Simplified and Rapid Workflow Enhances Throughput of De Novo Sequencing of COVID-19 Neutralizing Antibodies

During the current COVID-19 pandemic, precise antibody sequencing is crucial for the rapid development of broad-spectrum or pan-{beta}-coronavirus neutralizing antibodies (NAbs) to prevent new variants of the coronavirus and other highly pathogenic {beta}-coronaviruses. However, mass spectrometry-based de novo sequencing remains challenging due to its high cost, low throughput, and unexpected missing of certain ion information. We developed an innovative approach using solely bottom-up to provide a rapid, robust, and refined solution for the current de novo sequencing challenges. The methodology, referred to as SP-MEGD for Single-Pot and Multi-Enzymatic Gradient Digestion, capitalizes on a five-protease gradient digestion by sampling every two hours for a total of 6 hours in an integrative reactor. The SP-MEGD method could engender numerous missed cleavage events and produce various peptide products of diverse lengths with overlapping stretches of residues, enabling efficient database-free de novo sequencing. Antibody assembly for single or mixed COVID-19-NAbs with publicly available sequences and commercial antibodies with unknown sequences was efficiently deciphered using our previously proposed optimization method, Fusion assembler. Our innovative study represents the first successful simultaneous discrimination and achieving over 99% accurate sequence coverage of a mixture containing three humanized COVID-19-NAbs (S2P6LH, BD5514LH, and BD5840LH). Furthermore, we utilized SP-MEGD and Fusion to sequence commonly used anti-CD8 and anti-CD4, and successfully confirmed their impact on immune cell clearance through in vivo experiments. Overall, our developed workflow demonstrates promise in facilitating the discovery and development of vaccines and antibody therapeutics while providing deeper insights into monoclonal antibodies (mAbs).