Comparison of Sample Preparation Methods for the Resolution of Metal-Regulated Proteins in Helianthus Annuus by 2-Dimensional Gel-Electrophoresis

The Dwarf Sunflower, Helianthus annuus, is a hyperaccumulator of the heavy metals cadmium, nickel, and chromium. The molecular mechanism of hyperaccumulation and adapting to the resulting abiotic stress is largely unknown. Metal-binding and other proteins induced in response to stress conditions may play indispensable roles in allowing the metals to accumulate and the plant to adapt itself. However, the presence of large amounts of the large subunit of ribulose1, 5 bisphosphate carboxylase/oxygenase (Rubisco) in leaves hampers the detection of many and, therefore also differentially expressed proteins, in gels. The aim of this study was to improve on methods of two-dimensional gel electrophoresis with high enough resolution for the purpose of identifying differentially expressed proteins for use in liquid chromatography and tandem mass spectroscopy and matrix-assisted laser desorption and ionization time-of-flight mass spectrometric analysis. The Teddy Bear cultivar of H. annuus was exposed to a mixture of cadmium, nickel, chromium, plus lead and lead only. Soluble proteins were extracted from leaf samples and several sample preparation procedures were tested. Use of a phenol extraction method enabled better resolution of proteins compared to either acetone or Trichloroacetic acid (TCA/acetone) precipitation techniques. TCA/acetone precipitation combined with a phenol treatment step (double precipitation) improved gel resolution with regard to reducing background staining and horizontal streaks, however, the heavy streaks associated with high concentrations of Rubisco were still present. Treatment of the samples with polyethylene glycol (PEG) was the most effective in reducing these streaks.