Yadav Sandhya, Srivastava Akanksha, Biswas Subhankar, Chaurasia Neha, Singh Sushil Kumar, Kumar Sanjiv, Srivastava Vaibhav, Mishra Yogesh
Department of Botany, Centre of Advanced Study in Botany, Institute of Science, Banaras Hindu University, Varanasi, 221005, India.
Department of Biotechnology and Bioinformatics, North Eastern Hill University, Shillong, 793022, India.
BMC Res Notes. 2020 Feb 7;13(1):60. doi: 10.1186/s13104-020-4929-1.
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.
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.
地钱具有应对非生物胁迫的历史适应性策略，因为它们是首批从水生环境转移到陆地的植物。蛋白质组学是一种先进技术，能够捕捉许多生物体中蛋白质水平上发生的事件的快照。在此，我们重点比较和优化一种用于地钱二维凝胶电泳（2-DE）的有效蛋白质提取和沉淀方案。
我们比较了三种不同的蛋白质提取方法，即1.5 M Tris-HCl（pH 8.8）、50 mM Tris-HCl（pH 7.5）和聚乙烯聚吡咯烷酮（PVPP）提取法，随后又比较了三种沉淀方法，即80%乙醇、80%丙酮和20%三氯乙酸（TCA）-丙酮沉淀法，用于多毛地钱的提取。在这些方法中，50 mM Tris-HCl（pH 7.5）提取法，随后用20% TCA-丙酮沉淀法，似乎更适合2-DE。此外，我们在蛋白质洗涤、复溶到水化缓冲液以及等电聚焦（IEF）过程中进行了改进。这些改进在蛋白质产量、分辨率、斑点数量和强度方面为多毛地钱以及其他两种地钱，即浅色地钱和附枝平盘苔的2-DE凝胶提供了更好的结果。此外，我们从多毛地钱的2-DE凝胶中随机选择斑点并通过质谱进行鉴定，这证实了该方案在地钱蛋白质组学中的适用性。
