Integrated Workflow for Structural Proteomics Studies Based on Cross-Linking/Mass Spectrometry with an MS/MS...

Area of Science:

• Structural Biology
• Proteomics
• Biochemistry

Background:

• Cross-linking coupled with mass spectrometry (MS) is vital for determining protein structures and interactions.
• Current methods can be complex and time-consuming, limiting routine application.

Purpose of the Study:

• To develop an automated workflow for identifying cross-linked peptides using a novel cleavable cross-linker.
• To simplify and enhance the efficiency of the cross-linking/MS technique for structural proteomics.

Main Methods:

• Utilized a tandem mass spectrometry (MS/MS) cleavable cross-linker (BuUrBu) that generates characteristic doublet patterns.
• Employed an updated MeroX software for automated cross-link identification.
• Evaluated fragmentation methods on an Orbitrap Fusion mass spectrometer using purified proteins and E. coli lysate.

Main Results:

• The workflow achieved high confidence cross-link identification with a 0.5% false discovery rate (FDR) for purified proteins.
• The cleavable cross-linker simplifies analysis by providing necessary fragmentation data at the MS/MS level.
• Structural insights into the p53 protein were obtained, demonstrating the workflow's utility.

Conclusions:

• The developed automated workflow and cleavable cross-linker significantly streamline the cross-linking/MS process.
• This approach is applicable to various mass spectrometers with MS/MS capabilities.
• The method facilitates the routine application of cross-linking/MS in structural proteomics research.