A Handle on Mass Coincidence Errors in De Novo Sequencing of Antibodies by Bottom-up Proteomics
peer-reviewed · Journal of Proteome Research · 2024
| Date | 2024-08-02 |
| Type | peer-reviewed |
| Venue | Journal of Proteome Research |
| Publisher | American Chemical Society (ACS) |
| Contribution | post-processor |
| DOI | 10.1021/acs.jproteome.4c00188 |
| Citations (OpenAlex) | 17 |
| Venue 2-year citedness | 3.83 |
Abstract
Antibody sequences can be determined at 99% accuracy directly from the polypeptide product by using bottom-up proteomics techniques. Sequencing accuracy at the peptide level is limited by the isobaric residues leucine and isoleucine, incomplete fragmentation spectra in which the order of two or more residues remains ambiguous due to lacking fragment ions for the intermediate positions, and isobaric combinations of amino acids, of potentially different lengths, for example, GG = N and GA = Q. Here, we present several updates to Stitch (v1.5), which performs template-based assembly of de novo peptides to reconstruct antibody sequences. This version introduces a mass-based alignment algorithm that explicitly accounts for mass coincidence errors. In addition, it incorporates a postprocessing procedure to assign I/L residues based on secondary fragments (satellite ions, i.e. , w- ions). Moreover, evidence for sequence assignments can now be directly evaluated with the addition of an integrated spectrum viewer. Lastly, input data from a wider selection of de novo peptide sequencing algorithms are allowed, now including Casanovo, PEAKS, Novor.Cloud, pNovo, and MaxNovo, in addition to flat text and FASTA. Combined, these changes make Stitch compatible with a larger range of data processing pipelines and improve its tolerance to peptide-level sequencing errors.
Methods and tools
- Stitch: Assembles de novo peptides from Casanovo, PEAKS, pNovo and MaxNovo into full antibody sequences, and corrects the two error classes that assembly alone cannot: mass coincidences, where a different residue combination matches the same mass, and I/L ambiguity.
Data used
- Anti-FLAG-M2 antibody sequencing (as deposited) · PXD023419
- Bottom-up data - sequencing (as deposited) · 10.6084/m9.figshare.13194005
Cites (25)
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Cited by (14)
- Reference-free protein sequencing by consensus assembly of redundant de novo peptide reads (2026) both
- A large-scale unified deep learning model for peptide mass spectrum interpretation trained on multimodal data (2026) crossref
- XA-Novo: an accurate and high-throughput mass spectrometry-based de novo sequencing technology for monoclonal antibodies and antibody mixtures (2026) both
- AbNovoBench: a resource and benchmarking platform for monoclonal antibody de novo sequencing (2026) both
- Identification of Unknown Biological Toxin Proteins Using Mass Spectrometry: A Case Study on De Novo Sequencing of Ricin (2025) both
- Systematic benchmarking of mass spectrometry-based antibody sequencing reveals methodological biases (2025) both
- Framework for de novo sequencing of peptide mixtures via network analysis and two-dimensional tandem mass spectrometry (2025) crossref
- Limitations of de novo sequencing in resolving sequence ambiguity (2025) both
- Structural Basis for Postfusion-Specific Binding to the Respiratory Syncytial Virus F Protein by the Canonical Antigenic Site I Antibody 131–2a (2025) both
- Do-It-Yourself De Novo Antibody Sequencing Workflow that Achieves Complete Accuracy of the Variable Regions (2025) crossref
- Efficient Screening of α-Glucosidase Inhibitory Peptides From Seahorse Through the Innovative Joint Technique: De Novo Sequencing and Parallel SPOT Synthesis (2025) crossref
- Cumulating MS Signal enables polyclonal antibody analysis (2025) both
- Systematic benchmarking of mass spectrometry-based antibody sequencing reveals methodological biases (2024) crossref
- Comprehensive assembly of monoclonal and mixed antibody sequences (2024) semanticscholar