A proteomics strategy for protein expression profiling and by dyr60218

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									A proteomics strategy for protein
expression profiling and biomarker
discovery in wildlife: effects of
endocrine disrupting chemicals in
frog (Xenopus laevis)


  Anders Goksøyr 1,2, Christina C Tolfsen 1, Anneli Bohne Kjersem 1,
  Tina Søfteland, Torbjørn Midtun 1, Ralph Urbatzka, Werner Kloas 3,
  & Bjørn Einar Grøsvik 1,4
  1Department    of Molecular Biology, University of Bergen, Bergen, Norway
  2Biosense Laboratories AS, Bergen, Norway
  3Department of Inland Fisheries, Institute of Freshwater Ecology and Inland Fisheries, Berlin, Germany
  4Institute of Marine Research, Bergen, Norway
        Proteomics and the proteome
Proteomics = the study of all proteins expressed by the genome of
a given cell or tissue of an organism
                                                       DNA




                                                                                                                Complexity
                       A   C   G       G       T       A       T        C       A       A       C       G   G



                                                                       Transcription

                  mRNA             G       G       U       A       U        C       A       A       C



                                                                       Translation

        Unfolded protein               ALVASGAGINLVPEFTPGKT

                                                                        Folding and PTM

         Folded protein
     EASYRING biomarker discovery strategy
    (EASYRING - Environmental Agent Susceptibility Assessment Utilizing Existing and Novel Biomarkers as
    Rapid Non Invasive Testing Methods) - EU FP5 project associated with the CREDO cluster (2003-2005)


 Test species                     Aquaria/ cell culture exposures                          Sampling

                                                                                           - Mucus
   Common carp              EE2                            MDHT                            - Liver
  (Cyprinus carpio)                                                                        - Plasma
                                                                                           - Hepatocytes
                                                                                           - MVLN cells
                            TAM                            FLU                             - Culture medium
 African clawed frog
  (Xenopus laevis)

                            + Lambro river water (Italy)




Ab production and
assay development             Biomarker candidates                                 2-DE/MS
          Carp and Xenopus exposures




Control   EE2     Tamoxifen MDHT      Flutamide Lambro R.
         Carp: 4 doses, 2 weeks, continuous
     Xenopus: 1 dose (10-8M), 4 weeks, semi-static
                         Mini 2-DE of X. laevis plasma
                                                 5 µg protein, IPG pH 3-10 NL




                                                                                 Complement C3
                                                                                 Actin



                                                                                 Immunoglobulin
                                                                                 light chain
               Control                 10-8 M MDHT


Vitellogenin                                                                    Serotransferrin
                                                                                precursor

                                                                                Albumin


                                                                                Estrogen regulated
                                                                                protein Ep45

                                                                                Unidentified

               10-8 M EE2
               Proteins identified in X. laevis plasma

                                                • 123 identified proteins
                                                • Constitutive + differentially regulated
                                                • 19 protein families


                                                - Albumin                   - Serotransferrin precursor
                                                - Endodermin                - Triosephosphate isomerase
                                                - Alpha-2-macroglobulin     - Vimentin

                                                - Apolipoprotein A1         - Vitellogenin B1

                                                - Alpha-1-antitrypsin       - Ficolin-1

                                                - Complement C3/ C4         - Fetuin

                                                - Enolase 3                 - Hypotetical proteins

                                                - Creatine kinase
                                                - Estrogen regulated protein Ep45 precursor
                                                - Immunoglobulin heavy and light chain
                                                - Fibrinogen (alpha, beta, gamma)
Control, pooled male plasma, 500 ug, CBB-G250
                                                - Glyceraldehyde-3-phosphate dehydrogenase

   Differentially regulated proteins            - Fructose-1,6-bisphosphate aldolase
                                                - Actin
       Differential expression in X. laevis liver
Male, 10-8 M EE2, pooled/individual, ≥ 2-fold regulation
                                                             ↑, EE2 pooled and individual
                                                             ↑, EE2 individual only
                                                             ↑, EE2 pooled only
                                                             ↑↓, Contradictory results EE2
                                                             pooled/ individual
                                                             ↓, EE2 pooled and individual
                                                             ↓, EE2 individual only
                                                             ↓, EE2 pooled only


                                                           Complex pattern of expression

                                                           430 proteins differentially
                                                           regulated by EDC treatment

