Resurrection plant Ramonda serbica is a suitable model to investigate vegetative desiccation tolerance. However, the detailed study of these mechanisms at the protein level is hampered by the severe tissue water loss, high amount of phenolics and polysaccharide, and possible protein modifications and aggregations during the extraction and purification steps. When applied to R. serbica leaves, widely used protein extraction protocols containing polyvinylpolypyrrolidone and ascorbate, as well as the phenol/SDS/buffer–based protocol recommended for recalcitrant plant tissues failed to eliminate persistent contamination and ensure high protein quality. Here we compared three protein extraction approaches aiming to establish the optimal one for both hydrated and desiccated R. serbica leaves. To evaluate the efficacy of these protocols by shotgun proteomics, we also created the first R. serbica annotated transcriptome database, available at http://www.biomed.unipd.it/filearrigoni/Trinity_Sample_RT2.fasta. The detergent-free phenol-based extraction combined with dodecyl-β-d-maltoside-assisted extraction enabled high-yield and high-purity protein extracts. The phenol-based protocol improved the protein-band resolution, band number, and intensity upon electrophoresis, and increased the protein yield and the number of identified peptides and protein groups by LC-MS/MS. Additionally, dodecyl-β-d-maltoside enabled solubilisation and identification of more membrane-associated proteins. The presented study paves the way for investigating the desiccation tolerance in R. serbica, and we recommend this protocol for similar recalcitrant plant material.

Graphical abstract

中文翻译：

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从复活的Ramonda serbica植物的水合和干燥的叶片中有效提取protein枪蛋白质组学蛋白质

复活植物Ramonda serbica是研究营养干燥耐性的合适模型。但是，由于严重的组织水分流失，大量的酚类和多糖类以及提取和纯化步骤中可能的蛋白质修饰和聚集，阻碍了在蛋白质水平上对这些机制的详细研究。当应用于R时。塞尔维亚叶子上，广泛使用的包含聚乙烯基聚吡咯烷酮和抗坏血酸的蛋白质提取方案，以及推荐用于顽固植物组织的基于苯酚/ SDS /缓冲液的方案未能消除持久性污染并确保高蛋白质质量。在这里，我们比较了三种蛋白质提取方法，旨在为水合和干燥的R建立最佳的蛋白质提取方法。塞尔维亚叶子。为了评估这些协议通过鸟枪蛋白质组学的疗效，我们也创造了第一[R 。塞尔维亚语注释的转录组数据库，网址为http://www.biomed.unipd.it/filearrigoni/Trinity_Sample_RT2.fasta。不含去污剂的酚基萃取物与十二烷基-β- d的组合麦芽糖苷辅助提取可实现高产量和高纯度的蛋白质提取物。基于苯酚的方案提高了电泳时的蛋白条带分辨率，条带数和强度，并通过LC-MS / MS提高了蛋白产量以及鉴定的肽和蛋白基团的数量。另外，十二烷基-β - d-麦芽糖苷能够溶解和鉴定更多的膜相关蛋白。提出的研究为研究R的干燥耐性铺平了道路。serbica，我们建议此方案用于类似的顽强植物材料。

图形概要