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Updated: Jun 25, 2026
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Published on: February 7, 2022
Comparative Assessment of Sample Preparation Workflows and Mass Spectrometry Acquisition Strategies for Bottom-Up
Ji Soo Kim1, Na Won Park1, Gi Hyun Lee1
1Department of Plant Bioscience, Life and Industry Convergence Research Institute, Pusan National University, Miryang 50463, Republic of Korea.
Journal of Agricultural and Food Chemistry
|June 23, 2026
Summary
Optimizing plant proteome profiling requires efficient sample preparation and mass spectrometry (MS) acquisition. The SP3 workflow combined with 4D-DIA offers a sensitive, reproducible, and time-saving platform for plant bottom-up proteomics.
Area of Science:
- Plant science
- Proteomics
- Mass spectrometry
Background:
- Achieving robust and reproducible proteome coverage in plant bottom-up proteomics is challenging due to biochemical complexity and variability.
- Tissue-specific differences and inconsistencies in sample preparation and MS acquisition further complicate proteomic studies.
Purpose of the Study:
- To systematically evaluate and compare four distinct sample preparation workflows.
- To assess three different mass spectrometry (MS) acquisition strategies for plant proteome profiling.
- To identify an optimal combination for sensitive, reproducible, and time-efficient plant proteome analysis.
Main Methods:
- Evaluated four sample preparation workflows: in-solution digestion, FASP, S-trap, and SP3.
- Compared three MS acquisition strategies: data-dependent acquisition (DDA), data-independent acquisition (DIA), and 4D-DIA.
- Applied these methods across rice tissues (leaves, roots, and seeds).
Main Results:
- All workflows showed comparable protein extraction profiles across different rice tissues.
- The SP3 workflow demonstrated lower median coefficients of variation (10.5–15.1%) and reduced processing time.
- 4D-DIA identified 3- to 4-fold more proteins than DDA, outperforming conventional DIA with enhanced detection of low-abundance proteins and high quantitative reproducibility.
Conclusions:
- The SP3 sample preparation combined with the 4D-DIA acquisition strategy provides a highly sensitive and reproducible platform for plant bottom-up proteome profiling.
- This optimized workflow is particularly beneficial for samples with limited material or significant metabolite interference.
- It represents a time-efficient advancement for plant proteomic research.
