Protein Profiles in Zebrafish (Danio rerio) Embryos Exposed to Perfluorooctane Sulfonate

TOXICOLOGICAL SCIENCES110(2),334–340(2009)

doi:10.1093/toxsci/kfp111

Advance Access publication May27,2009

Protein Pro?les in Zebra?sh(Danio rerio)Embryos Exposed to

Per?uorooctane Sulfonate

Xiongjie Shi,*Leo W.Y.Yeung,?Paul https://www.sodocs.net/doc/307830979.html,m,?Rudolf S.S.Wu,?and Bingsheng Zhou*,1 *State Key Laboratory of Freshwater Ecology and Biotechnology,Institute of Hydrobiology,Chinese Academy of Sciences,Wuhan430072,China;and ?Department of Biology and Chemistry,City University of Hong Kong,HK SAR,China

Received March16,2009;accepted May15,2009

Per?uorooctane sulfonate(PFOS)is widely distributed and persistent in the environment and in wildlife,and it has the potential for developmental toxicity.However,the molecular mechanisms that lead to these toxic effects are not well known.In the present study,proteomic analysis has been performed to investigate the proteins that are differentially expressed in zebra?sh embryos exposed to0.5mg/l PFOS until192h postfertilization.Two-dimensional electrophoresis coupled with mass spectrometry was employed to detect and identify the protein pro?les.The analysis revealed that69proteins showed altered expression in the treatment group compared to the control group with either increase or decrease in expression levels(more than twofold difference).Of the69spots corresponding to the proteins with altered expression,38were selected and subjected to matrix-assisted laser desorption/ionization tandem time-of-?ight mass spectrometry(TOF/TOF)analysis;18proteins were identi?ed in this analysis.These proteins can be categorized into diverse functional classes such as detoxi?cation,energy metabo-lism,lipid transport/steroid metabolic process,cell structure, signal transduction,and apoptosis.Overall,proteomic analysis using zebra?sh embryos serves as an in vivo model in environ-mental risk assessment and provides insight into the molecular events in PFOS-induced developmental toxicity.

Key Words:PFOS;proteomics;embryo;mechanism;zebra?sh. The widespread use of per?uorinated chemicals(PFCs)in a variety of commercial and industrial applications,such as protective coatings for packaged foods,textiles,and carpets and as constituents of insecticides,has resulted in global contamination.Among the different types of PFCs,the levels of per?uorooctane sulfonate(PFOS)are routinely measured.It has been found that PFOS is a predominant contaminant in the environmental matrices and wildlife and has been shown to biomagnify in the aquatic food web(Kannan et al.,2005; Martin et al.,2004).For example,high concentrations of PFOS have been detected in?sh liver tissue(Giesy and Kannan, 2001;Hoff et al.,2003;Kannan et al.,2002).In addition, PFOS has also been detected in the eggs of the lake white?sh (Coregonus clupeaformis)from Michigan waters(Kannan et al.,2005),suggesting oviparous transfer of this compound to offspring.Increasing evidences show that exposure to PFOS can induce various toxic effects,such as hepatotoxicity in animals(Hoff et al.,2003;Lau et al.,2003),developmental and reproductive toxicity(Ankley et al.,2005;Du et al.,2009; Lau et al.,2003;Oakes et al.,2005;Shi et al.,2008; Thibodeaux et al.,2003),interferences in cell-cell communi-cation(Hu et al.,2002),and mitochondrial bioenergetics (Berthiaume and Wallace,2002;Kleszczyn′ski et al.,2009). Proteins are the actual functional molecules in the cell. Therefore,proteomics analysis may provide more direct insights into the mechanisms of effects.Such analysis has recently been employed to gain a better understanding of toxicity and the mechanisms of exposure to several toxicants,such as 2,2#,4,4#,5-pentabromodiphenyl ether in mice(Alm et al., 2006),per?uorooctanoic acid in rare minnow(Gobiocypris rarus)(Wei et al.,2008),polychlorinated biphenyls(PCBs) mixture Aroclor1254in African clawed frogs(Xenopus laevis) (Gillardin et al.,2009),microcystin in medaka(Oryzias latipes) (Mezhoud et al.,2008),and in zebra?sh liver treated with tetrabromobisphenol-A(De Wit et al.,2008).The importance of engaging in zebra?sh proteomics to reveal the potential mechanisms of the model of action has been highlighted in recent studies(Love et al.,2004;Tay et al.,2006).Zebra?sh embryos have been employed for the rapid and high-throughput screening of environmental chemicals for developmental toxicity and the mechanisms of toxicant exposure.A proteomic approach to zebra?sh embryos covers many different aspects of the changes in proteins during the early embryonic stages;thus,it may provide the means to?nally unravel the mechanisms that link patterning to the generation of embryonic forms(Reintsch and Mandato,2008).Furthermore,the application of embryo proteomics may lead to earlier and more sensitive toxicity analysis and also advance the zebra?sh embryo bioassay in ecotoxicological testing.A detailed proteomic protocol has been developed,from preparation to protein identi?cation in zebra?sh embryos and the global protein expression pro?les of zebra?sh embryogenesis(Link et al.,2006;Lucitt et al.,2008;Tay et al., 2006).For instance,previous reports on zebra?sh proteomics

1To whom correspondence should be addressed.Fax:t86-27-68780123.

E-mail:bszhou@https://www.sodocs.net/doc/307830979.html,.

óThe Author2009.Published by Oxford University Press on behalf of the Society of Toxicology.All rights reserved. For permissions,please email:journals.permissions@https://www.sodocs.net/doc/307830979.html, at Research Center of Eco-Environmental Sciences, CAS on December 15, https://www.sodocs.net/doc/307830979.html, Downloaded from

have concerned embryos administered with endocrine disrupted chemicals(Shrader et al.,2003)and ethanol(Gu¨ndel et al., 2007),thus suggesting certain expressed proteins as the sensitive stress indicators in zebra?sh embryos and re?ecting the overall ?tness of the intact organisms.

