XPlex: An Effective, Multiplex Cross-Linking Chemistry for Acidic Residues

Area of Science:

• Structural Biology
• Proteomics
• Chemical Biology

Background:

• Cross-linking/Mass spectrometry (XLMS) is vital for protein structure determination.
• Current XLMS methods predominantly use N-hydroxysuccinimide (NHS) esters, limiting cross-linking to lysine residues.
• Expanding XLMS applicability requires methods targeting a broader range of amino acids.

Purpose of the Study:

• To introduce a multiplex chemistry (XPlex) for XLMS that expands residue targeting.
• To demonstrate XPlex's ability to generate more cross-links, including for lysine-poor proteins.
• To present a computational solution for analyzing XPlex-generated data.

Main Methods:

• Developed XPlex, a multiplex cross-linking chemistry targeting Asp, Glu, Lys, and Ser residues.
• Applied XPlex to model proteins and the lysine-poor protein SalBIII.
• Updated the SIM-XL search algorithm to accommodate XPlex data analysis for multiple cross-linkers.

Main Results:

• XPlex generated significantly more cross-links compared to NHS ester-based methods.
• XPlex reactions occurred at lower temperatures, enabling targeting of previously inaccessible proteins.
• The updated SIM-XL algorithm successfully identified XPlex spectra.

Conclusions:

• XPlex provides a comprehensive chemistry and computational solution to increase distance constraints in mass spectrometry experiments.
• XPlex significantly enhances the number of obtainable cross-links, complementing existing XLMS approaches.
• XPlex offers a valuable tool for advancing structural proteomics studies, particularly for challenging protein targets.