Protease strategy
7 methods · 2019–2026
Protease strategy: Choosing and combining proteases so that overlapping peptides cover a whole protein, and so that each peptide fragments well enough to read de novo. The method is in the digestion, not the sequencing algorithm, which is why…
Choosing and combining proteases so that overlapping peptides cover a whole protein, and so that each peptide fragments well enough to read de novo. The method is in the digestion, not the sequencing algorithm, which is why antibody sequencing leans on it.
The earliest of its 7 methods is MELD (2019); 6 more have followed.
| Methods | 7 |
| Papers describing them | 9 |
| Authors | 54 |
| Active | 2019-06-07 to 2026-03-11 |
| Kinds | adjacent (4), downstream-application (3) |
| Acquisition | DDA (6) |
Methods (7)
Oldest first, by the paper that describes each one.
- MELD (2019): Multi-Enzymatic Limited Digestion uses a diluted, time-limited mix of proteases to produce overlapping peptides, with a custom assembly algorithm that turns them into de novo protein sequences.
- Multi-protease dual-fragmentation antibody sequencing (2021): Direct de novo sequencing of monoclonal IgG from the purified product with a panel of complementary proteases and both stepped HCD and EThcD on every precursor, demonstrated on the anti-FLAG-M2 antibody.
- Continuous non-specific digestion protein sequencing (2021): A continuous-digestion device with several non-specific proteases yields overlapping peptides that improve coverage of de novo full-length protein sequencing.
- Cathepsin middle-down antibody digestion (2021): Shows Cathepsins D and L cleave IgGs right after CDR3, producing sequencing-sized fragments for middle-down de novo antibody sequencing.
- MELD snake venomics (2023): Applies multi-enzymatic limited digestion to four snake venoms, increasing de novo sequenced peptides and toxin coverage in PEAKS.
- SP-MEGD antibody sequencing (2024): Single-Pot and Multi-Enzymatic Gradient Digestion: a bottom-up-only workflow for faster, cheaper de novo sequencing of COVID-19 neutralizing antibodies.
- HTA-protease de novo antibody sequencing (2026): Uses two hyperthermoacidic archaeal proteases in place of trypsin or chymotrypsin for de novo antibody sequencing, each yielding about five times more unique peptide reads and, with EAciD fragmentation on a ZenoTOF, complete unambiguous sequence coverage of all complementarity-determining regions.
Applied in
Papers describing them (9)
- Multi-Enzymatic Limited Digestion: The Next-Generation Sequencing for Proteomics? (2019, Journal of Proteome Research, peer-reviewed)
- Mass spectrometry-based de novo sequencing of the anti-FLAG-M2 antibody using multiple proteases and a dual fragmentation scheme (2021, bioRxiv, preprint)
- Full-length Protein Sequencing Based on Continuous Digestion Using Non-specific Proteases (2021, Acta Chimica Sinica, peer-reviewed)
- Mass Spectrometry-Based De Novo Sequencing of Monoclonal Antibodies Using Multiple Proteases and a Dual Fragmentation Scheme (2021, Journal of Proteome Research, peer-reviewed)
- The lysosomal endopeptidases Cathepsin D and L are selective and effective proteases for the middle‐down characterization of antibodies (2021, The FEBS Journal, peer-reviewed)
- Next-Generation Sequencing for Venomics: Application of Multi-Enzymatic Limited Digestion for Inventorying the Snake Venom Arsenal (2023, Toxins, peer-reviewed)
- De novo antibody sequencing based on mass spectrometry (2024, thesis)
- Simplified and Rapid Workflow Enhances Throughput of De Novo Sequencing of COVID-19 Neutralizing Antibodies (2024, bioRxiv, preprint)
- Deep coverage and extended sequence reads obtained with a single archaeal protease expedite de novo protein sequencing by mass spectrometry (2026, Cell Systems, peer-reviewed)