                                                           106 differentially regulated
                                                           spots represented identified
                                                           proteins
        Proteins identified in X. laevis liver
241 analyzed proteins                       10-8 M EE2, female, 500 µg pooled liver
                                                     Carbamoyl phosphate synthetase
                                                                                                ?
196 identities                               Hsp 90b
                                                                   p97 subunit of 15S Mg(2+) ATPase
131 unique protein spots                  Hsp 70                                         ?
                                                                          Serum albumin B precursor
                                                                            Glycyl t-RNA synthethase
17 % of the protein spots             Calreticulin
contained > 1 protein                                                    ?
                                                                                               Catalase
                                 Thioredoxin


  67 kDa laminin receptor precursor                                                          Aldehyde
  ATP synthase alpha/ beta                                                                   dehydrogenase
  Tubulin, beta 5 and 7 alpha
  Keratin
  Thioredoxin                                                                         Argininosuccinate
  Catalase                                                                            synthase
  DEAD box helicase
                                                                        Fructose-1,6-bisphosphatase
  GDP dissociation factor
  Glutaminyl t-RNA synthetase
  Fibrinogen gamma chain precursor               Vitellogenin        Ribosomal protein L-10
  Insulinase
                                           Fructose-1,6-bisphosphate aldolase/
                                           7-keto-8-aminopelargonate synthetase
        Proteins identified in X. laevis liver
241 analyzed proteins                       10-8 M EE2, female, 500 µg pooled liver
                                                     Carbamoyl phosphate synthetase
                                                                                                ?
196 identities                               Hsp 90b
                                                                   p97 subunit of 15S Mg(2+) ATPase
131 unique protein spots                  Hsp 70                                         ?
                                                                          Serum albumin B precursor
                                                                            Glycyl t-RNA synthethase
17 % of the protein spots             Calreticulin
contained > 1 protein                                                    ?
                                                                                               Catalase
                                 Thioredoxin


  67 kDa laminin receptor precursor                                                          Aldehyde
  ATP synthase alpha/ beta                                                                   dehydrogenase
  Tubulin, beta 5 and 7 alpha
  Keratin
  Thioredoxin                                                                         Argininosuccinate
  Catalase                                                                            synthase
  DEAD box helicase
                                                                        Fructose-1,6-bisphosphatase
  GDP dissociation factor
  Glutaminyl t-RNA synthetase
  Fibrinogen gamma chain precursor               Vitellogenin        Ribosomal protein L-10
  Insulinase
                                           Fructose-1,6-bisphosphate aldolase/
                                           7-keto-8-aminopelargonate synthetase
     Gene Ontology terms for X. laevis liver proteins

Molecular function; 97 identified, 62 unknown                                   Biological process; 76 identified, 83 unknown
                 1
                     1                        Antioxidant activity
               2                               Antioxidant activity
                   2                          Binding                                                                      Cellular process
                                                                                                                          Cellular process
           12                                                                           44
                         11                    Binding
       3
            12
                          11                  Catalytic activity                                                           Physiological process
                                                                                                                          Physiological process
                                                                                        33                     83
                                                                                                                83
           3                                   Catalytic activity
      3                              62       Enzyme regulator activity                                                    Biological process
                                                                                                                          Biological process
          3                           62       Enzyme regulator activity
                                              Signal transducer activity
                                                                                                                           Response stimuli
                                                                                                                          Response toto stimuli
                                               Signal transducer activity                                                  GO term not found
                                                                                                                          GO term not found
                                              Structural activity
                                               Structural activity               76
                                                                                  76
     66                               1       Regulation of transcription
      66                                  1    Regulation of transcription
                                              Translation regulator activity
                                               Translation regulator activity
                               51             Transport proteins
                                                                                                              61
                                                                                                             61
                                51             Transport proteins
                                              GO term not found
   # of proteins associated with term          GO term not found                  # of proteins associated with term



                                                                                Cellular component; 38 identified, 121 unknown
                                                                                                                                  Cell
                                                                                                       121                 Cell
                                                                                                                                  Organelle
   Gene ontology (GO), is a                                                                      121
                                                                                                                           Organelle
                                                                                                                                  Extracellular region
   controlled vocabulary used to                                                                                           Extracellular region
                                                                                                                                  Protein complex
                                                                                                                           Protein complex
   describe molecular functions,                                                                                                  GO term not found
                                                                                                                           GO term not found
   biological processes and the
   location of gene products                                                       11
                                                                                        11
                                                                                                                     34
                                                                                             4                 34
                                                                                        4
                                                                                                       23
                                                                                             23
                                                                                  # of proteins associated with term
  1-D SDS-PAGE and Vtg-western blot of plasma

