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This study introduces a novel crosslinking strategy to significantly enhance the depth of human interactome mapping using crosslinking mass spectrometry (XL-MS). The new method achieves a ~20-fold increase in detected protein-protein interactions (PPIs), outperforming existing techniques.

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Area of Science:

  • Proteomics
  • Biochemistry
  • Molecular Biology

Background:

  • Crosslinking mass spectrometry (XL-MS) is a powerful technique for mapping protein-protein interactions (PPIs).
  • Current XL-MS methods suffer from low sampling depth, limiting their ability to comprehensively analyze the interactome.
  • Existing methods like affinity pulldown MS are indirect and prone to errors.

Purpose of the Study:

  • To develop an advanced crosslinking strategy to overcome the low sampling depth limitation in XL-MS.
  • To significantly increase the number of detectable protein-protein interactions (PPIs) for high-resolution interactome mapping.
  • To provide a more accurate and comprehensive view of the spatial proteome.

Main Methods:

  • A two-step sequential and orthogonal crosslinking strategy was developed.
  • The method involves pre-stabilizing the spatial proteome using an immunofluorescence-inspired fixation protocol.
  • Surface-accessible lysines were labeled with N-hydroxysuccinimide (NHS)-modified click reagents, followed by copper-catalyzed azide-alkyne cycloaddition (CuAAC) for crosslinking.

Main Results:

  • The novel crosslinking strategy achieved crosslink levels approaching 30% of the total signal.
  • Protein-protein interactions (PPIs) were detected at levels approximately 20 times higher than conventional DSS-based methods.
  • The method demonstrated no detectable side reactions or distortions of the spatial proteome.

Conclusions:

  • The developed crosslinking strategy substantially enhances sampling depth in XL-MS.
  • This method offers a significant improvement over existing techniques for mapping the human interactome.
  • The approach provides high-fidelity, high-resolution mapping of protein-protein interactions with unprecedented depth.