The First Zero-Length Mass Spectrometry-Cleavable Cross-Linker for Protein Structure Analysis

Area of Science:

• Biochemistry
• Structural Biology
• Analytical Chemistry

Background:

• Protein structure analysis is crucial for understanding biological function.
• Cross-linking/mass spectrometry (MS) is a powerful technique for mapping protein interactions and structures.
• Existing cross-linking reagents often lack cleavability or introduce significant spacer lengths, complicating data analysis.

Purpose of the Study:

• To introduce 1,1 -carbonyldiimidazole (CDI) as a novel, zero-length, cleavable cross-linker for protein structure determination.
• To demonstrate the utility of CDI in conjunction with tandem mass spectrometry (MS/MS) for enhanced protein structural analysis.
• To provide a cost-effective and user-friendly reagent for obtaining short-distance information in protein 3D structures.

Main Methods:

• Utilizing 1,1 -carbonyldiimidazole (CDI) to cross-link primary amines and hydroxy groups in proteins under physiological conditions (pH 7.2-8).
• Employing tandem mass spectrometry (MS/MS) to induce cleavage of urea and carbamate cross-linked products.
• Analyzing characteristic product ions generated during MS/MS for unambiguous identification of cross-links.

Main Results:

• CDI functions as a zero-length cross-linker (ca. 2.6 Å spacer), bridging protein residues efficiently.
• Cross-linked products formed by CDI are readily cleaved by MS/MS, yielding specific product ions.
• The method was successfully applied to diverse proteins: bovine serum albumin (BSA), p53, and GCAP-2, demonstrating its broad applicability.

Conclusions:

• CDI is an effective and versatile reagent for protein structure analysis via cross-linking/MS.
• The cleavability of CDI-induced cross-links significantly enhances the accuracy and automation of data analysis.
• This approach provides valuable short-distance structural insights, particularly for complex and disordered proteins.