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

peer-reviewed · Journal of Proteome Research · 2022

peer-reviewed · Journal of Proteome Research · 2022. Michelle V. Gadush et al. In this study, we used multiple enzyme digestions, coupled with higher-energy collisional dissociation (HCD)…
Date 2022-07-01
Type peer-reviewed
Venue Journal of Proteome Research
Publisher American Chemical Society (ACS)
Contribution downstream-application
DOI 10.1021/acs.jproteome.1c00913
Citations (OpenAlex) 23
Venue 2-year citedness 3.83

Abstract

In this study, we used multiple enzyme digestions, coupled with higher-energy collisional dissociation (HCD) and electron-transfer/higher-energy 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.

Authors

  1. Michelle V. Gadush · University of Texas at Austin
  2. Giuseppe A. Sautto · University of Georgia
  3. Hamssika Chandrasekaran · University of Texas at Austin
  4. Alena Bensussan · University of Texas at Austin
  5. Ted M. Ross · University of Georgia
  6. Gregory C. Ippolito · University of Texas at Austin
  7. Maria D. Person · University of Texas at Austin

Methods and tools

  • Multi-enzyme template-assisted mAb sequencing: Complete monoclonal-antibody sequencing from multiple enzyme digests read by HCD and EThcD, with the de novo calls made template-assisted in Supernovo. Refined on an antibody of known sequence, then applied to 25 anti-haemagglutinin influenza antibodies of unknown sequence, recovering high-confidence sequence for over 99% of the CDRs; the resulting heavy and light chains were cloned and expressed, and the recombinant antibodies reproduced the original binding curves. Distinguishes Ile from Leu correctly on the known-sequence controls.

Methods it uses

  • Supernovo: Automated antibody de novo sequencing: finds the closest-matching germline V-J-C sequences by database search, then converges on the true heavy and light chains by iterative wildcard substitution against the MS/MS spectra. Commercial, shipped in Protein Metrics’ Byos platform.

Data deposited

  • De Novo Sequencing of SARS-CoV-2 and influenza monoclonal antibodies by mass spectrometry using HCD and EThcD fragmentat — as deposited · PXD030094

Cites (14)

Cited by (4)

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