We previously analyzed the Fab-1:VEGF (vascular endothelial growth factor) system described in this work, with both native top-down mass spectrometry and bottom-up mass spectrometry (carboxyl-group or GEE footprinting) techniques. This work continues bottom-up mass spectrometry analysis using a Fast Photochemical Oxidation of Proteins (FPOP) platform to map the solution binding-interface of VEGF and a fragment antigen-binding region of an antibody (Fab-1). In this study, we use FPOP to compare the changes in solvent accessibility by quantitating the extent of oxidative modification in the unbound versus bound states. Determining the changes in solvent accessibility enables the inference of the protein binding sites (epitope and paratopes) and a comparison to the previously published Fab-1:VEGF crystal structure. Using this method, we investigated peptide-level and residue-level changes in solvent accessibility between the unbound proteins and bound complex. Mapping these data onto the Fab-1:VEGF crystal structure enabled successful characterization of both the binding region and regions of remote conformation changes. These data, coupled with our previous higher order structure (HOS) studies, demonstrate the value of a comprehensive toolbox of methods for identifying the putative epitopes and paratopes for biotherapeutic antibodies.

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), electron-capture dissociation (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.

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A detailed study of the electrochemical behavior of the [Fe(CN)6]4-/3- redox couple was performed in water/AOT/n-heptane reverse micelles using a Pt microelectrode according to the high resistivity of the media. The results of varying the surfactant concentration at W 0 = [H2O]/[AOT] = 10 and constant [Fe(CN)6]4- concentration show an anomalous response of the limiting current value, i L. Despite the invariant reverse micelles size the i L value, expected to be constant, increases with the AOT concentration. These interesting results are explained as due to the ion pairing that [Fe(CN)6]4- may undergo with K+ counterions giving less charged species that tend to reside at the AOT reverse micelles interface and need more energy to undergo the oxidation process. The association process is favored when the molecular probe occupation number is greater than 2 because of the low water availability for ion solvation. As the water content increases, the ion solvation increases and reduces the degree of association. The free [Fe(CN)6]4- ion resides in the water pool where the electrochemical response is close to the one obtained in bulk water for the nonassociated species. Absorption spectroscopy experiments confirm these assumptions. As AOT reverse micelles can be considered monodisperse systems which make the entrance of the molecules or ions inside the reverse micelles a random effect and the [Fe(CN)6]4-/3- couple resides exclusively in their water pool due to Coulombic repulsion with the surfactant headgroups, we propose a model based in the Poisson distribution to account for different properties. The model and the measurements allow us to determine:  (i) the real concentration of the electroactive species, the one that it is not associated with the K+ counterions; (ii) the AOT reverse micelle diffusion coefficient, and (iii) the micellar hydrodynamic radius. The values are in very good agreement with results obtained by other techniques which clearly show that electrochemical measurements can be a powerful alternative approach to investigate organized media such as reverse micelles.

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