Female
         Control   EE2, 10-8 M     Control       EE2, 10-8 M
kDa

250
150
                                                                  Vtg
100
 75

 50

 37

25
                        7.5 % PA
                                        Anti-xenopus Vtg 1:2000
Male
kDa      Control   EE2, 10-8 M     Control       EE2, 10-8 M

250
150
                                                                  Vtg
100
 75

 50
 37
 25
                        7.5 % PA        Anti-xenopus Vtg 1:2000
Development of dipstick for
endocrine disruption
monitoring - non-disruptive
sampling of fish mucus
                  Detection of Vtg in carp mucus
Development and testing of Vtg LFIA

                                                  Oneway Analysis of log ELISA mucus By LFIA mucus
                                                                     2                                                +
                                                                                                                      Vtg
                                                                                                                      -
                                                                                                                      Vtg
                                                                     1




                                                   log ELISA mucus
                5                                                    0
                min


                                                                     -1




 Dip-stick in mucus/                                                 -2

    PBS extract                                                                                                    With Control
                                                                            0            1                     2
                                                                                                                   Dunnett's
                                                                                                                    0,05
                                                                                             LFIA mucus

                                                      Means               Comparisons
                                                           Dif=Mean[i]-Mean[j]
                                                                        2          1              0
                                                            2     0,0000      1,2143         1,7085
                                                            1    -1,2143      0,0000         0,4942
                                                            0    -1,7085     -0,4942         0,0000
    LFIA = Lateral Flow Immunoassay
                                                           Alpha= 0,05
    Rapid non-invasive testing method for detection of Vtg in carp mucus.
                                                           Comparisons with a control using Dunnett's Method
                                                           Control Group = 0
                                                                    |d|       Alpha
                                                              2,20712          0,05
Vtg in plasma vs. mucus
Vtg in plasma vs. mucus
                                Vtg in plasma vs. mucus
                  2




                  1
log ELISA mucus




                  0




                  -1




                  -2


                       -1   0   1         2        3   4   5
                                log ELISA plasma
                                 Vtg in plasma vs. mucus
                  2




                  1
log ELISA mucus




                  0




                  -1




                  -2


                       -1    0   1          2        3   4   5
                                  log ELISA plasma




                            EE2, MDHT, FLU
                                 Vtg in plasma vs. mucus
                  2




                                                                                   1
                  1




                                                                 log ELISA mucus
log ELISA mucus




                                                                                   0
                  0




                                                                                   -1
                  -1




                  -2                                                               -2


                       -1    0   1          2        3   4   5                          -1   0   1             2        3   4
                                  log ELISA plasma                                                   log ELISA plasma




                            EE2, MDHT, FLU
                                 Vtg in plasma vs. mucus
                  2




                                                                                   1
                  1




                                                                 log ELISA mucus
log ELISA mucus




                                                                                   0
                  0




                                                                                   -1
                  -1




                  -2                                                               -2


                       -1    0   1          2        3   4   5                          -1   0     1             2        3   4
                                  log ELISA plasma                                                     log ELISA plasma




                            EE2, MDHT, FLU                                                       TAM      Field
                                            Vtg in carp plasma vs. mucus
Oneway Analysis of log ELISA plasma By Group
                     5


                     4                                                                                                       Plasma Vtg                Mucus Vtg
                                                                                                                            µg/ml ±SD (N)             µg/ml ±SD (N)
log ELISA plasma




                     3


                     2


                                                                                            Untreated                                                       0.41±0.32
                     1
                                                                                                                              202±310 (10)
                                                                                            control                                                             (8)
                     0

                          C EE2   CE EE2    EE2 1      EE2 2      EE2 3     EE2 4
                                                                                            Solvent                                                         0.54±0.97
                                                                                                                               86±99 (10)
                                               Group
                                                                                            control                                                             (9)
Oneway Analysis of log ELISA mucus By Group
                                                                                                                                                            1.04±1.93
                   1,5                                                                      EE2 – 1 ng/l                      169±157 (10)
                                                                                                                                                                (9)
                     1
log ELISA mucus




                   0,5
                                                                                                                                                            5.25±6.11
                                                                                            EE2 – 4 ng/l                      775±1278 (9)
                     0
                                                                                                                                                               (9)*
                   -0,5

                    -1
                                                                                                                               5935±4453                    27.6±11.6
                                                                                            EE2 – 16 ng/l
                   -1,5                                                                                                           (8)*                         (7)*
                          C EE2    CE EE2      EE2 1           EE2 2      EE2 3     EE2 4