Although the effect of PFOS on gene expression of developing zebra?sh has been reported(Shi et al.,2008), there are no studies focusing on the potential effects of PFOS on the protein expression pro?le of developing zebra?sh.In order to understand the mechanisms underlying PFOS toxicity during zebra?sh development,we used two-dimensional gel electrophoresis(2-DE)and peptide mass?ngerprinting(PMF) to investigate the protein expression pro?les of zebra?sh embryos that had been exposed to PFOS.Proteomic analysis indicated that PFOS regulates a set of proteins that are involved in signal transduction,hormone activity regulation,structure formation,membrane transportation,and energy metabolism.

MATERIALS AND METHODS

Chemicals Heptadeca?uorooctanesulfonic acid potassium salt(PFOS,> 99%)was purchased from Tokyo Kasei Kogyo Co.Ltd(Tokyo,Japan).The stock solution(50,000mg/l)was prepared by dissolving the crystals in high-performance liquid chromatography grade dimethyl sulfoxide(DMSO)and storing it at4°C.All the chemicals used for electrophoresis were purchased from Amersham Biosciences(Piscataway,NJ).

Animals and Exposure Mature wild-type(AB strain)zebra?sh(about8 months old)were maintained at28±0.5°C in a01400h:1000h light:dark cycle in a continuous?ow-through system in charcoal-?ltered tap water.The?sh were fed twice daily with Artemia nauplii.Zebra?sh eggs were obtained from the spawning adults in groups of about20males and10females held in tanks overnight.At4–5h postfertilization(hpf),we randomly distributed approxi-mately300normally developed embryos into each beaker and exposed them to 0and0.5mg/l PFOS;the embryos were then grown up to192hpf.The exposure solution contained0.2mM Ca(NO3)2,0.13mM MgSO4,19.3mM NaCl,0.23mM KCl,and1.67mM N-2-hydroxyethylpiperazine-N#-2-ethanesulfonic acid buffer. The concentrations of PFOS were selected on the basis of our previous study(Shi et al.,2008).Exposure of the embryos to0.5mg/l PFOS did not cause a signi?cant increase in mortality over the entire exposure time.This indicated that the protein expression changing in our experiment may mostly are response to the toxic properties of chemical but not to acute biological damage of this compound.Our exposure time was selected because at192hpf,all the organs of the embryos,except gonads,are well developed.The control and treated embryos received0.001%DMSO,and three replicates for each treatment concentration were conducted.The zebra?sh embryos/larvae were examined at the end of exposure time.After192hpf,the toxicity end points,including morphological abnormalities,survival rates,and body length,were recorded,and zebra?sh larvae were snap frozen in liquid nitrogen and stored inà80°C.

Protein Extraction Protein extraction was performed using the method reported by Tay et al.(2006),with slight modi?cation.Brie?y,at192hpf, using a power homogenizer,approximately200zebra?sh larvae were homogenized in500l l lysis buffer containing7M urea,2M thiourea,4%3-[(3-chola-midopropyl)dimethylammonio]-1-propanesulfonate(CHAPS),1% wt/vol dithiothreitol(DTT),40mM Tris base,1%protease inhibitor cocktail, 0.5l l benzonase(25U/l l,>99%purity;Novagen,Madison,WI),and20l l/ml Bio-Lytes3/10.The samples were then disrupted by intermittent sonic oscillation for5min and incubated on a shaker for30min at4°C.Insoluble particles were removed by centrifugation at12,0003g for60min at4°C,after which the supernatants were collected.Four volumes of100%ice-cold acetone were added into1vol of supernatant.The samples were precipitated atà20°C for1h and then centrifuged at12,0003g for15min at4°C.The supernatants were then discarded,and the protein pellets were dissolved in a protein solution buffer(7M urea,2M thiourea,4%CHAPS,1%wt/vol DTT,and0.5% Immobilized pH Gradient(IPG)buffer).The protein concentration was determined using a2-D Quant Kit(GE Healthcare,Piscataway,NJ).

Two-Dimensional Electrophoresis In brief,the protein solution(350l g protein on analytical gels or1mg on preparative gels)was adjusted with a rehydration buffer(7M urea,2M thiourea,4%CHAPS,1%wt/vol DTT,0.5% IPG buffer,and a trace of bromophenol blue)for a?nal volume of350l l. Isoelectric focusing(IEF)was performed in IPG strips(pH3–10,18cm,GE Healthcare)at30V for12h,200V for1h,500V for1h,1000V for1h,8000V for1h,and then8000V constant for a total of56,000Vh on a Multiphor II system(GE Healthcare).After the IEF program,the strips were equilibrated in an IPG equilibration buffer(6M urea,2%SDS,30%glycerol,0.375M Tris,pH8.8, 20mg/ml DTT,and a trace of bromophenol blue)and then alkylated(25mg/ml iodoacetamide instead of DTT in an equilibration buffer)for15min each.The2-DE was performed in polyacrylamide gels(12.5%T[the total acrylamide and Bis monomer concentration in g/100ml]and2.6%C[the percentage of cross-linker in the total amount of acrylamide])with an Ettan DALT II system (GE Healthcare).Electrophoresis was carried out at20milliamperes per gel for 40min,followed by separation at30milliamperes per gel until the dye front had nearly reached the bottom.The protein spots were visualized via silver staining in analytical gels and via coomassie brilliant blue G-250staining in preparative gels.Two-dimensional gels were performed in triplicate and from three independent protein extractions for each group.

Image Acquisition and Analysis The gel images were captured on an ImageScanner(GE Healthcare).Image Master2D Platinum(GE Healthcare) software was used to match and analyze the images.The protein spots were detected automatically and then edited manually to remove streaks,speckles, and artifacts.The quanti?cation of the proteins was expressed as the volume of spots,which was determined in comparison with the total volume of all the spots within the gel.Only twofold increase or decrease changes between the PFOS-treated and control groups were considered as regulated spots.

