Tyrosine-Reactive Cross-Linker for Probing Protein Three-Dimensional Structures.

Area of Science:

• Biochemistry
• Chemical Biology
• Structural Biology

Background:

• Cross-linking mass spectrometry (XL-MS) is crucial for protein structure and complex elucidation.
• Current XL-MS methods are limited to specific amino acid residues like lysine, glutamic acid, aspartic acid, and cysteine.
• There is a need for novel cross-linkers targeting a broader range of amino acid residues.

Purpose of the Study:

• To design, synthesize, and characterize a novel cross-linker for selective targeting of tyrosine residues.
• To establish a new XL-MS strategy for protein structure analysis without photoirradiation or metal catalysts.

Main Methods:

• Development and synthesis of the novel cross-linker [4,4'-(disulfanediylbis(ethane-2,1-diyl))bis(1,2,4-triazolidine-3,5-dione)] (DBB).
• Application of DBB in an electrochemical click reaction for selective cross-linking of tyrosine residues in proteins.
• Analysis of cross-linked peptides before and after electrochemical reduction using mass spectrometry.

Main Results:

• DBB was successfully synthesized and characterized as a novel cross-linker.
• DBB demonstrated selective reactivity with tyrosine residues via an electrochemical click reaction.
• Characteristic peptides were observed post-electrochemical reduction, simplifying data analysis and enabling accurate identification of cross-linked products.

Conclusions:

• The novel DBB cross-linker enables selective targeting of tyrosine residues in proteins for the first time without photoirradiation or metal catalysts.
• This strategy offers a complementary tool for XL-MS, enhancing the ability to probe protein 3D structures, protein complexes, and protein-protein interactions.