open access publication

Article, 2024

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

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, ISSN 1878-2434, 1570-9639, 1878-1454, Volume 1872, 4, Page 141011, 10.1016/j.bbapap.2024.141011

Contributors

Larsen, Daniel Nyberg 0000-0002-2450-1833 [1] [2] Kaczmarek, Jakub Zbigniew 0000-0002-6602-8140 [3] Palarasah, Yaseelan 0000-0002-9337-0232 [2] Graversen, Jonas Heilskov 0000-0002-2411-9933 [2] Højrup, Peter 0000-0002-7838-6180 (Corresponding author) [2] [4]

Affiliations

  1. [1] 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.
    2. [NORA names: Denmark; Europe, EU; Nordic; OECD];
  2. [2] University of Southern Denmark
    3. [NORA names: SDU University of Southern Denmark; University; Denmark; Europe, EU; Nordic; OECD];
  3. [3] Ovodan Biotech A/S, Havnegade 36, DK-5000 Odense, Denmark.
    4. [NORA names: Denmark; Europe, EU; Nordic; OECD];
  4. [4] 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.
    5. [NORA names: Denmark; Europe, EU; Nordic; OECD]

Abstract

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.

Keywords

Delta variant, Fenton, Fenton chemistry, SARS-CoV-2, SARS-CoV-2 receptor-binding domain, SARS-CoV-2 spike protein, TMT, TMT labeling, X-ray, X-ray spectroscopy, alpha, alterations, analysis, antibodies, antibody control, antibody experiments, binding, binding region, binding residues, biological processes, biological replicates, biological triplicates, challenges, change region, chemistry, comparison, complex setup, conditions, control, control antibody, cost, cross-linking, cryoEM, data reliability, delta, depth, depth of analysis, design, domain, drug, drug design, enhance data reliability, epitope mapping, epitopes, exchange, exclusion, experiments, faces challenges, false positives, footprint, hydrogen-deuterium, hydrogen-deuterium exchange, hydroxyl, hydroxyl radical protein footprinting, identified binding regions, implementation, inclusion, initial cost, integration, interaction, investigate biological processes, labeling, laboratory, linear epitope mapping, literature, maps, mass spectrometry laboratories, mass spectrometry-based methods, mass spectrometry-based techniques, method, mixing, negative control antibody, neutralizing antibodies, nonspecific binding, observations, overlap, peptide, position, process, protein, protein footprinting, protein-protein interactions, quantification, receptor-binding domain, reduce false positives, region, reliability, replication, residues, samples, sampling conditions, setup, spectrometry-based methods, spectroscopy, spike protein, structural alterations, study, technical replicates, technique, triplicate, variants

Funders

  • Innovation Fund Denmark

Data Provider: Digital Science

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