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Sample preparation and cleanup methods for clinical top-down proteomics.

Amal Mohamed Kamal1,2, Amy Carfagno3, Cynthia Nagy3

  • 1Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK, USA.

Expert Review of Proteomics
|July 28, 2025
PubMed
Summary
This summary is machine-generated.

Top-down proteomics (TDP) enables disease-specific proteoform analysis for personalized medicine. This review covers sample preparation and cleanup methods to advance clinical TDP workflows and biomarker discovery.

Keywords:
Clinical proteomicsFilter-aided sample preparationNanoparticlePrecipitationSolid-phase extractionTop-down proteomicsserial size exclusion chromatographysingle-pot solid phase-enhanced sample preparation

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Area of Science:

  • Clinical proteomics
  • Biomarker discovery
  • Personalized medicine

Background:

  • Proteoform analysis in clinical samples aids disease mechanism elucidation and biomarker identification.
  • Top-down proteomics (TDP) offers potential for personalized medicine but faces sample preparation challenges.

Purpose of the Study:

  • To review state-of-the-art sample preparation and cleanup protocols for clinical top-down proteomics.
  • To inform the selection of effective and efficient sample processing approaches for clinical TDP.

Main Methods:

  • Summarizes targeted (affinity/non-affinity enrichment) and untargeted (gel-based fractionation) sample preparation protocols.
  • Reviews offline and online sample cleanup procedures including dialysis, SPE, FASP, precipitation, and SPPP for desalting and detergent removal.

Main Results:

  • Identifies various sample preparation and cleanup techniques applicable to clinical TDP.
  • Highlights the effectiveness of different methods for removing salts and detergents, crucial for proteoform analysis.

Conclusions:

  • Clinical TDP has significant potential for identifying disease-specific proteoforms missed by traditional assays.
  • Standardized guidelines for reproducible clinical TDP workflows are crucial for wider adoption and leveraging sample preparation advances.