylase (TfdB) [64]. Also, elongation factor 1 β and enolase 1 from Caco-2 cells were reported to be the biomarkers for the risk assessment of water and wastewater [86].

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The development of proteomics relies on the development of a series of technologies, from protein separation and identification to gel image capturing and comparing technologies, and any of them could be the bottle-neck of the development of proteomics. The main challenges of proteomics development are high sample throughput and reproducibility, which are also the limitation of the application of proteomic technology in environmental researches. 2-DE can analyze one gel each time, and can be replaced by DIGE to increase the separation throughput, although the cost is higher. Up to three dye signals can be detected in one gel using DIGE because the dyes have distinct excision and emission wavelengths. The other improved separation technologies all rely on automation. The identification step also faces the high throughput challenge, because the current MS-based techniques cannot completely cover a proteome of a cell or an organism with more than 1000 proteins [47]. The low expression level of proteins is the other limit of identification, as the separated proteins cannot be enhanced in quantity. And even worse, under some stressed conditions, some proteins’ expression is depressed. Data analysis steps have to analyze high throughput data, including the separation data and the identification data. The developed computer technologies and bioinformatics could provide the technological support, and a lot of established proteomic databases could provide the platform, such as the SWISS-PROT database, which is the best starting point for protein sequence searches due to its large amounts of information and the multitude of cross-referencing. The development of protein microarray technology provides another possibility to increase the throughput, which does not depend on gel electrophoresis, and MS technologies and the super protein detection limit enable it to detect the level of protein. Unlike genomic level study, where the ultimate optimized technologies have been developed so that the findings of different laboratories can be compared and referenced easily, proteomic technologies do not have an ultimate platform yet. Researchers using different technological platforms always face the difficulties when they compare their results with other studies. Even when the same platform is used, the results will be not comparable either, due to the different set of proteins or biomarkers chosen by researchers. Moreover, even one researcher using the same platform for protein identification cannot guarantee the good reproducibility every time. Good reproducibility is the key step for environment monitoring, especially for the PESs depending monitoring tasks, and the reproducibility needs intensive labor and skillful hands. However, the organisms’ responses at the proteomic level to other environmental changes such as pH, atmosphere, light, and temperature, besides the chemical stresses make the poor reproducibility even worse. To solve the reproducibility problem, the ultimate platform needs to be developed, which relies on technology development

and commercial kits. In addition, an automatic process is another way to increase reproducibility, which could avoid man-made interfering errors. The study of proteomics is more complicated than genomics, because the numbers and types of proteins in different cells of one individual organism are not the same, and even one cell contains different proteins in its diverse growth stages or under various conditions and stresses. In addition, the limitation of the current technology makes the proteomic study more challenging. Despite such challenges, the application of proteomic approaches in environmental science developed rapidly in recent years due to its advantages of a global approach to understanding the complex mechanisms of environmental stresses and sensitive capability of monitoring an individual or a group of pollutants. With the development of high reproducibility and throughput technologies, wide applications, and increased demand in environmental studies, the cost is expected to be reduced in the future, which would in return promote proteomics applications in environmental sciences.

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