Comparison of in-solution digestion, FASP, S-trap, and SP3-based sample preparation for bottom-up proteomic study in plant tissues

ABSTRACT: Efficient and reproducible sample preparation remains a major challenge in bottom-up plant proteomics because plant tissues require extensive solubilization and careful removal of salts, detergents, and metabolite derived impurities before liquid chromatography tandem mass spectrometry analysis. These requirements often lead to lengthy and complex cleanup procedures. To identify a practical approach for diverse plant tissues, we compared in solution digestion, filter aided sample preparation, suspension trapping, and the single pot solid phase enhanced sample preparation method using rice leaves, roots, and seeds. All workflows produced broadly comparable extraction profiles. The single pot solid phase enhanced method provided the shortest processing time because it bypasses precipitation and performs protein binding, washing, and bead-based digestion in one tube. It also reduced handling steps and showed overall low median coefficients of variation across tissues. Peptides prepared by this method were analyzed using data dependent acquisition, conventional data independent acquisition, and four-dimensional data independent acquisition on a timsTOF platform. Four-dimensional data independent acquisition identified three to four times more proteins than data dependent acquisition and modestly more than conventional data independent acquisition. This strategy also enhanced detection of low abundance proteins while retaining reproducible quantitative performance. Together, these findings indicate that the single pot solid phase enhanced workflow with four-dimensional data independent acquisition provides a sensitive and time efficient platform for plant proteome profiling, particularly in studies involving limited sample amounts or substantial metabolite interference.

INSTRUMENT(S):

ORGANISM(S): Oryza Sativa (rice)

TISSUE(S): Root, Seed, Leaf

SUBMITTER: Sun Tae Kim  

LAB HEAD: Sun Tae Kim

PROVIDER: PXD071817 | Pride | 2026-10-07

REPOSITORIES: Pride