Expanding the Crosslinking Mass Spectrometry Toolbox With Vinyl Sulfone Crosslinkers

Area of Science:

• Proteomics
• Biochemistry
• Structural Biology

Background:

• Crosslinking mass spectrometry (MS) is vital for protein structure determination.
• Existing N-hydroxysuccinimide (NHS) ester crosslinkers have limited reactivity (lysine, N-termini) and buffer incompatibilities (Tris, ATP).

Purpose of the Study:

• Introduce novel vinyl sulfone crosslinkers to overcome limitations of NHS esters.
• Expand the range of crosslinkable amino acid residues and improve MS-based structural analysis.

Main Methods:

• Synthesized and characterized two vinyl sulfone-based crosslinkers: Alkyne-BVSC (homobifunctional, acid-cleavable alkyne) and VSD (heterobifunctional, diazirine photo-crosslinking).
• Evaluated reactivity with cysteine, histidine, and lysine residues.
• Developed a stub-based post-search filtering strategy for enhanced crosslink identification.

Main Results:

• Vinyl sulfone crosslinkers demonstrate reactivity with cysteine, histidine, and lysine, broadening residue accessibility.
• Reagents are compatible with amine-rich biochemical environments.
• The new filtering strategy significantly improves crosslink identification sensitivity.

Conclusions:

• Vinyl sulfone crosslinkers provide versatile and complementary tools for structural proteomics.
• These reagents expand the scope of crosslinking mass spectrometry.
• The developed methods enhance the ability to probe protein structures.