Protein Identi?cation The proteins of interest were selected and destained two times using200mM ammonium bicarbonate in50%acetonitrile/water for 45min at37°C.The gels were then dehydrated using acetonitrile and spun dry. The dried gel bands were rehydrated in a minimal volume of25mM ammonium bicarbonate buffer that contained10ng/l l modi?ed trypsin(Promega, Madison,WI)and incubated overnight at37°C.Matrix-assisted laser desorption/ionization-time of?ight(MALDI)-TOF experiments were per-formed on an Ultra?ex TOF-TOF instrument(Bruker Daltonic,Bremen, Germany).The instrument was set in re?ector mode.Protein identi?cation was carried out using a combination of PMFs and peptide fragmentation patterns as inputs to search the National Center for Biotechnology Information(NCBI) nonredundant(nr)database using the Mascot search engine(https://www.sodocs.net/doc/307830979.html,).The classi?cation and functions of the proteins identi?ed were obtained by searching Gene Ontology(https://www.sodocs.net/doc/307830979.html,).

Statistical Analysis The normality of the data was veri?ed using the Kolmogorov-Smirnov test,and the homogeneity of variances was checked with Levene’s test.A two-tailed Student’s t-test was used to determine the signi?cant differences between the control and exposure groups.Statistical analysis was performed using SPSS13.0software(SPSS,Chicago,IL),and p <0.05was considered to be a statistically signi?cant difference.

RESULTS

Mortality and Growth There was no signi?cant difference in the survival rate between the control(81.11±4.01%)and the treatment(83.33±1.93%)groups.Some developmental abnormalities,such as yolk sac edema and spinal curvature, were recorded in the treatment group(5.56±1.12%),but there

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was no signi?cant difference in the percentage of abnormalities observed in the treatment group and that in the control group (1.11±1.92%).There was a signi?cant reduction in body length of the PFOS-exposed group(3.92±0.02mm)compared to the control group(3.79±0.03mm;p<0.001).

Two-DE and mass spectrometry Identify Proteins The separated proteins were stained with silver(Fig.1),and,after automatic marking with the software and manual editing,more than1200protein spots were detected on each gel.These spots were quantitatively measured,and comparisons were made with Image Master2D Platinum software.In this study,a twofold change cutoff was used as the criterion for differential expression. Compared with the gels from the controls,69protein spots were found to have been altered by the effects of PFOS.

Identical gels were run and used for in-gel tryptic digestion, followed by mass spectrometry.A total of38protein spots were selected for protein identi?cation based on spot intensity and spot integrity(Fig.1).After careful manual extraction,the protein spots were subjected to MALDI-TOF-mass specrom-etry(MS)/MS analysis.After a PMF search in the NCBI nr database,18spots were successfully identi?ed.PFOS exposure resulted in8proteins being upregulated and10being downregulated.The protein codes,accession numbers,descrip-tions,and fold changes are listed in Table1.Spots1,11,25, 30,and32resulted in the identi?cation of the proteins involved in the nucleotide and ATP metabolic process,including nucleoside diphosphate kinase(NDPK)-Z2,cytidylate kinase, adenylate kinase(AK)2,Ckmb protein(one of the creatine kinases[CKs]),and citrate synthase(CS).Spot2was identi?ed as oxysterol-binding protein-like1A(OSBP1A),which is related to lipid transport and the steroid metabolic process.The program identi?ed Spot3as crystallin,gamma MX that plays a role in the neurological system process.PMF analysis of Spot 4identi?ed it as F-box protein44,which is implicated in the regulation of cellular metabolism progress.Spot12was identi?ed as peroxiredoxin2,which is known as an antioxidation protein.Mass spectrometry identi?cation found Spot15to be vertebrate microtubule-actin cross-linking factor 1(MACF1).This protein is thought to take part in multiple biological processes,such as signal transduction,cell motion, anatomical structure formation,cell cycle arrest,and in-tracellular protein transport.Spot20was identi?ed as phosphoglycerate mutase1,a transferase enzyme.Spot22 was identi?ed as sulfotransferase(SULT),which plays a critical

role in the steroid metabolic process.Spots23and28were identi?ed as embryonic lethality and abnormal visual(ELAV)-like4and annexin A4(Anx4),respectively,and Spot33was identi?ed as glycine C-acetyltransferase,which belongs to the family of transferases and participates in glycine,serine,and threonine metabolism.The database search results showed that Spot34is eukaryotic translation elongation factor1gamma,an essential nucleotide-binding protein that catalyzes transloca-tion.Spot35was identi?ed as RNA-binding protein4(Rbp4),which is known to play a role in the RNA metabolic process. Finally,Spot38was identi?ed as tyrosine recombinase,which participates in diverse cellular biopolymer metabolic processes.

DISCUSSION

Proteomics is an ef?cient method to identify new proteins as well as to investigate the ecological risk assessments.It may

be FIG. 1.Representative2-DE gels of the protein expression pro?les obtained from the zebra?sh larvae after exposure to0.5mg/l until196h.(A) 2D gel image with proteins expressed in the control condition;(B)2D gel image with proteins expressed in the0.5mg/l PFOS exposure condition.The proteins of the samples were separated on a pH3–10liner IPG strip,followed by12.5%SDS-polyacrylamide gel and silver staining.The circle and number allocated by the Image Master2D Platinum software indicate spots with signi?cant changes in intensity(p<0.01,Student’s t-test in three independent gels).Each experiment was conducted independently at least three times,and an image taken from one representative experiment is shown.

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useful in providing insights into the molecular mechanisms underlying PFOS-induced responses in developing zebra?sh embryos.Most interestingly,among the proteins that showed differential expression at 192hpf after PFOS exposure,those involved in energy and cholesterol metabolism,detoxi?cation,membrane integrity,and cytoskeleton maintenance were pre-dominantly affected.

