Extraction of Proteins from Green Tissues of Plants and Phyllosphere.

The proteomic approach plays a key role to characterize a biological system at any given time. In recent years, advances in proteomics have led to an increasing application in all biological fields, including plant matrices and associated microbiome studies. However, extracting adequate protein samples remains the most critical step for any plant proteomics study. The protein extraction protocols proposed for the phyllosphere involve an initial leaf washing step; however, this is an approach only applicable if interest is restricted to epiphytes. A metaproteomic approach is required to obtain an overall picture and consequently an extraction that considers proteins derived from the plant, epiphytic and endophytic microorganisms. The most commonly used extractions for plant tissue involve the use of phenol or TCA-acetone. However, for efficient protein recovery is essential to remove interfering components abundant in plant tissues, such as polysaccharides, lipids, and phenolic compounds. A well-proven protocol on the basis of a combination of TCA-acetone and phenol extraction is presented here, obtaining some cleaned protein pellets, suitable for electrophoresis and subsequent proteomics studies. Important points for the success of this protocol are (i) a proper sampling and sample preparation, (ii) maintaining samples at a low temperature during extraction and using protease inhibitors, (iii) an initial step in TCA-acetone to remove part of the interfering substances, and (iv) careful recovery of the phenolic phase. Furthermore, the protocol is timesaving and can be completed in one working day.