Comparative Assessment of Sample Preparation Workflows and Mass Spectrometry Acquisition Strategies for Bottom-Up Proteomics in Rice Tissues

Robust and reproducible proteome coverage remains a major challenge in plant bottom-up proteomics because of tissue-specific biochemical complexity and variability during sample preparation and mass spectrometry (MS) acquisition. Here, we systematically evaluated four sample preparation workflows (in-solution digestion, FASP, S-trap, and SP3) combined with three MS acquisition strategies, including data-dependent acquisition (DDA), data-independent acquisition (DIA), and ion mobility-enabled four-dimensional DIA (4D-DIA), across rice leaves, roots, and seeds. All workflows generated comparable extraction profiles across tissues; however, SP3 exhibited lower median coefficients of variation (10.5 to 15.1%) while requiring substantially reduced processing time. Notably, 4D-DIA identified 3-fold to 4-fold more proteins than DDA in each tissue and modestly outperformed conventional DIA, with improved detection of low-abundance proteins and high quantitative reproducibility. Collectively, SP3 with the 4D-DIA workflow provides a sensitive, reproducible, and time-efficient platform for plant bottom-up proteome profiling, particularly for samples with limited material or high metabolite interference.