Peroxiredoxin 2belongs to a family of small antioxidant proteins that catalyze the reduction of hydrogen peroxide and other reactive oxygen species (ROS).It is thought to be involved in eliciting cellular defenses against ROS (Chaudhary et al.,2007).In the present study,a 3.11-fold increase in peroxiredoxin 2(see Table 1)expression was observed.A previous study showed that the expression of p53and Bax genes and oxidative stress was induced in zebra?sh embryos exposed to 0.1–0.5mg/l of PFOS (Shi et al.,2008).Induction of the expression of the detoxi?cation gene has also been reported in zebra?sh exposed to per?uorododecanoic acid (Liu,Wang,et al.,2008).PFOS

TABLE 1

The Differentially Altered Proteins Successfully Identi?ed in the Zebra?sh Larvae following 0.5mg/l PFOS Exposure

No.on gel

Accession no.

Identi?cation Fold change

Mw (kDa)

pI Match rate

Sequence coverage %

Score Functional description

Detoxi?cation 12gi j 50539996

Peroxiredoxin 2 3.1126.60 5.3910/4767108

Anti-apoptosis,oxidation reduction,

activation of MAPK activity,response to lipopolysaccharide,cell redox homeostasis 22gi j 47550819SULT 3.5743.537.4310/464460

Steroid metabolic process,catecholamine metabolic process Energy metabolism 1gi j 41053595NDPK-Z2

0.4018.157.2315/4878137UTP biosynthetic process,GTP biosynthetic process,CTP biosynthetic process 11gi j 150383502UMP-CMP kinase (cytidylate kinase)0.1225.49 5.0014/4675112Pyrimidine nucleotide metabolic process 25gi j 47086835AK 20.3544.42 5.7513/445377Nucleobase,nucleoside,nucleotide,and nucleic acid metabolic process 30gi j 31419240Ckmb protein 0.3145.008.0612/463248Phosphocreatine biosynthetic process 32gi j 41054571CS

0.25105.708.1319/4732114Cellular carbohydrate metabolic process,response to stress,tricarboxylic acid cycle 20

gi j 38488700

Phosphoglycerate mutase 1

0.33

38.83

7.74

14/45

57

97

Alcohol catabolic process,regulation of glycolysis,respiratory burst,regulation of pentose-phosphate shunt Membranes 28gi j 32401412Anx40.3544.71 6.7620/4865125Transport,anti-apoptosis,signal transduction Cholesterol metabolism 2gi j 113682341OSBP1A

0.2821.808.1313/441342Lipid transport,steroid metabolic process Cytoskeleton 3gi j 61651684Crystallin,gamma MX

0.4622.287.3013/4769152Neurological system process

15gi j 94732601

Vertebrate MACF1

2.08

33.26

8.11

34/47

17

69

Signal transduction,cell motion,anatomical structure formation/cell cycle arrest,intracellular protein transport Acyltransferases 33gi j 189517050Glycine C-acetyltransferase 0.1094.547.5414/483166Biosynthetic process Translation 34gi j 27545277

Eukaryotic translation

elongation factor 1gamma

3.49

123.73

8.21

14/46

33

63

Translational elongation

Transiently bind covalently to DNA 38gi j 189528745Tyrosine recombinase 2.05121.16 5.8811/463252Cellular biopolymer metabolic process RNA-binding protein 35gi j 62511030Rbp4 2.33

105.707.7417/4537105RNA splicing,mRNA processing 23gi j 18858617ELAV-like 4 2.1041.18 6.2410/462361Unkown

Mediates protein-protein interactions 4gi j 189535873F-box protein 44

2.23

23.36

6.19

12/45

29

45

Protein catabolic process

Note .UMP-CMP,uridine monophosphate/cytidine monophosphate;No.on gel,spot number as noted on the 2D gels;accession no.,the Mascot results of the MALDI-TOF-MS/MS search of the NCBI nr database;fold change,the average fold changes as compared to the controls;Mw,molecular weight;pI,isoelectric point;match rate,the percentage of the number of mass values matched to the number of mass values searched;sequence coverage,the percentage sequence coverage of the hit;score,Mascot probability based on the Mowse score calculated for the MS/MS results;functional description:the biological processes in Gene Ontology terms.The average fold changes as compared to the controls.Signi?cance is reached at a score !40.A number >1indicates upregulation,and a number <1indicates downregulation.

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exposure in cultured tilapia and Atlantic salmon(Salmo salar) hepatocytes led to the generation of ROS and the induction of detoxifying phase II enzymes such as glutathione-S-transferase (Kr?vel et al.,2008;Liu et al.,2007).To the best of our knowledge,this is the?rst study indicating an association between PFOS and the upregulation of activated detoxi?cation proteins.The upregulation of antioxidant proteins is in agreement with the observation that PFOS can induce oxidative stress.These results suggest that the elicitation of cellular responses to PFOS toxicity may result in the activation of the detoxi?cation pathway,indicating the disruption of the normal cellular homeostasis and induction of proteins involved in protective response in order to reduce oxidative damage.

The expression of SULTs was signi?cantly upregulated in the zebra?sh larvae exposed to PFOS.SULTs represent the major phase II enzymes.They are involved in the bio-transformation of endogenous chemicals such as steroid/ thyroid hormones and in the detoxi?cation of environmental xenobiotics in animals(Gamage et al.,2006).Zebra?sh SULT enzymes were recently cloned and were found to be signi?-cantly expressed in1-week-old larvae that displayed differen-tial sulfating activities toward xenobiotics such as phenolic compounds and PCBs(Liu,Bhuiyan,et al.,2008).This is the ?rst report that shows the SULT protein is signi?cantly upregulated in the PFOS-treated zebra?sh larvae.To date,there has been no evidence that PFOS can be metabolized in vivo; therefore,it is unlikely that the upregulation of SULTs could be due to the formation of a metabolic product of PFOS. However,whether the increase in the level of SULTs contributes to the depression in the expression of the thyroid hormone or sex hormone remains to be elucidated.Neverthe-less,the sensitivity induction of SULTs in response to xenobiotics may provide suf?cient basis to investigate the importance of SULTs in chemical risk assessment.

