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

Autor: Larsen DN; Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark; Ovodan Biotech A/S, Havnegade 36, DK-5000 Odense, Denmark., Kaczmarek JZ; Ovodan Biotech A/S, Havnegade 36, DK-5000 Odense, Denmark., Palarasah Y; Department of Inflammation, Institute of Molecular Medicine, Faculty of Health and Medical Sciences, University of Southern Denmark, Odense, Denmark., Graversen JH; Department of Inflammation, Institute of Molecular Medicine, Faculty of Health and Medical Sciences, University of Southern Denmark, Odense, Denmark., Højrup P; Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark; Ovodan Biotech A/S, Havnegade 36, DK-5000 Odense, Denmark. Electronic address: php@bmb.sdu.dk.
Jazyk: angličtina
Zdroj: Biochimica et biophysica acta. Proteins and proteomics [Biochim Biophys Acta Proteins Proteom] 2024 Jul 01; Vol. 1872 (4), pp. 141011. Date of Electronic Publication: 2024 Mar 17.
DOI: 10.1016/j.bbapap.2024.141011
Abstrakt: Understanding protein-protein interactions is crucial for drug design and investigating biological processes. Various techniques, such as CryoEM, X-ray spectroscopy, linear epitope mapping, and mass spectrometry-based methods, can be employed to map binding regions on proteins. Commonly used mass spectrometry-based techniques are cross-linking and hydrogen‑deuterium exchange (HDX). Another approach, hydroxyl radical protein footprinting (HRPF), identifies binding residues on proteins but faces challenges due to high initial costs and complex setups. This study introduces a generally applicable method using Fenton chemistry for epitope mapping in a standard mass spectrometry laboratory. It emphasizes the importance of controls, particularly the inclusion of a negative antibody control, not widely utilized in HRPF epitope mapping. Quantification by TMT labelling is introduced to reduce false positives, enabling direct comparison between sample conditions and biological triplicates. Additionally, six technical replicates were incorporated to enhance the depth of analysis. Observations on the receptor-binding domain (RBD) of SARS-CoV-2 Spike Protein, Alpha and Delta variants, revealed both binding and opening regions. Significantly changed peptides upon mixing with a negative control antibody suggested structural alterations or nonspecific binding induced by the antibody alone. Integration of negative control antibody experiments and high overlap between biological triplicates led to the exclusion of 40% of significantly changed regions. The final identified binding region correlated with existing literature on neutralizing antibodies against RBD. The presented method offers a straightforward implementation for HRPF analysis in a generic mass spectrometry-based laboratory. Enhanced data reliability was achieved through increased technical and biological replicates alongside negative antibody controls.
Competing Interests: Declaration of competing interest Daniel Nyberg Larsen reports financial support was provided by Innovation Fund Denmark grant number 9065-00215B. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2023. Published by Elsevier B.V.)
Databáze: MEDLINE
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