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PEPPI-MS: gel-based sample pre-fractionation for deep top-down and middle-down proteomics

Nature Protocols volume 20, pages 1413–1438 (2025)Cite this article

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Abstract

Top-down analysis of intact proteins and middle-down analysis of proteins subjected to limited digestion require efficient detection of traces of proteoforms in samples, necessitating the reduction of sample complexity by thorough pre-fractionation of the proteome components in the sample. SDS–PAGE is a simple and inexpensive high-resolution protein-separation technique widely used in biochemical and molecular biology experiments. Although its effectiveness for sample preparation in bottom-up proteomics has been proven, establishing a method for highly efficient recovery of intact proteins from the gel matrix has long been a challenge for its implementation in top-down and middle-down proteomics. As a much-awaited solution to this problem, we present an experimental protocol for efficient proteoform fractionation from complex biological samples using passively eluting proteins from polyacrylamide gels as intact species for mass spectrometry (PEPPI-MS), a rapid method for extraction of intact proteins separated by SDS–PAGE. PEPPI-MS allows recovery of proteins below 100 kDa separated by SDS–PAGE in solution with a median efficiency of 68% within 10 min and, unlike conventional electroelution methods, requires no special equipment, contributing to a remarkably economical implementation. The entire protocol from electrophoresis to protein purification can be performed in <5 h. By combining the resulting PEPPI fraction with other protein-separation techniques, such as reversed-phase liquid chromatography and ion mobility techniques, multidimensional proteome separations for in-depth proteoform analysis can be easily achieved.

Key points

  • Sample pre-fractionation is essential to reduce complexity before top- and middle-down proteomics. PEPPI-MS uses passive-elution from SDS–PAGE to recover proteins below 100 kDa. The workflow can be applied to intact proteins (top-down proteomics) or to partially digested proteins (middle-down proteomics). Analysis on size-selected fractions (targeted middle-down proteomics) is also possible.

  • This protocol uses standard electrophoresis equipment and reagents, reducing costs and easing barriers to implementation.

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Fig. 1: Rapid passive extraction of intact proteins in SDS–PAGE gels by PEPPI-MS.
Fig. 2: Sample pre-fractionation workflow for in-depth TDP or MDP by PEPPI-MS.
Fig. 3: Sample preparation with AnExSP.
Fig. 4: HCPE fractionation for in-depth TD proteomics.
Fig. 5: MD proteomics of HCPE by the global approach.

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Data availability

All MS raw data have been uploaded to the ProteomeXchange Consortium via the PRIDE partner repository with the accession PXD034353 (TDP data in Fig. 4)22 and PXD041358 (MDP data in Fig. 5)12.

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Acknowledgements

This work was supported by JSPS KAKENHI 22KK0077 and 23K04963 (to N.T.) and the Deutsche Forschungsgemeinschaft (DFG), within the Cluster of Excellence ‘Precision Medicine in Inflammation (PMI)’-RTF-V (to A. Tholey). The authors thank Erika Teraoka for editing and reviewing this manuscript for use of the English language and helpful discussion.

Author information

Authors and Affiliations

  1. Advanced Research Support Center, Ehime University, Ehime, Japan

    Ayako Takemori & Nobuaki Takemori

  2. Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, Kiel, Germany

    Philipp T. Kaulich & Andreas Tholey

Authors
  1. Ayako Takemori
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  2. Philipp T. Kaulich
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  3. Andreas Tholey
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  4. Nobuaki Takemori
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Contributions

Protocols for PEPPI-MS and AnExSP were developed by A. Takemori and N.T.; protocols for LC-FAIMS-MS analysis were developed by P.T.K. and A. Tholey.

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Correspondence to Nobuaki Takemori.

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Nature Protocols thanks Si Wu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references

Takemori, A. et al. J. Proteome Res. 19, 3779–3791 (2020): https://doi.org/10.1021/acs.jproteome.0c00303

Takemori, A. et al. Anal. Chem. 94, 12815–12821 (2022): https://doi.org/10.1021/acs.analchem.2c02777

Takemori, A. et al. Proteomics 24, e2200431 (2024): https://doi.org/10.1002/pmic.202200431

Takemori, A. et al. Chem. Commun. 58, 775–778 (2022): https://doi.org/10.1039/d1cc05529a

Supplementary information

Supplementary Information

Supplementary Figures 1–3 and Supplementary Table 1

Reporting Summary

Supplementary Video 1

PEPPI-MS: gel fractionation

Supplementary Video 2

PEPPI-MS: gel homogenization no. 1

Supplementary Video 3

PEPPI-MS: gel homogenization no. 2

Supplementary Video 4

PEPPI-MS: passive extraction

Supplementary Video 5

AX StageTip

Supplementary Video 6

AnExSP

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Takemori, A., Kaulich, P.T., Tholey, A. et al. PEPPI-MS: gel-based sample pre-fractionation for deep top-down and middle-down proteomics. Nat Protoc 20, 1413–1438 (2025). https://doi.org/10.1038/s41596-024-01100-0

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