Energy metabolism is considered to be essential for the early development of larvae.Our results show that PFOS exposure signi?cantly suppresses the expression of many proteins involved in the tricarboxylic acid cycle and ATP biosynthesis, namely,CS,NDPKs,CK,and AK.NDPK is an indicator of biosynthesis and is a crucial in the early embryogenic development and growth(Murphy et al.,2000).On the other hand,CK and AK play important roles in the regulation of cellular energy homeostasis and energy transfer,which is a vital process in tissues with high and rapidly changing energy demand,such as brain,heart,and liver and the early stages of fetal development(Dzeja et al.,1998).Thus,suppression of CK and AK would result in cellular dysfunction and pathological changes in the cellular energy state.In a previous study,exposure of common carp(Cyprinus carpio)to waterborne PFOS led to the downregulation of the genes in the liver that were mainly involved in energy metabolism and oxidative phosphorylation(Hagenaars et al.,2008).A more recent study indicated that PFOS exposure resulted in the disruption of mitochondrial bioenergetics and ATP loss in rat hepatocytes(Kleszczyn′ski et al.,2009).Thus,the down-regulation of energy metabolism observed in our study is in agreement with the previous reports on decreased ATP bio-synthesis in vitro.This observation proves the hypothesis that PFOS can affect energy metabolism.The metabolic cost hypothesis predicts that nonvital processes such as growth and reproduction are generally more affected(Rowe et al.,2001). This indicates that an increase in energy expenditure negatively affects the processes vital to the survival of organisms such as growth.Hence,PFOS exposure results in the reduction of stored energy that is required for the maintenance of growth and survival of organisms.In the present study,we observed that the average body length of the zebra?sh larvae was signi?cantly reduced after exposure to0.5mg/l PFOS;this observation is in agreement with our previous study indicating that PFOS exposure affects the growth of zebra?sh larvae(Shi et al.,2008).The reduction in the growth of PFOS-exposed zebra?sh larvae may be associated with an impairment of the bioenergetic pathways that are induced in response to an increased energy demand under stress conditions.

OSBP1is a member of the lipid-binding protein family. OSBP is known to play an important role in the regulation of intracellular sterol levels.When sterols accumulate in cells, oxysterols,which are a group of oxidized derivatives of sterols, act as potent secondary messengers that trigger the suppression of cholesterol synthesis.OSBP1is the only protein known to recognize oxysterols,and its activity is known to be associated with the regulation of cholesterol metabolism(Levine and Munro,2001).In our study,OSBP1expression was found to be signi?cantly downregulated,which suggests that cholesterol metabolism was possibly affected in PFOS-exposed zebra?sh larvae.In the early stages of development,animals need enor-mous amounts of cholesterol to maintain membrane growth, which is an essential step in cellular proliferation,growth,and differentiation.Cholesterol is the precursor of steroid hormones and oxysterols and is known to play critical role in a variety of biological processes,including homeostasis,sexual develop-ment,and reproduction(Miller,1988;Woollett,2008).Sup-pression of OSBP1may affect cholesterol synthesis and, thereby,contribute to the decrease in the synthesis of steroid hormones.Therefore,it might be considered that PFOS affects the development and reproduction of animals by suppressing cholesterol levels,although this probability needs to be further investigated.In addition,cholesterol is a key component of every membrane,and it helps maintain normal membrane ?uidity.Downregulation of OSBP1protein would result in the suppression of cholesterol synthesis;thus,cell membrane ?uidity might be affected,which has also been observed after PFOS exposure(Hu et al.,2003).

This is the?rst report showing the downregulation of annexins in zebra?sh larvae exposed to PFOS.It has been suggested that annexin complexes may be involved in the modi?cation of membrane structure,including?uidity and permeability,anchoring of cytoskeletal elements,aggregation

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of vesicles,and regulation of ion conductance(Gerke and Moss,2002).Annexin genes are known to be expressed in a wide range of tissues in zebra?sh during the embryonic and larval stages.On the other hand,the temporal and spatial expression patterns of Anx4in zebra?sh suggest that it is involved in pronephric tubule development(Farber et al., 2003).In the present study,we observed that Anx4protein expression was downregulated.Given its amphiphillic nature and that it is structurally homologous to the free fatty acids of PFOS,it could primarily affect cell membrane integrity.It has been hypothesized that Anx4is highly active at the lipid/ protein interfaces within the membranes and has been found to increase the permeability and?uidity of cell membranes in?sh leukocytes and to affect mitochondrial membrane potential(Hu et al.,2003).A recent study showed the dissipation of plasma membrane potential leading to the acidi?cation of the cytosol in cultured cells exposed to PFCs(Kleszczyn′ski and Sk?adanowski,2009).Therefore,the downregulation of Anx4 may be associated with PFOS-mediated dysregulation of the cell membrane function.The involvement of Anx4in many membrane-related events,such as the regulation of ion?uxes across membranes and membrane permeability,need to be elucidated,and further research is warranted to demonstrate this association.

We also observed that PFOS exposure affected the expression of a few proteins such as crystallin,vertebrate MACF1,and Rbp4that are known to be associated with structure formation,signal transduction,and RNA splicing. MACF1is a cytoskeletal linker protein.It can connect both the micro?laments and microtubules that are vital for controlling microtubule dynamics and plays an important role in signal transduction,protein transportation,and embryonic develop-ment(Chen et al.,2006).The function of MACF1in zebra?sh is yet unknown;however,since PFOS is known to affect cell membrane?uidity and communication,it can be considered that the cell skeleton integrity may also be affected.The cytoskeletal network integrity is important for maintaining cell morphology and cell locomotion.Since MACF1is known to play a critical role in the intracellular transport as well as in embryo reorganization,the changes in the levels of MACF1 may affect the cellular functions associated with the cytoskel-eton;further studies are required in this regard.

