Electron capture dissociation as structural probe for noncovalent gas-phase protein assemblies

Area of Science:

• Mass Spectrometry
• Structural Biology
• Biochemistry

Background:

• Electron capture dissociation (ECD) typically causes charge reduction and backbone cleavage in proteins.
• ECD usually preserves labile, intramolecular noncovalent interactions.
• Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) is a powerful tool for analyzing protein complexes.

Purpose of the Study:

• To investigate the dissociation pathways of the 84-kDa noncovalent heptameric gp31 complex using ECD.
• To compare ECD with sustained off-resonance irradiation collisionally activated dissociation (SORI-CAD).
• To understand the role of charge state and gas-phase structure in ECD fragmentation.

Main Methods:

• Electron capture dissociation (ECD) was applied to the heptameric gp31 complex at various charge states.
• Sustained off-resonance irradiation collisionally activated dissociation (SORI-CAD) was used for comparison.
• Mass spectrometry data were analyzed to determine dissociation products and charge states.

Main Results:

• The 21+ charge state of the gp31 oligomer unexpectedly dissociated into a hexamer and monomer via ECD, disrupting intermolecular noncovalent bonds while keeping the backbone intact.
• Charge distribution in ECD products was proportional to molecular weight, indicating minimal charge redistribution.
• Lower charge states of gp31 did not show dissociation of noncovalent bonds under ECD.
• ECD of the 21+ charge state resulted in different fragmentation pathways compared to SORI-CAD.

Conclusions:

• ECD can disrupt intermolecular noncovalent bonds in protein complexes, contrary to typical observations.
• The fragmentation pathway in ECD is dependent on the initial gas-phase structure of the protein complex, particularly the charge state.
• The observed dissociation suggests that the monomeric product retains more of its original structure in ECD compared to SORI-CAD.