Simultaneous polyclonal antibody sequencing and epitope mapping by cryo electron microscopy and mass spectrometry
- Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University, Padualaan, Netherlands
- Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, Royal Institute of Technology – KTH, Sweden
Figures
Figure 1
Schematic workflow to estimate de novo antibody sequences by the integration of cryoEM and LC-MS/MS data with Stitch.
Figure 2
Determining de novo antibody sequences from cryoEM data with ModelAngelo.
(A) Exemplary map (with top10% alignment scores of 1076/1174 for HC/LC) from the benchmark dataset, representing an Influenza B virus neuraminidase (NA) in complex with four copies of a neutralizing Fab, at global FSC resolution of 2.3 Å. Shown are the deposited map, model, and the de novo model generated by ModelAngelo, along with a detailed view of CDRH3. (B) Consensus sequences for heavy and light chains as generated by Stitch compared to the true sequences. Sequencing errors are indicated by an asterisk (*).
Figure 3
V-gene assignment from ModelAngelo data.
(A) Correlation between Stitch alignment score and sequence identity between the top-scoring V-gene of the ModelAngelo vs PDB sequence of the heavy and light chain variable domains, as indicated with the non-parametric Spearman correlation coefficient. (B) Distribution of V-gene sequence identity for progressive alignment score cutoffs, compared to the pairwise V-gene sequence identity in the IMGT repertoire.
Figure 4
Analysis of de novo CDR3 modeling in ModelAngelo-Stitch.
(A) Coverage of CDR3 for the heavy and light chain. CDR3 was counted for coverage if the de novo sequence spanned the flanking cysteine on the V gene and the tryptophan or phenylalanine on the J gene. Proportion of maps with CDR3 coverage in red/blue, maps with missing CDR3 in grey. (B) Difference in length between de novo modelled CDR3 vs. true sequence. (C) Sequence identity of de novo modelled CDR3 vs. true sequence.
Figure 5
Correlation between global FSC resolution and Stitch alignment score (A) or inferred V-gene identity (B).
The non-parametric Spearman correlation coefficient is indicated for heavy and light chain.
Figure 6
Sequencing CR3022 with integrated cryoEM and LC-MS/MS data.
(A) Shown are the deposited cryoEM map (global FSC resolution 4.1 Å), model, and de novo ModelAngelo output for the CR3022 Fab in complex with the SARS-CoV-2 Spike S1 subunit. The sequences were extracted from the de novo model and used as input for Stitch, resulting in the identification of the indicated V-genes and CDR3 sequences. These variable domains were used as templates in Stitch to assemble the LC-MS/MS derived de novo peptides. (B) Consensus sequences for CR3022 from the integrated cryoEM-MS data in Stitch compared to the true heavy and light chain sequences. Sequencing errors are indicated with an asterisk (*) .
Figure 7
Targeted sequencing of CR3022 against a complex background of other antibodies.
Plotted are the de novo consensus sequence identities derived from the LC-MS/MS data using either the true sequences, the full IMGT repertoire, or the ModelAngelo-derived variable domains as templates. We compare the output from the CR3022 dataset alone (‘No backgr.’) with the output after adding either a diffuse polyclonal IgG background from a COVID-19 patient (‘+whole IgG’) or full datasets from five additional anti-Influenza-HA monoclonals (‘+5 mAbs’). Use of decoy sequences as indicated by dark/light colors.
Figure 8
Inferring V-genes from published EMPEM data.
(A) Plotted are all non-zero alignment scores in Stitch from published EMPEM maps. (B) Views of the variable domains of EMPEM maps with alignment scores >50 for both heavy and light chains. The EMDB identifiers are indicated at each panel.
Additional files
-
Supplementary file 1
Benchmark of experimental cryoEM maps of monoclonal antibody-antigen complexes from EMDB.
Overview and results of maps for benchmark.
Name: PDB-ID of deposited model for corresponding map
Actual: corresponding to true VH/VL sequence as deposited in PDB model
Built: corresponding to de novo sequences determined by ModelAngelo
Segment: inferred V-gene from top-scoring hit in Stitch alignment
Score: alignment score in Stitch
Identity: sequence identity between Actual-Built based on de novo seq. or inferred Segments
Resolution: Global FSC resolution of corresponding map
HC: heavy chain
LC: light chain
- https://cdn.elifesciences.org/articles/101322/elife-101322-supp1-v1.csv
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Supplementary file 2
Benchmark of EMPEM maps downloaded from EMDB for de novo modelling in ModelAngelo, with alignment scores in Stitch.
Overview and results of maps for EMPEM benchmark.
- https://cdn.elifesciences.org/articles/101322/elife-101322-supp2-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/101322/elife-101322-mdarchecklist1-v1.pdf
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Simultaneous polyclonal antibody sequencing and epitope mapping by cryo electron microscopy and mass spectrometry
eLife 14:RP101322.
https://doi.org/10.7554/eLife.101322.3
