Identification of protein from Eurycoma Longifolia root extract

Abstract

This study was carried out to identify plant protein from the root samples of Eurycoma longifolia harvested from Pahang and Perak, Malaysia. Protein usually presents in small quantity which is only 0.001% in plant, therefore it is critical to determine the extraction method for high yield and good quality of protein for the subsequent process of protein identification spectrometrically. Four extraction methods, namely water, Triton X-100 (non-ionic detergent), phenol-SDS precipitation and TCA-acetone precipitation were investigated for plant protein extraction. The yield of protein extracted from the plant samples was determined using Bradford assay. Both water extracts (water and Triton X-100) methods contained slightly higher protein content (0.2-0.53 mg protein/ml crude protein) than the extracts of precipitation (phenol and TCA) methods (0.12-0.29 mg protein/ml crude protein). The water extraction method also produced the highest resolution of 15% polyacrylamide gel with six and five protein bands for E. longifolia Pahang and Perak, respectively. However, the number of protein bands decreased from five to three for the extraction method of Triton X-100, phenol-SDS and TCA-acetone, respectively. After trypsin digestion of the protein bands, the presence of protein was analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The mass spectra matched with the databases showed that the addition of Triton X-100 could assist the extraction of mitochondrial protein, namely superoxide dismutase. Proteins that involved in energy metabolism such as phosphoenolpyruvate carboxykinase and plant protein inhibitor were also detected in the phenol-SDS buffer extraction because SDS acted as ionic detergent for cell lysis in this method. In line with previous studies, TCA-acetone did not exhibit clear gel image and subsequently less peptides were detected spectrometrically. In the present study, the addition of detergent (Triton X-100 and SDS) could enhance plant protein extraction from E. longifolia root from 46.7 to 72.7% w/w, but the use of TCA did not improve protein precipitation effectively.