Comparison and optimization of protein extraction and two-dimensional gel electrophoresis protocols for liverworts

Abstract

Objective: Liverworts possess historical adaptive strategies for abiotic stresses because they were the first plants that shifted from water to land. Proteomics is a state-of-the-art technique that can capture snapshots of events occurring at the protein level in many organisms. Herein, we highlight the comparison and optimization of an effective protein extraction and precipitation protocol for two-dimensional gel electrophoresis (2-DE) of liverworts. Results:We compared three different protein extraction methods, i.e.,1.5 M Tris-HCl (pH 8.8), 50 mM Tris-HCl (pH 7.5), and polyvinylpolypyrrolidone (PVPP) extraction, followed by three precipitation methods, i.e., 80% ethanol, 80% acetone, and 20% tricholoroacetic acid (TCA)-acetone, in a liverwort Dumortiera hirsuta. Among these methods, 50 mM Tris-HCl (pH 7.5) extraction, followed by 20% TCA-acetone precipitation, appeared to be more suitable for 2-DE. Furthermore, we performed modifications during protein washing, re-solubilization in rehydration buffer and isoelectric focusing (IEF). The modifications provided us better results in terms of protein yield, resolution, spot numbers, and intensities for 2-DE gels of D. hirsuta and other two liverworts, i.e., Marchantia paleacea and Plagiochasma appendiculatum. Furthermore, we randomly selected spots from the 2-DE gel of D. hirsuta and identified using mass spectrometry, which confirms the applicability of this protocol for liverworts proteomics.

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

“…As with peanut, the TCA/acetone extract of walnut samples showed a low protein density in terms of composition compared to SDS, PVPP or Ambi/urea extracts. Similar results were obtained by Yadav et al [ 27 ] who found that after precipitation in 20% TCA-acetone, the amount of soluble protein was lower than in 50 mM Tris-HCl (pH 7.5) and 1.5 M Tris-HCl (pH 8.8) solutions. In contrast, when testing different extraction methods for tomato pollen proteome analysis, Sheoran et al [ 47 ] found the TCA-acetone protein extraction method to be more appropriate.…”

“…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.…”