Our results show that a broad spectrum of proteins involved in zebra?sh larvae development are affected by PFOS exposure.Besides the proteins that have been previously reported to be affected by PFOS exposure,other important proteins,including uridine monophosphate/cytidine monophos-phate kinase(cytidylate kinase),phosphoglycerate mutase1, eukaryotic translation elongation factor1c,and c-crystallins, that are involved in cellular processes were found to be induced in response to PFOS.For instance,c-crystallins,which is typically found in vertebrates,are expressed in zebra?sh lens (Xu et al.,2000).Recently,injections of c-crystallins were found to strongly enhance the regeneration of axons in the retinal ganglion cells(Fischer et al.,2008).The down-regulation of c-crystallin protein in PFOS-exposed zebra?sh larvae appears to affect lens development.These?ndings provide the prospects of identifying new toxic mechanisms. In summary,the present study has demonstrated that PFOS exposure in zebra?sh embryos/larvae causes signi?cant changes in the abundance of69proteins,as observed on2D gels.We successfully identi?ed18proteins that are involved in energy metabolism,cholesterol disruption,oxidative stress,and mem-brane integrity and can be assigned to functional groups that are known to be affected by PFOS toxicity.However,the other proteins that showed differential expression and their functional roles yet remain to be identi?ed and hence require further investigation.Alterations in protein expression in zebra?sh larvae can provide new clues to the molecular mechanisms underlying PFOS toxicity in developing zebra?sh.Our study also indicates that zebra?sh embryo proteomics can be used as a good model for investigating environmental risk assessment.

FUNDING

National Nature Science Foundation of China(20890113, 20877094);State Key Laboratory of Freshwater Ecology and Biotechnology(2008FBZ10).

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第35卷　第1期2011年1月 南京林业大学学报(自然科学版) J o u r n a l o f N a n j i n g F o r e s t r y U n i v e r s i t y (N a t u r a l S c i e n c e E d i t i o n ) V o l .35,N o .1 J a n .,2011 h t t p ://w w w .n l d x b .c o m [d o i :10.3969/j .i s s n .1000-2006.2011.01.024] 　收稿日期:2009-12-31 修回日期:2010-10-26 　基金项目:国家自然科学基金项目(31000287);江苏省高校自然科学基础研究项目(10K J B 220002)　作者简介:甄艳(1976—),副教授,博士。＊施季森(通信作者),教授。E -m a i l :j s h i @n j f u .e d u .c n 。 　引文格式:甄艳,施季森.质谱技术在蛋白质组学研究中的应用[J ].南京林业大学学报:自然科学版,2011,35(1):103-108. 质谱技术在蛋白质组学研究中的应用 甄　艳,施季森 ＊ (南京林业大学,林木遗传与生物技术省部共建教育部重点实验室,江苏　南京　210037) 摘要:随着蛋白质组学研究的迅速发展,质谱技术已成为应用于蛋白质组学研究中的强有力工具和核心技术。质谱技术的先进性在于为蛋白质组学研究提供的通量和分子信息。笔者重点概述了基于质谱路线的蛋白质组学研究,介绍了基于质谱的定量蛋白质组学﹑翻译后修饰蛋白质组学、定向蛋白质组学、功能蛋白质组学以及基于串联质谱技术的蛋白质组学数据解析的研究 进展。 关键词:质谱;蛋白质组学;定量蛋白质组学;翻译后修饰;定向蛋白质组学;功能蛋白质组学中图分类号:Q 81 文献标志码:A 文章编号:1000-2006(2011)01-0103-06 A p p l i c a t i o n o f m a s s s p e c t r o m e t r y i n p r o t e o m i c s s t u d i e s Z H E NY a n ,S H I J i s e n ＊ (K e y L a b o r a t o r y o f F o r e s t G e n e t i c s a n d B i o t e c h n o l o g y M i n i s t r y o f E d u c a t i o n , N a n j i n g F o r e s t r y U n i v e r s i t y ,N a n j i n g 210037,C h i n a ) A b s t r a c t :W i t ht h e r a p i d d e v e l o p m e n t o f p r o t e o m i c s ,m a s s s p e c t r o m e t r y i s m a t u r i n g t o b e a p o w e r f u l t o o l a n dc o r e t e c h -n o l o g y f o r p r o t e o m i c s s t u d i e s d u r i n g t h e r e c e n t y e a r s .T h e s u p e r i o r i t y o f m a s s s p e c t r o m e t r y l i e s i n p r o v i d i n g t h e t h r o u g h -p u t a n d t h e m o l e c u l a r i n f o r m a t i o n ,w h i c hn o o t h e r t e c h n o l o g y c a n b e m a t c h e di np r o t e o m i c s .I nt h i s r e v i e w ,w e m a d e a g l a n c e o n t h e o u t l i n e o f m a s s s p e c t r o m e t r y -b a s e d p r o t e o m i c s .A n dt h e nw e a d d r e s s e d o n t h e a d v a n c e s o f d a t a a n a l y s i s o f m a s s s p e c t r o m e t r y -b a s e dp r o t e o m i c s ,q u a n t i t a t i v em a s ss p e c t r o m e t r y -b a s e dp r o t e o m i c s ,p o s t -t r a n s l a t i o n a l m o d i f i c a t i o n s b a s e d m a s s s p e c t r o m e t r y ,t a r g e t e d p r o t e o m i c s a n df u n c t i o n a l p r o t e o m i c s b a s e d -m a s s s p e c t r o m e t r y . K e yw o r d s :m a s ss p e c t r o m e t r y ;p r o t e o m i c s ;q u a n t i t a t i v ep r o t e o m i c s ;p o s t -t r a n s l a t i o n m o d i f i c a t i o n ;t a r g e t e d p r o -t e o m i c s ;f u n c t i o n a l p r o t e o m i c s 蛋白质组学(P r o t e o m i c s )是从整体水平上研究细胞内蛋白质的组成、活动规律及蛋白质与蛋白质的相互作用,是功能基因组学时代一门新的学科。目前蛋白质组学的研究主要有两条路线:一是基于双向电泳的蛋白质组学;二是基于质谱的蛋白质组学,其中基于双向电泳的蛋白质组学研究路线最终也离不开质谱技术的应用。自20世纪80年代末,两种质谱软电离方式即电喷雾电离(e l e c t r o s p r a y i o n i z a t i o n ,E S I )和基质辅助激光解析离子化(m a -t r i x a s s i s t e d l a s e r d e s o r p t i o n i o n i z a t i o n ,M A L D I )的发明和发展解决了极性大、热不稳定蛋白质和多肽分 析的离子化和分子质量大的测定问题[1] ,蛋白质组学研究中常用的质谱分析仪包括离子阱(i o n t r a p ,I T ),飞行时间(t i m e o f f l i g h t ,T O F ),串联飞行时间(T O F -T O F ),四级杆/飞行时间(q u a d r u p o l e /T O F h y b r i d s ),离子阱/轨道阱(I T /o r b i t r a ph y b r i d ) 和离子阱/傅里叶变换串联质谱分析仪(I T /F o u r i e r t r a n s f o r m i o n c y c l o t r o nr e s o n a n c em a s s s p e c t r o m e t e r s h y b r i d s ,I T /F T M S ),这些质谱仪具有不同的灵敏度、分辨率、质量精确度和产生不同质量的M S /M S 谱[2] 。质谱作为蛋白质组学研究的一项强有力的工具日趋成熟,并作为样品制备及数据分析的信息学工具被广泛地应用。因此,有学者指出质谱技术 已在蛋白质组学研究中处于核心地位[3] 。目前在通量及所包含的分子信息内容上,基于质谱的蛋白质组学技术在细胞生物学研究中可以鉴定和量化

