The Protein Extraction from Longan Pulp (Dimocarpus longan Lour. cv. Daw) for Proteomic Analysis Using One-Dimensional Electrophoresis

摘要

Three procedures for protein extraction in longan pulp had been applied to analyze protein pattern and quality of Longan pulp (Dimocarpus longan Lour. cv. Daw) during fruit growth to increase protein expression in proteomic analysis at Maejo university’s farm. There were data points to compare between normal and physiological disorder syndromes during fruit growth (5,10, 15, 20, 25 and 30 weeks, respectively) by one dimensional electrophoresis (1-D gel) technique in reducing condition. The first protein extraction, M1 (95% ethanol) showed obviously 15 protein bands which molecular weights were 14.97, 17.90, 18.30, 21.63, 28.54, 31, 33.96, 35.02, 42, 51.69, 65.69, 71.54, 88.02, 106.86 and 130 kDa, respectively. While M2 extraction (phenol-methanol/ammonium acetate) and M3 extraction (1.5 mM tris-HCl pH 8.0, 5 mM EDTA, 2% SDS) had low protein expression and no sharpness (13 and 12 protein bands, respectively). In different extraction conditions, therefore, M1 was a suitable method because of highest protein bands and obvious protein expression on longan pulp for proteomic analysis. Proteomic analysis of M1 extraction method was used in protein analysis by using LC-MS / MS techniques. It was found that the heat shock protein 83 (81.0 kDa), a family of proteins that was produced by cells in response to exposure on stressful conditions, the elongation factor 1-alpha (49.45 kDa), a selective regulator of translation, and the peroxidase 4 (39.74 kDa), a protein that is involved in the degeneration or aging of cells. These proteins exhibited a darker appearance of the protein bands at 30 weeks. Moreover, a partial glyceraldehyde-3-phosphate dehydrogenase (34.06 kDa), the protein involved in metabolic processes in glucose degradation, was also founded a darker appearance at 25 weeks and low appearance at 30 weeks of abnormal longan. However, higher proteomic techniques should be studied to confirm this biomarker protein in the further.