Establishment of the optimal conditions for two-dimensional gel electrophoresis of papaya seed proteome

Papaya seeds are considered as a recalcitrant material for protein preparation in two-dimensional gel electrophoresis (2-DE) because of abundance of interfering compounds. We examined two protein extraction methods, the classical trichloroacetic acid (TCA) / acetone precipitation (TCA-A) and phenol ammonium acetate-methanol precipitation (P-AA-M) methods, to evaluate their compatibility and efficiency of extracting papaya seed proteins in SDS-PAGE and two-dimensional (2D) gel electrophoresis analysis. Compared with TCA-A method, P-AA-M method showed better results, i.e., it increased at least five distinguished bands in SDS-PAGE, reduced problems of streaking and smearing in 2D gel, and enhanced resolution of detectable spots from 124±27 to 717±32. Comparison between traditional Coomassie Brilliant Blue R-250 staining and zinc-reverse staining was also conducted. The result showed that seven times more protein spots could be identified from 2D gel stained with zinc-reverse stain procedure. The resolution of 2D gel analysis for papaya seed proteome was further improved by utilizing pH 4-7 isoelectric focusing gel instead of pH 3-10 one. The protein spots picked from 2D gel through the protocol that we established were compatible with identification assessments of LC-MS/MS. Our data will serve as a platform and reference for future research in proteomics of papaya seeds or other recalcitrant plant materials.