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PEPPI-SP3: A Gel-Based High-Resolution Sample Preparation Workflow for In-Depth Top-Down Analysis of Intact
Ayako Takemori1, Jake T Kline2, Luca Fornelli2,3
1Advanced Research Support Center, Ehime University, Toon, Ehime, 791-0295, Japan.
Methods in Molecular Biology (Clifton, N.J.)
|August 1, 2026
Summary
This study introduces the PEPPI-SP3 workflow, a novel method enhancing top-down proteomics. It improves the identification of intact protein forms by combining gel electrophoresis with bead-based purification.
Area of Science:
- Proteomics
- Biochemistry
- Analytical Chemistry
Background:
- Top-down proteomics identifies intact proteoforms using mass spectrometry.
- Current methods face limitations in analytical depth and sample preparation efficiency.
Purpose of the Study:
- To present the PEPPI-SP3 workflow, a high-resolution sample prefractionation method.
- To enhance the analytical depth of top-down proteomics.
- To improve the efficiency of intact protein analysis.
Main Methods:
- Combining in-gel protein extraction after SDS-PAGE separation.
- Utilizing SP3 (Single-Pot Solid-Phase-peptide, -protein, -purification) bead-based purification.
- Integrating passive extraction with bead purification for intact proteins.
Main Results:
- The PEPPI-SP3 workflow significantly enhances analytical depth in top-down proteomics.
- Achieved high-resolution prefractionation of intact proteins.
- Demonstrated efficient passive extraction and purification of proteins.
Conclusions:
- PEPPI-SP3 is an effective workflow for improving top-down proteomics.
- This method increases the identification of proteoforms.
- Offers a robust approach for high-resolution intact protein analysis.
