Native MS and ECD Characterization of a Fab-Antigen Complex May Facilitate Crystallization for X-ray Diffraction

Area of Science:

• Biochemistry
• Structural Biology
• Mass Spectrometry

Background:

• Characterizing large protein complexes is crucial for understanding biological function.
• Antibody-protein interactions are key targets for therapeutic development.
• Native mass spectrometry (MS) and electron-capture dissociation (ECD) offer powerful tools for studying intact protein assemblies.

Purpose of the Study:

• To investigate the near-native structure of an antibody Fab (Fab-1)-VEGF complex.
• To determine the stoichiometry and identify flexible regions within the complex.
• To demonstrate the utility of native MS coupled with top-down ECD for protein complex analysis.

Main Methods:

• Native Electrospray Ionization (ESI) coupled with Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry.
• Top-down fragmentation techniques including collisionally activated dissociation (CAD), ECD, and infrared multiphoton dissociation (IRMPD).

Main Results:

• VEGF was confirmed to be a dimer in solution.
• The Fab-1-VEGF complex exhibited a 2:2 binding stoichiometry.
• Fragmentation analysis identified flexible regions within the complex, providing insights into its structural dynamics.

Conclusions:

• Native MS and top-down ECD are effective for characterizing large protein complexes like the Fab-1-VEGF assembly.
• The identified flexible regions may guide future crystallization and X-ray diffraction studies.
• This approach provides a comprehensive structural overview of protein-protein interactions in a near-native state.