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De Novo Sequencing of Polyclonal Antibodies by Integrating Intact Mass, Top-Down and Bottom-Up Mass Spectrometry

ABSTRACT: Antibodies are specialized proteins produced by the adaptive immune system to identify and neutralize harmful antigens. Antibody-based diagnostics and therapeutics have advanced rapidly, with recombinant antibodies derived from monoclonal antibodies (mAbs) becoming the preferred standard due to their high reproducibility and effectiveness. However, mAbs are limited to binding a single antigen epitope. Polyclonal antibodies (pAbs), on the other hand, are produced by multiple B cells and can target multiple epitopes, making them more robust against antigen variations. Despite their advantages, pAbs are notoriously challenging to sequence due to their high complexity. Current de novo sequencing methods based on mass spectrometry have shown limitations when applied to pAb mixtures, often requiring germline databases or yielding incomplete or inaccurate sequences. Here we propose PolySeq, a fully de novo sequencing workflow that integrates bottom-up, top-down, and intact mass spectrometry data to accurately sequence pAb samples without relying on external databases. Our workflow automates the entire sequencing process from peptides to full proteins, achieving complete and accurate antibody profiles. Evaluation results on a mixture of four known mAbs and a pAb sample derived from mouse myeloma demonstrate that our de novo antibody sequences achieved up to 100% coverage and accuracy, with strong supporting evidence from bottom-up, top-down, and intact mass data. Furthermore, antibodies recombinantly expressed using de novo sequencing results showed expected properties, such as binding affinity and antigen neutralization, thus confirming the accuracy and efficacy of our pAb de novo sequencing workflow.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Ngoc Hieu Tran

LAB HEAD: Baozhen Shan

PROVIDER: PXD063526 | Pride | 2025-10-31

REPOSITORIES: Pride

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			Action	DRS
	1-10_whole_intact.raw	Raw
	1-3_whole_intact.raw	Raw
	1-5_whole_intact.raw	Raw
	20240325_4-anti_Chymo_ETHCD_SH_Ec.raw	Raw
	20240325_4-anti_Chymo_HCD_SH_Ec.raw	Raw

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De Novo Sequencing of Polyclonal Antibodies by Integrating Intact Mass, Top-Down and Bottom-Up Mass Spectrometry

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