A cell-sorting-based nano-scale pipeline for cell-type-specific proteomics in plants
Multicellular organisms such as plants contain different types of cells with specialized functions. Analyzing the characteristics of each type of cells will reveal specific cell functions and enhance understanding of how an organism organizes and works. Due to the lack of a method to enrich the particular type of cells and the limited sensitivity and capability of mass spectrometry-based technologies, comprehensive analyses of the global proteome in a single plant cell are still impossible. Here we report a high-sensitive and efficient single-cell-proteomic pipeline, combining simplified flow cytometry-based fluorescent cell-sorting for fluorescent protoplasts and an optimized nano-scale proteomics method, which allows us to identify more than 1,000 unique proteins from 500 guard cell protoplasts. This method was applied to profile the proteome of guard cells and mesophyll cells in Arabidopsis leaves, and epidermal cells in root tips of Arabidopsis and rice seedlings. We also performed an in-depth, quantitative comparison between the proteomics of guard cells and mesophyll cells and revealed that the enrichment of signal transduction-related proteins enables guard cells to respond to various environmental stimuli quickly. This method is applicable to other types of cells in plant or non-plant systems to acquire systemic knowledge of how cells work specifically and in highly organized multiple cell organisms.