An Improved Plant Protein Extraction Method Based On Trichloroacetic Acid/acetone Precipitation

Plant tissues contain large amounts of secondary metabolites that significantly interfere with protein extraction and proteomics analysis.Thus,a simple,rapid and efficient protein extraction method is always required in plant proteomics.To date,trichloroacetic acid(TCA)/acetone precipitation method is the most widely used protocol for plant protein extraction due to its high ability of removing interfering compounds.However,this method greatly depends on the fineness of the tissue powder,and the precipitated proteins are often difficult to be re-solubilized.In addition,a prolonged incubation of tissue powder in TCA/acetone or acetone can lead to the modifications of proteins by acetone and the proportion of modified peptide increased over time,thus affecting the outcome of MS/MS analysis.To overcome these limitations,this paper developed a modified TCA/acetone precipitation method.Total proteins were directly extracted using SDS extraction buffer from maize tissues(e.g.,embryos,leaves and roots)and then precipitated by 20% TCA/acetone.Compared with routine TCA/acetone precipitation method,the modified protocol developed in this study can effectively extract and purify proteins from plant tissues with an increase yield,and the resolution of proteins in 2D gel was improved.Moreover,the final protein pellets were easy to be re-solubilized in 2D buffer for electrophoretic analysis.Designed to run on a small scale,this method can be completed within 1 h.This modified method can be applied in various plant tissues for proteomic analysis.