A Simple Cross-Linking/Mass Spectrometry Workflow for Studying System-wide Protein Interactions
Michael Götze1, Claudio Iacobucci2, Christian H Ihling2
1Institute for Biochemistry and Biotechnology, Charles Tanford Protein Center , Martin Luther University Halle-Wittenberg , Kurt-Mothes-Strasse 3a , D-06120 Halle (Saale) , Germany.
Analytical Chemistry
|July 9, 2019
Summary
We developed a rapid, week-long proteome-wide cross-linking/mass spectrometry workflow. This method maps protein-protein interactions and protein conformations, offering insights into cellular networks.
Area of Science:
- Proteomics
- Biochemistry
- Molecular Biology
Background:
- Understanding protein-protein interactions and conformations is crucial for deciphering cellular mechanisms.
- Existing methods for proteome-wide analysis can be time-consuming and complex.
Purpose of the Study:
- To present an optimized, rapid cross-linking/mass spectrometry workflow for proteome-wide analysis.
- To enable comprehensive mapping of protein-protein interaction networks and protein conformations.
- To provide an updated, automated software tool for reliable data analysis.
Main Methods:
- Utilized a mass spectrometry-cleavable cross-linker, disuccinimidyl dibutyric urea.
- Implemented an updated version 2.0 of the freeware software MeroX for automated data analysis.
- Applied the workflow to analyze protein-protein interactions in Drosophila melanogaster embryos.
Main Results:
- Detected 29,931 cross-link spectrum matches, identifying 7,436 unique cross-linked residues at a 1% false discovery rate.
- Identified 1,611 interprotein cross-linking sites, revealing novel protein-protein interactions.
- Characterized 5,825 intraprotein cross-links, providing insights into protein conformational dynamics.
Conclusions:
- The presented workflow significantly accelerates proteome-wide cross-linking analysis.
- This approach provides a powerful tool for mapping complex protein interaction networks and conformational landscapes.
- The MeroX software facilitates reliable and automated analysis of cross-linking mass spectrometry data.
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