                                                                                                                              35355±20978                   30.8±25.5
                                               Group
                                                                                            EE2 – 64 ng/l
                                                                                                                                  (10)*                        (2)*
                                                                              * Significantly different from untreated control, Dunnetts test of log-transformed data (p<0.05)


                                                                                            Carp Vtg ELISA kit (Biosense)
  Estrogen regulated protein Ep45 precursor (Ep45)
                                                                  CONTROL, M

• Belongs to the serpin superfamily of proteinase inhibitors

• Similarity to Hu alpha-1-antitrypsin, the major plasma
  serpin2

• Absent in control, 6-fold increase in expression within 8
  days by E2 exposure1                                            EE2, 10-8 M, M


• Induction parallels that of Vtg1

• Possible role in female reproduction by protecting Vtg
  from proteolytic cleavage during transport2

                                                                  EE2, 10-8 M, F




    1Holland LJ, Wangh LJ. 1987. Mol. Cell. Endocrinol. 49:63-7
    2Holland et al. 1992. Journ. of Biol.Chem. 267:7053-7059
                                                                           Ep45
    Xenopus plasma: Anti-Ep45-peptide western blots

             EE2       FLU    MDHT       TAM   Lambro




♀

           EE2          FLU    MDHT      TAM    Lambro




♂
    Xenopus plasma: Anti-Ep45-peptide western blots

             EE2       FLU    MDHT       TAM   Lambro




♀

           EE2          FLU    MDHT      TAM    Lambro




♂
    Xenopus plasma: Anti-Ep45-peptide western blots

             EE2       FLU    MDHT       TAM   Lambro




♀

           EE2          FLU    MDHT      TAM    Lambro




♂
    Xenopus plasma: Anti-Ep45-peptide western blots

             EE2       FLU    MDHT       TAM   Lambro




♀

           EE2          FLU    MDHT      TAM    Lambro




♂
Xenopus plasma: Anti-serotransferrin-peptide western blots

             EE2         FLU   MDHT        TAM   Lambro




♀

           EE2           FLU    MDHT       TAM    Lambro




♂
Xenopus plasma: Anti-serotransferrin-peptide western blots

             EE2         FLU   MDHT        TAM   Lambro




♀

           EE2           FLU    MDHT       TAM    Lambro




♂
    Xenopus plasma: Anti-fibrinogen -peptide western blots

                EE2        FLU   MDHT       TAM   Lambro




♀

              EE2          FLU    MDHT      TAM    Lambro




♂
    Xenopus plasma: Anti-fibrinogen -peptide western blots

                EE2        FLU   MDHT       TAM   Lambro




♀

              EE2          FLU    MDHT      TAM    Lambro




♂
    Xenopus plasma: Anti-fibrinogen -peptide western blots

                EE2        FLU   MDHT       TAM   Lambro




♀

              EE2          FLU    MDHT      TAM    Lambro




♂
Proteome changes in Atlantic cod
larvae exposed to produced water
    Utvikling av metoder for overvåking av
    effekter av utslipp fra oljevirksomheten
                                 Photo: J. Skadal and I. Rønnestad




                  Anders Goksøyr
                 Molekylærbiologisk institutt
                   Universitetet i Bergen
Biomarker discovery in Atlantic cod fry liver after
    continuous exposure to produced water



                                              P2




                                          L
                                              C
                               P3
                                                   H

                                         M
                                              P1

                                              E




                                     Kjersem (2007), PhD thesis
Rifai et al.
Conclusions
                       Conclusions

A toxicoproteomic strategy has been established to identify biomarker
candidates under various exposure regimes in different species
Higher identification success rates are obtained in species with better
genomic coverage (e.g. X. laevis > carp > cod)
Proteome changes linked to annotated databases and Gene Ontology
terms may help elucidates toxicological mechanisms and modes of
action
In general, responses specific to a single chemical are interesting as
biomarker candidates, however suites of biomarkers may prove more
informative in field studies targeting emerging pollutants - e.g. applied
in protein/antibody arrays
 Acknowledgements

           EASYRING partners (2003-2005):
           Alberta Mandich, University of Genova, Italy
           Luigi Viganó, IRSA-CNR, Milano, Italy
           Emilio Benfenati, Mario Negri Institute, Milano, Italy
           Anne Van Cauwenberge, UMH, Mons, Belgium
           Mark Cronin, LJMU, Liverpool, UK

           The Institute of Marine Research, Bergen, Norway
           Computational Biology Unit, University of Bergen


Support: EU (FP5): EASYRING project, Total E&P,
       the Norwegian Research Council

								
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