Epitope mapping of SARS-CoV-2 RBDs by hydroxyl radical protein footprinting reveals the importance of including negative antibody controls

Abstract

Epitope mapping methods using hydroxyl radical protein footprinting (HRPF) are currently hampered by high initial cost and complex experimental setup. Here we set out to present a generally applicable method using Fenton chemistry for mapping of epitopes and paratopes in a standard laboratory setting. Furthermore, the described method illustrates the importance of controls on several levels when performing mass spectrometry-based epitope mapping. In particular, the inclusion of negative antibody controls has not previously been widely utilized in epitope mapping by HRPF analysis. In order to limit the false positives, we further introduced quantification by TMT labelling, thereby allowing for direct comparison between sample conditions and biological triplicates. Lastly, up to six technical replicates were incorporated in the experimental setup in order to achieve increased depth of the final analysis.Both binding and openings of regions on receptor binding domain (RBD) from SARS-CoV-2 Spike Protein, Alpha and Delta variants, were observed. The negative control antibody experiment, combined with the high overlap between biological triplicates, resulted in the exclusion of 40% of the significantly changed regions, including both binding and opening regions. The identified binding region agrees with the literature for neutralizing antibodies towards SARS-CoV-2 Spike Protein.The presented method is an easily implementable technique for the analysis of HRPF in a generic MS-based laboratory. The high reliability of the data was achieved by increasing the number of technical and biological replicates combined with negative antibody controls.