4.7 Article

Template-Assisted De Novo Sequencing of SARS-CoV-2 and Influenza Monoclonal Antibodies by Mass Spectrometry

Journal

JOURNAL OF PROTEOME RESEARCH
Volume -, Issue -, Pages -

Publisher

AMER CHEMICAL SOC
DOI: 10.1021/acs.jproteome.1c00913

Keywords

de novo sequencing; monoclonal antibodies; EThcD; SARS-CoV-2; influenza virus

Funding

  1. NIH NIAID under a NIH NCI COVID-19 SeroNet grant [75N93019C00052, U54-CA260543]
  2. University of Texas System Proteomics Network Pilot Project
  3. Center for Biomolecular Research Support Biological Mass Spectrometry Facility [RRID: SCR_021728]

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In this study, a mass spectrometric method was developed to determine the complete protein sequence of monoclonal antibodies. The method was refined on known sequences and successfully determined high confidence sequences for unknown antibodies. This method has significant practical implications for studying antibody sequences.
In this study, we used multiple enzyme digestions, coupled with higher-energy collisional dissociation (HCD) and electron-transfer/higherenergy collision dissociation (EThcD) fragmentation to develop a mass spectrometric (MS) method for determining the complete protein sequence of monoclonal antibodies (mAbs). The method was refined on an mAb of a known sequence, a SARS-CoV-1 antireceptor binding domain (RBD) spike monoclonal antibody. The data were searched using Supernovo to generate a complete template-assisted de novo sequence for this and two SARS-CoV-2 mAbs of known sequences resulting in correct sequences for the variable regions and correct distinction of Ile and Leu residues. We then used the method on a set of 25 antihemagglutinin (HA) influenza antibodies of unknown sequences and determined high confidence sequences for >99% of the complementarity determining regions (CDRs). The heavy-chain and light chain genes were cloned and transfected into cells for recombinant expression followed by affinity purification. The recombinant mAbs displayed binding curves matching the original mAbs with specificity to the HA influenza antigen. Our findings indicate that this methodology results in almost complete antibody sequence coverage with high confidence results for CDR regions on diverse mAb sequences.

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