Fast Photochemical Oxidation of Proteins (FPOP) Maps the Epitope of EGFR Binding to Adnectin

Abstract

Epitope mapping is an important tool for the development of monoclonal antibodies, mAbs, as therapeutic drugs. Recently, a class of therapeutic mAb alternatives, adnectins, has been developed as targeted biologics. They are derived from the tenth type III domain of human fibronectin (10Fn3). A common approach to map the epitope binding of these therapeutic proteins to their binding partners is X-ray crystallography. Although the crystal structure is known for Adnectin 1 binding to human EGFR, we seek to determine complementary binding in solution and to test the efficacy of footprinting for this purpose. As a relatively new tool in structural biology and complementary to X-ray crystallography, protein footprinting coupled with mass spectrometry is promising for protein-protein interaction studies. We report here the use of fast photochemical oxidation of proteins (FPOP) coupled with MS to map the epitope of EGFRAddnectin 1 at both the peptide and amino-acid residue levels. The data correlate well with the previously determined epitope from the crystal structure and are consistent with HDX MS data, which are presented in an accompanying paper. The FPOP-determined binding interface involves various amino-acid and peptide regions near the N terminus of EGFR. The outcome adds credibility to oxidative labeling by FPOP for epitope mapping and motivates more applications in the therapeutic protein area as a stand-alone method or in conjunction with X-ray crystallography, NMR, site-directed mutagenesis, and other orthogonal methods.

Imaging short‐lived reactive oxygen species (ROS) with endogenous contrast MRI

“…[8][9][10][11][12][13] Instead of developing an MRI technique with submicrosecond temporal resolution to study its MRI properties, we alternatively established an imaging platform that sustainably produces ROS. Fresh egg white treated with H 2 O 2 prolongs ROS production for hours, with quantifiable ROS concentrations over time.…”

“…Based on this simple model, we estimated the detection sensitivity of ROS MRI is at the pM level under the assumption of a 1 ls lifetime for hydroxyl radicals. [8][9][10][11][12][13] Under shorter lifetimes (eg, 1 ns), the detection sensitivity will be proportionally higher (eg, 10 23 pM level). Given that the physiological concentrations of ROS is in the lM level 14 and their concentration can be elevated by over 100 times under conditions such as inflammation or stroke and reperfusion, 14,31,32 the pM detection sensitivity provides the capability to detect ROS under both physiological and pathological conditions.…”

“…According to Rosen criteria, requiring CNR >5, the detection sensitivity of ROS imaging was around 10 pM assuming a lifetime of 1 ls for the hydroxyl radicals. [8][9][10][11][12][13] With the estimated ROS concentrations over time and the corresponding T 1 relaxation times, the T 1 relaxivity of ROS was computed to be 3.4 3 10 7 s 21 ÁmM 21 , which is much larger than that of Gd-DTPA (0.9 s 21 ÁmM 21 ) (Fig. 5).…”

“…For example, the lifetime of singlet oxygen, superoxide, and hydroxyl radical in aqueous solutions is within the ls regime. [8][9][10][11][12][13] In addition, The power of MRI resides in providing superb softtissue structural contrast for clinical diagnosis. In addition to the anatomical information provided by MRI, molecular information may also be obtained.…”

“…For example, the lifetime of singlet oxygen, superoxide, and hydroxyl radical in aqueous solutions is within the ls regime. [8][9][10][11][12][13] In addition, the in vivo concentration of ROS is generally low (lM level), 14 making the detection more challenging. In order to measure ROS, several methods have been developed including optical redox scanning 15 and exogenous contrast magnetic resonance imaging (MRI)-based methods.…”