Evaluation of six different protocols for protein extraction from rice young panicles by two-dimensional electrophoresis

Abstract

Background: For most reported proteomics approaches, protein extraction are of crucial importance for optimal results. However, extraction of protein from plant tissues still exist great challenges due to low protein content and many secondary metabolites that prominently interfering with isoelectric focusing. Up to now, no attempts are focused on comparison of protein extraction from rice young panicles.Methods: To establish a higher efficiency protein extraction protocol suited for two-dimensional electrophoresis in rice young panicles, six protocols for protein preparation were evaluated in terms of protein concentration, the molecular weight range of protein, gel image resolution, the number of protein spots: 1) Phenol extraction; 2) Mg/Nonidet P-40 (NP-40) extraction; 3) Tris-Base/acetone extraction; 4) SDS extraction; 5) trichloroacetic acid (TCA)/acetone/phenol extraction; 6) TCA/acetone precipitation.Results: The result explicitly demonstrated that TCA/acetone/phenol method provided a high-enhanced protein extraction efficacy from rice young panicles than other protocols in terms of the protein concentration (9.79±0.23 SD), the most comprehensive proteins (10 KDa to 150 KDa), the maximum number of protein spots (450±53 SD), the greater gel image resolution and spot abundance. In addition, these methods also generated remarkably differential 2-DE protein patterns. Twenty-nine of 30 visible differentially extracted proteins were identified by MS analysis and were divided into eight categories. Prediction for protein subcellular localization and grand average of hydropathy (GRAVY) analysis showed that certain special proteins respectively necessitate different extraction methods due to different physicochemical properties of each protocol.Conclusions: Overall, this paper will facilitate to provide a cornerstone of comparative proteomic analysis from rice young panicles, including other complicated plant tissues.

Effect of Sample Preparation on the Detection and Quantification of Selected Nuts Allergenic Proteins by LC-MS/MS

“…It is therefore usually essential to remove such compounds prior to the analysis, making the procedure more challenging. The standard and primary protocols generally used for protein extraction from plant tissues are the conventional buffer extraction such as ammonium bicarbonate, Tris-HCl, and sodium phosphate [ 27 , 28 ], TCA precipitation [ 29 , 30 , 31 , 32 ], phenol extraction methods [ 33 , 34 , 35 ], polyvinylpolypyrrolidone (PVPP) extraction [ 27 , 28 , 36 ], or using the sodium dodecyl sulfate (SDS) buffer [ 33 , 37 , 38 ]. Doellinger et al [ 39 ] presented a sample preparation method by easy extraction and digestion (SPEED) using neither detergents nor chaotropic agents but pure trifluoroacetic acid for protein extraction.…”