Optimized Cross-Linking Mass Spectrometry for in Situ Interaction Proteomics

Area of Science:

• Biochemistry
• Proteomics
• Molecular Biology

Background:

• Large-scale cross-linking mass spectrometry (XL-MS) enables protein interaction mapping.
• Optimizing XL-MS is crucial for studying native protein complexes in vivo.

Purpose of the Study:

• To optimize the use of cleavable disuccinimidyl sulfoxide (DSSO) cross-linker for large-scale XL-MS in live human cells.
• To improve the sensitivity and specificity of XL-MS for protein interaction mapping.

Main Methods:

• Utilized cleavable DSSO cross-linker for labeling native protein complexes in live human cells.
• Applied a generalized linear mixture model to calibrate cross-link peptide-spectra matching (CSM) scores.
• Employed specific CSM score thresholds to control false discovery rate and explored HCD/ETD fragmentation methods.
• Incorporated multiple proteases and focused sample-specific search databases to enhance map coverage and specificity.

Main Results:

• Optimized DSSO cross-linking and spectral identification for large-scale XL-MS in human cells.
• Demonstrated that both HCD and ETD fragmentation are effective for protein interaction mapping.
• Showed improved protein-protein interaction map coverage using multiple proteases.
• Confirmed enhanced specificity of cross-linked peptide spectral matching with focused databases.

Conclusions:

• The optimized XL-MS approach effectively maps native protein interactions in situ, as shown in human chromatin studies.
• This method successfully identified known and novel protein interactions within chromatin-associated complexes.
• The refined XL-MS technique offers a valuable tool for diverse biological investigations of protein interactions.