A cell-sorting-based nano-scale pipeline for cell-type specific proteomics in plants

Abstract 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 proteome in a single plant cell are still impossible. Here we report a high-sensitive and efficient cell-type-specific 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. By this method, we 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 was also applied to profile the proteome of different cells in root tips of Arabidopsis and rice seedlings. Thus, this pipeline is applicable to various 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.