Evaluation of three types of protein extraction methods for tetraploid black locust (Robinia pseudoacacia L.) phloem tissue proteome analysis by two-dimensional electrophoresis

Abstract

Sample preparation is a decisive step in a two-dimensional gel electrophoresis (2-DE) proteome approach and is essential for good experimental results. For recalcitrant plant tissues, choosing a suitable protein extraction method is very important for follow-up research. Many articles about protein extraction methods have been published that have mainly focused on plant leaves, fruits, and seeds, whereas few of them have evaluated protein extraction methods using phloem tissue. This study was performed to compare three precipitation protocols for protein extraction for 2-DE proteomics analysis of tetraploid black locust phloem tissue as follows: TCA/acetone precipitation, Tris-HCl extraction, and phenol extraction-ammonium acetate/methanol precipitation. The results showed that classic TCA/acetone precipitation was the most effective method for protein extraction. Compared to the Tris-HCl and phenol extraction methods, TCA/acetone precipitation had the highest total protein yield and the greatest number of protein spots on the 2-DE gels. To verify the quality of the 2-DE analysis, several gel spots were identified by MALDI TOF/TOF MS/MS. These data demonstrated the characteristic features of each extraction protocol. In conclusion, the TCA/acetone precipitation extraction method is suitable for proteomic analyses of recalcitrant tissues from woody plants.