2-DE PROTOCOL OPTIMIZATION AND EVALUATION FOR PROTEOME ANALYSIS OF GENUS CLEMATIS TAXA (RANUNCULACEAE)

Abstract

An approach was conducted to optimize two-dimensional gel electrophoresis (2-DE) method for leaf proteome analysis of genus Clematis species, as a molecular approach to explore its taxonomy and differentially expressed genome patterns. During establishment and optimization of protocol we extracted proteins by three extraction protocols, viz., phenol-SDS (PS) method, TCA/acetone (TA) method and lysis buffer (LB) method, and PS was the best one with 2.35±0.05 µg protein yield. For protein solubilization two lysis buffers (LB-1 & LB-2) were prepared, used and comparatively LB1 depicted better resolution. Proteins were by quantified by the Bio-Rad protein assay (Hercules, CA, USA) with bovine serum albumin as standard and purified by 2-D clean-up Kit (Amersham Biosciences). 2-DE analysis was conducted on pH 3~10, non-linear gradient strips (24cm) as first step, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 13% polyacrylamide gels as the second phase. For spot visualization gels were stained for with silver stain. The gels were scanned using Powerlook 2100XL scanner and gel images were analyzed by ImageMaster 2-D Platinum. Validation of experiment was performed by measuring analytical variance (AV) and biological variance (BV) for replicate spots. AV was calculated for 60 protein spots present in three replicate 2-DE gels of the same protein extract and BV was determined for the same protein spots from independent tissue extracts corresponding to leaves from different plants, or the same tree at different orientations or sampling times during a day. Values of 26% for the analytical variance and 58.6% for the biological variance among independent sampled species were obtained. This provided a threshold values for the evaluation of protein expression changes in comparative proteomic investigations with this species. Some spots were selected and subjected to liquid chromatography mass spectrometry (LC-MS) for identification purpose. Due to absence of Clematis DNA or protein sequences databases, FASTA and BLAST similarity searches were performed against other plant species databases were used for protein identification. The significance of 2-DE proteome analysis in predicting evolutionary trend of Clematis (liana) species and its potential significance in taxonomic identification for Traditional Chinese Medicine (TCM) pharmacopeia is described.