期刊
JOURNAL OF PROTEOME RESEARCH
卷 20, 期 7, 页码 3559-3566出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.jproteome.1c00169
关键词
mass spectrometry; antibody; de novo sequencing EThcD; stepped HCD; herceptin; FLAG-tag; anti-FLAG-M2
资金
- Protein Metrics Inc.
- Dutch Research Council NWO Gravitation 2013 BOO, Institute for Chemical Immunology (ICI) [024.002.009]
The study demonstrates a method for direct de novo sequencing of monoclonal antibodies, using a panel of multiple complementary proteases and dual fragmentation schemes. This method achieves full sequence coverage of monoclonal antibodies, providing robust and reliable sequences for further research and therapeutic purposes.
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.
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