Two-Dimensional Fractionation Method for Proteome-Wide Cross-Linking Mass Spectrometry Analysis

Area of Science:

• Proteomics
• Biochemistry
• Molecular Biology

Background:

• Cross-linking mass spectrometry (XL-MS) is crucial for studying protein-protein interactions (PPIs) and protein complex structures.
• Current XL-MS methods face limitations in scope and depth due to proteome complexity and dynamic range, hindering low-abundance cross-linked peptide identification.
• Existing chromatographic enrichment methods like SEC and SCX are effective but require further improvement for deeper PPI mapping.

Purpose of the Study:

• To develop and validate a novel two-dimensional (2D) separation strategy for enhanced proteome-wide cross-linking mass spectrometry (XL-MS) analysis.
• To improve the identification of cross-linked peptides in complex biological samples.
• To expand the coverage and depth of protein-protein interaction network mapping.

Main Methods:

• Integration of peptide size exclusion chromatography (SEC) with tip-based high pH reverse-phase (HpHt) fractionation.
• Utilized the MS-cleavable cross-linker DSSO (Disuccinimidyl sulfoxide).
• Applied the 2D separation strategy to HEK293 cell lysates for *in vitro* PPI mapping.

Main Results:

• The developed 2D separation strategy significantly improved the identification of cross-linked peptides compared to existing methods.
• Successfully mapped *in vitro* protein-protein interactions from HEK293 cell lysates.
• Demonstrated enhanced coverage and depth in XL-MS analysis.

Conclusions:

• The integrated 2D SEC and HpHt fractionation method is effective for expanding proteome-wide XL-MS coverage.
• This approach offers improved identification of cross-linked peptides, advancing PPI studies.
• The method is generalizable for cross-linking studies in complex biological samples.