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蛋白质结构预测

实习 5 ：蛋白质结构预测 学号20090***** 姓名****** 专业年级生命生技**** 实验时间2012.6.21 提交报告时间2012.6.21 实验目的： 1.学会使用GOR和HNN方法预测蛋白质二级结构 2.学会使用SWISS-MODEL进行蛋白质高级结构预测 实验内容： 1.分别用GOR和HNN方法预测蛋白质序列的二级结构，并对比异同性。 2.利用SWISS-MODEL进行蛋白质的三级结构预测，并对预测结果进行解释。 作业： 1. 搜索一条你感兴趣的蛋白质序列，分别用GOR和HNN进行二级结构预测，解释预测结果，分析两个方法结果有何异同。 答：所选用蛋白质序列为>>gi|390408302|gb|AFL70986.1| gag protein, partial [Human immunodeficiency virus] （1）GOR预测结果： 图1 图1是每个氨基酸在序列中所处的状态，可以看出序列的二级结构预测结果为： 1到9位个氨基酸为无规卷曲，10到33位氨基酸为α螺旋,34到37位为β折叠，38到45位为无规卷曲，46到49位为α螺旋，50到53位为无规卷曲，54到65为α螺旋，66到72位为无规卷曲，73到95位为α螺旋，96到101位为无规卷曲，102到108为β折叠，109到115位为无规卷曲，117位为β折叠。 图2 图2为各种结构在序列中所占的比例，其中Alpha helix占53.85%，Extended strand占11.11%，Random coil占35.04%，无他二级结构。

图3 图3为各个氨基酸在序列中的状态以及二级结构在全序列中二级结构分布情况。 （2）HNN预测： 图4 图4是每个氨基酸在序列中所处的状态，可以看出序列的二级结构预测结果为： 1到6位个氨基酸为无规卷曲，7到34位氨基酸为α螺旋,35到37位为β折叠，38位为α螺旋，39到44位为无规卷曲，45到49位为α螺旋，50到55位为无规卷曲，56到65为α螺旋，66到71位为无规卷曲，72到83位为α螺旋，84到86位为无规卷曲，87到95位为α螺旋，96到102为无规卷曲，103到108位为β折叠，108到117位为无规卷曲。 图5 图5为各种结构在序列中所占的比例，其中Alpha helix占55.56%，Extended strand占7.69%，Random coil占36.75%，无他二级结构。

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蛋白质组学及其研究方法与进展 蛋白质是生命活动的体现者，基因的表达最后是通过蛋白质来体现的，在这个过程中，蛋白质起了连接基因与表现的功能。蛋白质是有氨基酸组成的，组成蛋白质的氨基酸的种类及排列顺序构成了蛋白质的一级结构，而在一级机构基础上的多肽链本身的折叠和盘绕方式构成了蛋白质的二级结构，考虑到多肽链上原子在空间的分布，由二级结构进一步形成了蛋白质的三级结构，对于有多个亚基的蛋白质还存在四级结构。 蛋白质的一级结构决定了高级结构，而高级结构则决定着蛋白质的生物学功能。如今对于蛋白质研究已经单独形成了一个活跃的生物学分支学科―――蛋白质组学，在蛋白质的研究中发挥着很重要的作用，下面将介绍蛋白质组学的一些基本内容及研究进展。 一.产生背景[1] 在20世纪中后期随着DNA双螺旋结构的提出和蛋白质空间结构的解析，生命科学研究进入了分子生物学时代，对遗传信息载体DNA和生命功能的体现者蛋白质的研究，成为了其主要内容。90年代初期启动的庞大的人类基因组计划．在经过各国科学家多年的努力下，已经取得了巨大的成就。10多种低等模式生物的基因组序列测定L三完成；第一个多细胞生物一线虫基因组的DNA全序列测定也在1998年年底完成；人类所有基因的部分序列测定(EST)已经完成；人类基因组的全序列测定有可能提前到2003年完成。生命科学已跨入了后基因组时代。在后基因组时代，研究重心将从揭示生命的所有遗传信息转移到在整体水平上对功能的研究。这种转向的第一个标志是产生了功能基因组学这一新学科，即从基因组整体水平上对基因的活动规律进行阐述。如在mRNA 水平上，通过DNA 芯片(DNA chips)和微阵列(Microarray)法等技术检测大量基因的表达模式，并取得了很好的进展。但是，mRNA的表达水平(包括mRNA的种类和含量)由于mRNA储存和翻译调控以及翻译后加工等的存在．并不能直接反映蛋白质的表达水平}蛋白质自身特有的活动规律，如蛋白质的修饰加工、转运定位结构形成、代谢、蛋白质与蛋白质及其他生物大分子的相互作用等．均无法从在基因组水平上的研究获知。因此，对生物功能的主要体现者或执行者一蛋白质的表达模式和功能模式的研究就成为生命科学发展的必然。在此背景下．80年代中期，国际上葫发了一门研究细胞内垒部蛋白质的组成及其活动规律的新兴学科- 蛋白质组学(Proteomic)。 蛋白质组(proteome)一词是马克.威尔金斯(Marc Wilkins)最先提出来的, 最早见诸于1995年7月的“Electrophoresis”杂志上它是指一个有机体的全部蛋白质组成及其活动方式。蛋白质组研究虽然尚处于初始阶段, 但已经取得了一些重要进展。当前蛋白质组学的主要内容是, 在建立和发展蛋白质组研究的技术方法的同时, 进行蛋白质组分析。对蛋白质组的分析工作大致有两个方面。一方面,通过二维凝胶电泳得到正常生理条件下的机体、组织或细胞的全部蛋白质的图谱, 相关数据将作为待检测机体、组织或细胞的二维参考图谱和数据库。一系列这样的二维参考图谱和数据库已经建立并且可通过联网检索。二维参考图谱

