Toward simple, rapid, and deep plant proteome analysis with an in-cell proteomics strategy.

While liquid chromatography-mass spectrometry (LCMS) has revolutionized plant proteomics over the past decade, plant sample preparation remains a major challenge due to rigid cell walls, abundant secondary metabolites, and wide dynamic range of protein abundance. These hurdles demand laborious tissue disruption, complex precipitation, and extensive cleanup prior to LCMS analysis, limiting the widespread adoption of proteomic technologies within the plant biology community. To overcome these barriers, we introduced an "in-cell proteomics" strategy that bypasses cell lysis and protein extraction by performing digestion directly inside methanol-fixed cells. We systematically benchmarked this strategy against conventional lysate-based workflows across 4 model plants (Arabidopsis thaliana, Nicotiana benthamiana, Zea mays, and Sorghum bicolor) and 3 tissue types (leaves, pollen, and seeds). Combined with minimal input material and single-shot LCMS, the in-cell approach consistently identified 9,000 to 12,000 proteins from leaves, 7,000 to 9,000 from pollen grains, and approximately 8,000 from seeds. Our comprehensive dataset demonstrates that this in-cell digestion approach substantially simplifies plant sample preparation while delivering proteomic performance equivalent to established workflows. Finally, to demonstrate the biological utility of this approach, we characterized the proteomes of N. benthamiana leaves infected with 2 fungal strains that exhibit different host specificities. Our in-depth proteomic data revealed distinct host response signatures differentiating the host-adapted Colletotrichum destructivum from the nonhost-adapted Colletotrichum sublineola strain. Overall, this study provides a simple, unbiased alternative for plant proteomic analysis that can be readily applied to tackle complex agricultural and physiological challenges in plant biology.