Expanding the Cross-Link Coverage of a Carboxyl-Group Specific Chemical Cross-Linking Strategy for Structural...

Area of Science:

• Proteomics
• Structural Biology
• Biochemistry

Background:

• Amine-specific cross-linking is common in structural proteomics.
• Carboxyl-group specific cross-linking offers complementary structural information.
• Dihydrazide linkers and DMTMM enable neutral pH cross-linking.

Purpose of the Study:

• To systematically investigate how experimental conditions affect the balance of carboxyl-carboxyl and carboxyl-amine cross-links.
• To optimize protocols for carboxyl-group specific chemical cross-linking.
• To enhance cross-link coverage in structural mass spectrometry.

Main Methods:

• Utilizing a combination of pimelic dihydrazide and the coupling reagent DMTMM.
• Systematically varying the ratios and concentrations of the two reagents.
• Applying the optimized strategy to model proteins, bovine 20S proteasome, and E. coli 70S ribosome.

Main Results:

• The ratio of pimelic dihydrazide to DMTMM can be adjusted to favor specific cross-linking products.
• Optimized reaction conditions more than doubled the number of identified cross-linked peptides in model proteins.
• The strategy demonstrated complementarity and increased cross-link coverage on large protein complexes.

Conclusions:

• Experimental conditions significantly influence the outcome of carboxyl-group specific cross-linking.
• Optimized protocols enhance the efficiency and coverage of structural proteomics studies.
• This approach provides a valuable tool for elucidating protein structures and interactions.