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Identification of Abiotic Stress Proteins in Cryopreservation via 2D PAGE Analysis

This study focuses on the analysis of abiotic stress proteins correlated with cryopreservation using 2D PAGE. The research delves into the comparative study of sample preparation methods and future perspectives in the field of proteomics. Various techniques like TCA precipitation, phenol extraction, and no-precipitation/fractionation are evaluated for their advantages and disadvantages. The work emphasizes the importance of reliable methods for analyzing meristematic tissues and highlights the efficiency of the phenol/precipitation technique in removing interfering substances. Furthermore, the study identifies genes associated with dehydration tolerance and their roles in cell growth, defense mechanisms, metabolism, and signal transduction. The research also discusses current and future endeavors in proteomic approaches, sucrose pre-treatment experiments, and Western blot analyses to identify specific proteins. Acknowledgements are extended to CMPG.

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Identification of Abiotic Stress Proteins in Cryopreservation via 2D PAGE Analysis

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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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