Native MS and ECD Characterization of a Fab-Antigen Complex May Facilitate Crystallization for X-ray Diffraction.
Native mass spectrometry (MS) and top-down electron-capture dissociation (ECD) combine as a powerful approach for characterizing large proteins and protein assemblies. Here, we report their use to study an antibody Fab (Fab-1)-VEGF complex in its near-native state. Native ESI with analysis by FTICR mass spectrometry confirms that VEGF is a dimer in solution and that its complex with Fab-1 has a binding stoichiometry of 2:2. Applying combinations of collisionally activated dissociation (CAD), ECD, and infrared multiphoton dissociation (IRMPD) allows identification of flexible regions of the complex, potentially serving as a guide for crystallization and X-ray diffraction analysis. Graphical Abstract ᅟ.
Department of Chemistry, Washington University in St. Louis, One Brookings Dr., St. Louis, MO, 63130, USA.|Analytical Research and Development, Pfizer Inc., Chesterfield, MO, 63017, USA.
Ying
Y
Zhang
Department of Chemistry, Washington University in St. Louis, One Brookings Dr., St. Louis, MO, 63130, USA.
Weidong
W
Cui
Protein Analytical Chemistry, Genentech, a Member of the Roche Group, South San Francisco, CA, 94080, USA.
Aaron T
AT
Wecksler
Department of Chemistry, Washington University in St. Louis, One Brookings Dr., St. Louis, MO, 63130, USA.
Hao
H
Zhang
Protein Analytical Chemistry, Genentech, a Member of the Roche Group, South San Francisco, CA, 94080, USA.
Patricia
P
Molina
Protein Analytical Chemistry, Genentech, a Member of the Roche Group, South San Francisco, CA, 94080, USA.
Galahad
G
Deperalta
Department of Chemistry, Washington University in St. Louis, One Brookings Dr., St. Louis, MO, 63130, USA. mgross@wustl.edu.
Michael L
ML
Gross
D003460:Crystallization; D004583:Electrons; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D013058:Mass Spectrometry; D011506:Proteins; D014961:X-Ray Diffraction
D007140:Immunoglobulin Fab Fragments; D011506:Proteins
Electron capture dissociation (ECD); Fab-Antigen Complex; Fourier transform ICR mass spectrometry; High-order structure (HOS); Native MS; VEGF; X-ray crystallography