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Sebastien Carpentier

Proteomics: identification of abiotic stress proteins correlated with cryopreservation via 2D PAGE analysis. Sebastien Carpentier K.U.Leuven Department of Applied Plant Sciences Laboratory of Tropical Crop Improvement Kasteelpark Arenberg 13 - 3001 Leuven Belgium. Overview.

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Sebastien Carpentier

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  1. Proteomics: identification of abiotic stress proteins correlated with cryopreservation via 2D PAGE analysis Sebastien Carpentier K.U.Leuven Department of Applied Plant Sciences Laboratory of Tropical Crop Improvement Kasteelpark Arenberg 13 - 3001 Leuven Belgium

  2. Overview • Introduction: 2D principle • Comparative study of sample prep methods • Future perspectives

  3. 2 D PAGE Amersham biosciences principles and methods

  4. pH 10 pH 3 Iso-Electric Focusing - +

  5. Second dimension pI 10 pI 3

  6. Plant material: Sample preparation • It does not provide a ready source of proteins for investigation by 2D: • rich in interfering compounds (vacuole  salts, organic acids, phenolics, proteases, pigments, terpenes,...) • low protein content (low concentration) • Solution: Precipitation of the proteins  removinginterfering compounds and concentrating the proteins

  7. Damerval et al. Electrophoresis 1986, 7, 52-54 Cell disruption (liquid N2 crush) Precipitation with 20 % TCA in ice cold aceton 0.2% DTT Wash twice with ice cold aceton 0.2% DTT Dry Dissolve in lysis buffer (4% CHAPS; 1% DTT, 7M urea, 2M thiourea; 0.8 % IPG-buffer) TCA precipitation

  8. TCA precipitation

  9. Disadvantages • Not all proteins precipitate • Proteins might get lost during washing • Proteins may be difficult to resolubilize • Extended exposure may cause protein degradation or modification

  10. TCA / fractionation Jacobs et al. Proteomics 2001, 1,1345-1350 • Dry • Dissolve in lysis buffer I (4% CHAPS; 1% DTT, 9M urea, 0.8 % IPG-buffer)  fraction I • Wash • Dissolve remaining pellet in lysis buffer II (4% CHAPS; 1% DTT, 7M urea, 2M thiourea; 0.8 % IPG-buffer)  fraction II

  11. TCA / fractionation

  12. TCA / fractionation I

  13. TCA / fractionation II

  14. Disadvantages • Cross contamination of both TCA fractions is too strong to be considered as a standard sample prep method

  15. Phenol extraction / precipitation Hurkman and Tanaka Plant Physiol. (1986) 81, 802-806 • Cell disruption (liquid N2 crush) and add extraction buffer (Tris-HCL, KCl, DTT, EDTA, prot. inh. cocktail, PVPP) • Extraction of proteins with phenol • Wash with extraction buffer (Tris-HCL, KCl, DTT, EDTA, prot. inh. cocktail,PVPP) • Precipitation with AmAce in methanol • Wash twice with ice cold aceton 0.2% DTT • Dry • Dissolve in lysis buffer (4% CHAPS; 1% DTT, 7M urea, 2M thiourea, 0.8 % IPG-buffer)

  16. Phenol extraction / precipitation

  17. Disadavantages • Proteins not present in the phenolic phase are lost • Time consuming • Protease inhibitors needed

  18. No-precipitation / fractionation Giavalisco et al. Electrophoresis 2003, 24, 207-216 • Liquid nitrogen crush • Extraction in water based buffer (Tris-HCL, KCl, glycerol, prot.inh.cocktail fraction I • Liquid nitrogen crush • Extraction/solubilization in lysis buffer fraction II

  19. No-precipitation / fractionation I

  20. No-precipitation / fractionation II

  21. Disadvantages • This method is not suitable for analyzing our material • Extraction efficiency is too low (starting material is limited) • Bad reproducibility

  22. Conclusions • TCA-precipitation and phenol/precipitation are reliable and are suitable as standard method for meristematic tissue (150 mg FW) • The phenol/precipitation is the most efficient in removing interfering substances • Both methods show a bias towards certain proteins

  23. TCA Phenol

  24. TCA Phenol

  25. TCA Phen

  26. Dehydration tolerance: genes with up or down regulated expression • Cell growth and division • Cell rescue and defence • Radical scavenging and detoxification, osmotic adjustment, structural adjustment, LEA • Metabolism • Sec metabolites, sugars, PA • Protein destination • Chaperones, heat shock, dhn • Signal transduction • Transcription • Transport facilitation • Water channels, ion transporters, sugar and carbohydrate transporters

  27. Current and future research • Proteomic approach • sucrose pre-treated meristems vs control meristems (phenol extraction, 24 cm strips 3-10 and 4-7) • Picking of interesting spots and identifying them • Known candidate proteins • Western blot: identifying and quantifying known candidate proteins (DHNs, HSPs, Enzymes involved in ROS scavenging) 1D and 2 D

  28. CRPGL Acknowledgements CMPG

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