Resurrection of a clinical antibody: Template proteogenomic de novo proteomic sequencing and reverse engineering of an anti‐lymphotoxin‐α antibody
peer-reviewed · PROTEOMICS · 2011
| Date | 2011-02-01 |
| Type | peer-reviewed |
| Venue | PROTEOMICS |
| Publisher | Wiley |
| Contribution | downstream-application |
| DOI | 10.1002/pmic.201000487 |
| Citations (OpenAlex) | 35 |
Abstract
A mouse hybridoma antibody directed against a member of the tumour necrosis factor (TNF)-superfamily, lymphotoxin-alpha (LT-α), was isolated from stored mouse ascites and purified to homogeneity. After more than a decade of storage the genetic material was not available for cloning; however, biochemical assays with the ascites showed this antibody against LT-α (LT-3F12) to be a preclinical candidate for the treatment of several inflammatory pathologies. We have successfully rescued the LT-3F12 antibody by performing MS analysis, primary amino acid sequence determination by template proteogenomics, and synthesis of the corresponding recombinant DNA by reverse engineering. The resurrected antibody was expressed, purified and shown to demonstrate the desired specificity and binding properties in a panel of immuno-biochemical tests. The work described herein demonstrates the powerful combination of high-throughput informatic proteomic de novo sequencing with reverse engineering to reestablish monoclonal antibody-expressing cells from archived protein sample, exemplifying the development of novel therapeutics from cryptic protein sources.
Methods and tools
- Template proteogenomic antibody resurrection: Recovers the sequence of an anti-lymphotoxin-alpha mouse hybridoma antibody whose genetic material was lost after a decade in storage, by template proteogenomic de novo sequencing of the stored ascites, then reverse-engineers the gene.
Cites (4)
- Template Proteogenomics: Sequencing Whole Proteins Using an Imperfect Database (2010) both
- Automated de novo protein sequencing of monoclonal antibodies (2008) both
- De novo proteomic sequencing of a monoclonal antibody raised against OX40 ligand (2006) both
- InsPecT: Identification of Posttranslationally Modified Peptides from Tandem Mass Spectra (2005) both
Cited by (13)
- Simultaneous polyclonal antibody sequencing and epitope mapping by cryo electron microscopy and mass spectrometry (2025) crossref
- A Handle on Mass Coincidence Errors in De Novo Sequencing of Antibodies by Bottom-up Proteomics (2024) both
- Template-Based Assembly of Proteomic Short Reads For De Novo Antibody Sequencing and Repertoire Profiling (2022) both
- Template-Assisted De Novo Sequencing of SARS-CoV-2 and Influenza Monoclonal Antibodies by Mass Spectrometry (2022) crossref
- Template-based assembly of proteomic short reads for de novo antibody sequencing and repertoire profiling (2022) crossref
- Mass Spectrometry-Based De Novo Sequencing of Monoclonal Antibodies Using Multiple Proteases and a Dual Fragmentation Scheme (2021) both
- Mass spectrometry-based de novo sequencing of the anti-FLAG-M2 antibody using multiple proteases and a dual fragmentation scheme (2021) both
- Antibody de novo Sequencing (2017) crossref
- Peptidomics of Acanthoscurria gomesiana spider venom reveals new toxins with potential antimicrobial activity (2017) crossref
- De Novo Sequencing and Resurrection of a Human Astrovirus-Neutralizing Antibody (2016) both
- Combining phage display with de novo protein sequencing for reverse engineering of monoclonal antibodies (2016) both
- Sequencing-Grade De novo Analysis of MS/MS Triplets (CID/HCD/ETD) From Overlapping Peptides (2013) both
- The spectral networks paradigm in high throughput mass spectrometry (2012) both