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Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography
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Developing a new cleavable crosslinker reagent for in-cell crosslinking.

Fränze Müller1, Bogdan R Brutiu2, Iakovos Saridakis2

  • 1Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC), Campus-Vienna-Biocenter 1, 1030, Vienna, Austria.

Communications Chemistry
|June 23, 2025
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Summary
This summary is machine-generated.

A new MS-cleavable crosslinker, DiSPASO, enables rapid in-cell protein-protein interaction (PPI) analysis. While promising for mapping cellular networks, optimizing its design is crucial for improving crosslinking yield in complex biological environments.

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Area of Science:

  • Biochemistry
  • Proteomics
  • Chemical Biology

Background:

  • Crosslinking mass spectrometry (XL-MS) is vital for protein structure and interaction studies.
  • Existing cleavable crosslinkers have limitations in fragmentation analysis and backbone behavior.
  • System-wide protein-protein interaction (PPI) mapping in cells requires advanced XL-MS tools.

Purpose of the Study:

  • Introduce DiSPASO, a novel lysine-selective, MS-cleavable crosslinker.
  • Evaluate DiSPASO's efficiency in cell membrane permeability and crosslinking with minimal cellular perturbation.
  • Assess DiSPASO's utility for mapping PPIs in complex cellular systems.

Main Methods:

  • Developed DiSPASO with an alkyne handle for affinity enrichment and MS-cleavable properties.
  • Utilized three copper-based enrichment strategies for testing.
  • Employed model systems ranging from purified proteins to live HEK 293 cells.
  • Conducted in-cell crosslinking experiments with fluorescence microscopy.

Main Results:

  • DiSPASO demonstrated rapid uptake into HEK 293 cells within 5 minutes.
  • Successful application in crosslinking studies using model systems of increasing complexity.
  • Confirmed MS-cleavable properties and lysine selectivity of DiSPASO.

Conclusions:

  • DiSPASO shows potential as an improved tool for in-cell PPI analysis.
  • Limitations in crosslinking yield highlight the need for further optimization of crosslinker design.
  • Continuous innovation is essential for accurate PPI network mapping in dynamic cellular environments.