Novel Urazole-Derived MS-Cleavable Cross-Linkers Targeting Tyrosine/Lysine Residues for Protein Structural Analysis

Area of Science:

• Biochemistry
• Structural Biology
• Analytical Chemistry

Background:

• Chemical cross-linking mass spectrometry (XL-MS) is vital for understanding protein structure and interactions.
• Existing lysine-reactive cross-linkers are widely used but have limitations in coverage.
• There is a need for novel cross-linkers to expand the scope of XL-MS.

Purpose of the Study:

• To design and synthesize novel urazole-derived, MS-cleavable cross-linkers.
• To evaluate the reaction efficiency and applicability of these new cross-linkers.
• To enhance the capabilities of XL-MS for protein structural analysis.

Main Methods:

• Design and synthesis of two urazole-derived cross-linkers: a homobifunctional (SBT) targeting tyrosine and a heterobifunctional (SCT) targeting lysine and tyrosine.
• Evaluation of cross-linker reaction efficiencies using peptides, proteins, and protein complexes.
• Analysis of Bovine Serum Albumin (BSA) structures using AlphaFold 3 and comparison with crystal structures.

Main Results:

• Successful synthesis of two novel urazole-derived, MS-cleavable cross-linkers (SBT and SCT).
• Demonstrated reaction efficiency of the cross-linkers with various biological molecules.
• Uptake of urazole-derived cross-linkers enhances recognition coverage in XL-MS analysis.

Conclusions:

• The developed urazole-derived cross-linkers are valuable additions to the XL-MS toolkit.
• These cross-linkers enable accurate identification of cross-linked peptides via MSⁿ.
• The new reagents improve structural analysis and the study of protein-protein interactions.