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High-throughput proteome integral solubility alteration assay for low cell input using One-Tip.

Maico Lechner1, Pierre Sabatier2,3, Jesper V Olsen4

  • 1Department of Cellular and Molecular Medicine, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Communications Chemistry
|September 27, 2025
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Summary
This summary is machine-generated.

We streamlined drug-target engagement analysis using proteome integral solubility alteration (PISA) with the One-Tip method. This approach significantly reduces cell requirements and processing time, enhancing efficiency for broader laboratory application.

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Area of Science:

  • Biochemistry
  • Proteomics
  • Chemical Biology

Background:

  • Cellular thermal shift assay (CETSA) and its mass spectrometry-based variants like proteome integral solubility alteration (PISA) are crucial for monitoring drug-target engagement.
  • Current PISA methods face limitations in throughput, scalability, and sample volume, restricting their widespread use.

Purpose of the Study:

  • To develop a streamlined and scalable sample preparation workflow for PISA by integrating the One-Tip method.
  • To reduce sample input requirements and enhance the efficiency and accessibility of CETSA-based drug-target engagement profiling.

Main Methods:

  • Combined PISA with the One-Tip method for simplified sample preparation.
  • Utilized n-Dodecyl-β-D-Maltoside (DDM) for cell lysis compatible with mass spectrometry and One-Tip processing.
  • Reduced cell input requirements to 200 cells per µL.
  • Applied the One-Tip-PISA workflow in a 96-well plate format for profiling kinase inhibitors.

Main Results:

  • Achieved significantly lower cell requirements (down to 200 cells/µL) for PISA.
  • Demonstrated comparable proteomic depth and improved reproducibility with the One-Tip-PISA method.
  • Reduced material and solvent usage, with faster proteolytic digestion compared to conventional protocols.
  • Enabled a complete workflow from cell treatment to mass spectrometry injection within 12 hours.

Conclusions:

  • The One-Tip-PISA method offers a cost-effective, fast, and user-friendly approach for drug-target engagement studies.
  • This integrated workflow enhances scalability and accessibility, making advanced proteomic profiling feasible for more laboratories.
  • The optimized workflow accelerates drug discovery and development by improving the efficiency of target engagement assessment.