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A Streamlined Approach for Mass Spectrometry-Based Proteomics Using Selected Tissue Regions
Published on: April 18, 2025
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Efficient and robust proteome-wide approaches for cross-linking mass spectrometry
Oleg Klykov1,2, Barbara Steigenberger1,2, Sibel Pektaş1,2
1Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Utrecht, the Netherlands.
Nature Protocols
|November 18, 2018
Summary
This study presents an optimized protocol for cross-linking mass spectrometry (XL-MS) to identify thousands of protein-protein interactions (PPIs) and their spatial information. The method uses disuccinimidyl sulfoxide (DSSO) and simplifies data analysis for high-confidence results within 10 days.
Area of Science:
- Biochemistry
- Proteomics
- Structural Biology
Background:
- Cross-linking mass spectrometry (XL-MS) is a powerful technique for unbiased investigation of protein-protein interactions (PPIs) in complex biological samples.
- XL-MS offers direct spatial information about interacting proteins, a unique advantage over other PPI detection methods.
- Recent advancements in cross-linking chemistry and mass spectrometry (MS) platforms have enhanced XL-MS capabilities for complex sample analysis.
Purpose of the Study:
- To provide a detailed, optimized protocol for XL-MS using disuccinimidyl sulfoxide (DSSO) for native protein preparation and cross-linking.
- To demonstrate simplified data analysis using XlinkX in Proteome Discoverer and subsequent protein structure investigations with DisVis and HADDOCK.
- To enable high-confidence identification of thousands of PPIs and their spatial arrangements within a ~10-day timeframe.
Main Methods:
- Optimized native protein preparation and cross-linking using the gas-phase cleavable cross-linker disuccinimidyl sulfoxide (DSSO).
- Extensive sample fractionation to simplify complex proteomic data.
- Utilized XlinkX software within Proteome Discoverer for streamlined data analysis and DisVis/HADDOCK for structural investigations.
Main Results:
- Successful application of the optimized XL-MS protocol to identify a large number of protein-protein interactions.
- Demonstrated simplified and efficient data analysis pipeline, reducing complexity.
- Enabled subsequent protein structure investigations, providing spatial context to identified interactions.
Conclusions:
- The presented XL-MS protocol, utilizing DSSO, offers a robust and efficient method for comprehensive PPI analysis.
- The streamlined workflow, including data analysis and structural investigation, yields high-confidence results.
- This protocol facilitates deeper understanding of protein complex organization and function within biological systems.
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