In vivo crosslinking and effective 2D enrichment for proteome wide interactome studies

Area of Science:

• Proteomics
• Biochemistry
• Molecular Biology

Background:

• Cross-linking mass spectrometry (XMS) is vital for studying protein-protein interactions (PPIs) and protein structures.
• Challenges in XMS include low reaction efficiency and complex sample matrices, hindering system-wide crosslink analysis.

Purpose of the Study:

• To improve and streamline an Azide-A-DSBSO based in vivo crosslinking workflow.
• To enhance the enrichment of crosslinked peptides and reduce background noise.
• To generate a comprehensive PPI network and identify novel interactions, particularly in nuclear extracts.

Main Methods:

• Implemented an Azide-A-DSBSO based in vivo crosslinking approach.
• Employed two orthogonal enrichment strategies: affinity enrichment and size exclusion chromatography (SEC).
• Analyzed a single SEC fraction for efficient crosslink identification and throughput.

Main Results:

• The optimized workflow yielded over 5000 crosslinks from K562 cells, constructing a comprehensive PPI network.
• Identified 56 novel PPIs within the nucleus out of 393 total.
• Demonstrated increased crosslink identification on low-abundance proteins, such as DDX39B, using DSBSO on nuclear extracts.

Conclusions:

• The streamlined workflow significantly enhances the efficiency and throughput of in vivo crosslinking mass spectrometry.
• The method enables the discovery of novel protein-protein interactions and provides insights into protein complex formation.
• The findings highlight the utility of DSBSO crosslinking for analyzing nuclear proteins and their interactions, including potential monomeric and dimeric forms of DDX39B.