生态毒理基因组学和生态毒理蛋白质组学研究进展_戴家银

第26卷第3期2006年3月生 态 学 报ACTA EC OLOGI CA SI NICA Vol .26,No .3Mar .,2006生态毒理基因组学和生态毒理蛋白质组学研究进展 戴家银,王建设 (中国科学院动物研究所,北京　100080) 基金项目:中国科学院知识创新工程重要方向性资助项目(KSCX2-SW -128) 收稿日期:2005-08-30;修订日期:2005-12-05 作者简介:戴家银(1965～),男,安徽怀宁人,博士,研究员,主要从事生态毒理学和生物化学研究.E -mail :daijy @ioz .ac .cn Foundation item :The project was supported by the Innovation Project of Chines e Academy of Sciences (No .KSCX2-SW -128) Received date :2005-08-30;Accepted date :2005-12-05 Biography :DAI Jia -Yin ,Ph .D .,Professor ,mainly engaged in ecotoxicology and biochemis try .E -mail :daijy @ioz .ac .cn 摘要:将基因组学和蛋白质组学知识整合到生态毒理学中形成了生态毒理基因组学和生态毒理蛋白质组学。通过生态毒理基因组学和生态毒理蛋白质组学的研究能够在基因组和蛋白质组水平更深入理解毒物的作用机制,寻找更敏感、有效的生物标记物,形成潜在的强有力的生态风险评价工具。介绍了生态毒理基因组学和生态毒理蛋白质组学的研究进展,以及DNA 芯片技术和2D -凝胶电泳技术在持久性有毒污染物的生态毒理学研究中的应用。 关键词:生态毒理基因组学;生态毒理蛋白质组学;DNA 芯片技术;2D -凝胶电泳;持久性有机污染物 文章编号:1000-0933(2006)03-0930-05　中图分类号:X171　文献标识码:A Progress in ecotoxicogenomics and ecotoxicoproteomics DAI Jia -Yin ,WANG Jian -She (Institut e of Zoology ,C hines e Acade my of Sci ence s ,Beijing 100080,C hina )..Acta Ecologica Sinica ,2006,26(3): 930～934.Abstract :Ec otoxicogeno mics and ecotoxic oproteo mics are integration of genomics and proteomics into ec otoxicology .Ecotoxic ogenomics is defined as the study of gene and pr otein expr ession in non -target organisms that is impor tant in responses to environmental toxicant exposures .Ecotoxic ogenomic toolsmay provide us with a better mechanistic understanding of ec otoxicology ,and they are likely to provide a vital r ole in ecological risk assessment .Pr ogress in ec otoxicogenomics and ecotoxicoprote omics are discussed in this paper .DNA gene c hip and 2D -gel usually used in ecotoxicogeno mics and ecotoxicoproteomics ar e also e xpounded by exa mples . Key words :ec otoxicogeno mics ;ecotoxic oproteo mics ;D NA micr oarra y ;2D -gel ;persistent organic pollutants 随着生态学和环境科学的深入发展,生态毒理学已成为生态学和环境科学前沿研究领域,正从基因、蛋白质、器官和整体水平深入开展研究工作。 在人类基因组计划实施的短短几年间,以“组学(-omics )”构成的学科及其相关研究如雨后春笋般在生命科学界迅速蔓延、蓬勃发展。在环境科学领域中也出现了环境基因组学(environmental genomics )、毒理基因组学(toxicogenomics )等学科。Snape 等人[1～3]将基因组学知识整合到生态毒理学中,于2004年提出了“生态毒理基因组学(ecotoxicogenomics )”的概念,通过生态毒理基因组学研究确认一系列毒物效应基因,从而在基因组水平更深入理解毒物的作用机制,并在基因和蛋白质水平寻找更敏感、有效的生物标记物(biomarkers ),形成潜在的强有力的生态风险评价工具。 持久性有机污染物(Persistent Organic Pollutants ,POPs )是指能持久存在于环境中、通过食物链蓄积、逐级传递,经直接或间接途径进入人体的化学物质。POPs 具有致癌、致畸、致突变性、内分泌干扰等毒作用。POPs 对人体健康和生态环境带来的危害受到全社会的普遍关注,引起世界各国的决策者和科学家的高度重视,也成为环境科学和生态毒理学研究的热点课题之一[4,5]。我国已于2001年5月签署了控制12种P OPs 对人类健康