Covalent Cross-Linking as an Enabler for Structural Mass Spectrometry

Area of Science:

• Biophysical Chemistry
• Structural Biology
• Analytical Chemistry

Background:

• Native mass spectrometry (MS) elucidates biomolecular structures by analyzing gas-phase conformations.
• This technique faces limitations with dynamic systems and non-ideal conditions, potentially disrupting native folds.
• Preserving solution-like conformations during electrospray ionization and gas-phase analysis is crucial.

Purpose of the Study:

• To evaluate a cross-linking strategy for enhancing protein stability in mass spectrometry.
• To investigate if cross-linking preserves native protein conformations under challenging mass spectrometry conditions.
• To assess the structural integrity and gas-phase behavior of cross-linked proteins.

Main Methods:

• Proteins (cytochrome c, myoglobin, β-lactoglobulin) were chemically cross-linked using BS³.
• Far-ultraviolet circular dichroism (UV-CD) assessed structural integrity in solution.
• Ion mobility mass spectrometry (IM-MS) evaluated gas-phase stability and conformational preservation.
• Molecular dynamics (MD) simulations provided insights into structural changes.

Main Results:

• Cross-linking was structurally nonintrusive in solution, as confirmed by UV-CD.
• Cross-linked proteins exhibited enhanced resilience to denaturation and preserved folded conformations in the gas phase.
• Ion mobility data showed collision cross sections consistent with native folds.
• MD simulations corroborated the preservation of secondary and tertiary structures.

Conclusions:

• Protein cross-linking is a viable strategy to improve gas-phase stability in mass spectrometry.
• This method ensures the preservation of native-like conformations under demanding experimental conditions.
• Cross-linking offers a promising approach for future structural mass spectrometry workflows.