Comparison of methods for the extraction of proteins from root and leaf tissue of sugarcane (Saccharum spp.) for proteomic analysis

摘要

The extraction of proteins from plants is a crucial procedure for successful protein determination such as purification, separation, and mass spectrometry.Protein extraction from plant tissues is generally difficult due to the presence of various molecules (cell wall, polysaccharides, and lipids) and interfering compounds.For this reason, the step of separation of proteins is a big challenge in obtaining good results in plant proteomic studies, notably from sugarcane.The current study assesses three extraction methods to prepare protein samples for proteomic analysis.Method 1 (control): TCA/acetone, method 2: TCA/acetone modified and Method 3: Phenol/SDS/ammonium acetate.Plants of cultivar RB92579 were grown in 10L pots under ideal humidity conditions in a greenhouse for 60 days.Samples collected on leaves +1 and roots were carried out using nitrogen and stored in an ultra-freezer at -80oC for later use in proteome assays.For the tested methods, a comparison was made between the quantitative and qualitative data obtained from the tissue of sugarcane leaves and roots.According to the results obtained, methods 2 and 3 produced the best yield in the extraction of total proteins from the leaves and roots of sugarcane, when compared to (control) method 1 (TCA/acetone).This can be observed when comparing the quantitative and qualitative data obtained using the different extraction methods.By comparing methods 2 and 3, the latter showed a massive gain of extracted proteins much greater than the first method, mainly when the extraction of total proteins from the roots are compared.Similarly, the 2-DE gels run after using method 3 showed less background, compared to method 2.Another observation was the presence of different “spots” in the 2-DE gels between the samples extracted using methods 2 and 3.Method 3 (phenol / SDS / ammonium acetate) presented better results for extraction of proteins and in the 2-DE gels, with a greater number of total and specific “spots”, greater reproducibility and less background.This method could be utilized as the standard method for proteomic studies in sugarcane.