engleski

Evaluation of protein extraction methods for proteomic analysis of non-model recalcitrant plant tissues

Plant tissues contain relatively low amounts of proteins whose extraction is often difficult due to the presence of interfering compounds such as rigid cellulosic cell wall, storage polysaccharides, lipids and other contaminants that can cause protein degradation or modification. Therefore it is important to optimize protein extraction and to establish a robust protocol for two-dimensional gel electrophoresis (2-DE) and downstream processing. In this study, acetone, trichloroacetic acid/acetone and Tris-buffered phenol/methanol extraction protocols were evaluated on non-model and recalcitrant plants: a Beta vulgaris L. in vitro cell line, Mammillaria gracilis Pfeiff. in vitro plants and Sempervivum tectorum L. leaves. Although phenol extraction was more time-consuming than the other two methods, it gave almost two-fold higher protein yield, and spectral analysis showed less contamination. SDS-PAGE, 2-DE and bioinformatic analysis showed that protein extraction using phenol was superior to the other two methods, providing more protein bands or spots on the gels and less proteolysis. These results lead to the conclusion that the phenol method is the most suitable protein extraction method for these non-model and recalcitrant plant tissues.

electrophoresis; mass spectrometry; phenol; plant proteomics; protein extraction; trichloroacetic acid

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