A stepwise ionic liquid extraction method for plant leaf protein extraction

Background Protein extraction from plant leaves is a key step in plant proteomics analysis. However, traditional methods often face challenges, including low efficiency, incomplete protein coverage, and environmental concerns. These limitations highlight the need for developing more efficient and environmentally friendly extraction techniques. Ionic liquids (ILs) have shown promise in protein extraction due to their excellent solubility and enzyme compatibility, offering a potential solution to these issues. In this study, we explore the use of short and long alkyl chain ILs to improve protein extraction efficiency from plant leaves. Results Using tobacco K326 as a model plant, a stepwise extraction system was designed with 10% 1-ethyl-3-methylimidazolium chloride ([C 2 MIM]Cl) and 4% 1-dodecyl-3-methylimidazolium chloride ([C 12 MIM]Cl). Both [C 2 MIM]Cl and [C 12 MIM]Cl effectively disrupted the cellulose hydrogen-bond network, facilitating protein release from the plant cell wall, while [C 12 MIM]Cl also enhanced membrane protein solubility, significantly improving the overall protein extraction efficiency. The relative protein concentration achieved with this method was 2.28, which is notably higher than traditional methods using urea (1.21) and SDS (1.47). The method was applied to proteomic analysis of healthy and brown spot disease-affected tobacco leaves, identifying 6858 proteins in total. Among these, 758 proteins were differentially expressed, with 414 upregulated and 344 downregulated in diseased leaves, providing insight into disease-related protein expression. Significance This novel protein extraction method offers a highly efficient alternative to traditional approaches. It significantly enhances protein yield and solubility, providing valuable support for in-depth plant proteomics research. This method is particularly useful for studying plant disease responses, facilitating the identification of disease-related proteins and contributing to the understanding of plant-pathogen interactions.