Mass Spectrometry-Based De Novo Sequencing of Monoclonal Antibodies Using Multiple Proteases and a Dual Fragmentation Scheme
| Autor: | Peng, Weiwei, Pronker, Matti F, Snijder, Joost, Sub Biomol.Mass Spectrometry & Proteom., Biomolecular Mass Spectrometry and Proteomics |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Proteases stepped HCD Chemistry(all) FLAG-tag medicine.drug_class Peptide anti-FLAG-M2 Computational biology Mass spectrometry Monoclonal antibody Biochemistry 03 medical and health sciences EThcD de novo sequencing Western blot antibody medicine mass spectrometry chemistry.chemical_classification 030102 biochemistry & molecular biology biology medicine.diagnostic_test herceptin General Chemistry 030104 developmental biology chemistry biology.protein Target protein Antibody |
| Zdroj: | Journal of Proteome Research, 20(7), 3559. American Chemical Society |
| ISSN: | 1535-3907 1535-3893 |
| DOI: | 10.1021/acs.jproteome.1c00169 |
| Popis: | Antibody sequence information is crucial to understanding the structural basis for antigen binding and enables the use of antibodies as therapeutics and research tools. Here, we demonstrate a method for direct de novo sequencing of monoclonal IgG from the purified antibody products. The method uses a panel of multiple complementary proteases to generate suitable peptides for de novo sequencing by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a bottom-up fashion. Furthermore, we apply a dual fragmentation scheme, using both stepped high-energy collision dissociation (stepped HCD) and electron-transfer high-energy collision dissociation (EThcD), on all peptide precursors. The method achieves full sequence coverage of the monoclonal antibody herceptin, with an accuracy of 99% in the variable regions. We applied the method to sequence the widely used anti-FLAG-M2 mouse monoclonal antibody, which we successfully validated by remodeling a high-resolution crystal structure of the Fab and demonstrating binding to a FLAG-tagged target protein in Western blot analysis. The method thus offers robust and reliable sequences of monoclonal antibodies. |
| Databáze: | OpenAIRE |
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