7BG1

Structure of anti-FLAG M2 Fab domain remodeled based on proteomic sequencing

Summary for 7BG1

• Entry DOI: 10.2210/pdb7bg1/pdb
• Descriptor: anti-FLAG M2 heavy chain, anti-FLAG M2 light chain, SULFATE ION, ... (5 entities in total)
• Functional Keywords: antibody, immunoglobulin, igg, fab, monoclonal, m2, protein purification, flag, anti-flag, flag-tag, immunohistochemistry, immunocytochemistry, immunoprecipitation, elisa, western blot, research tool, affinity tag, immune system
• Biological source: Mus musculus; More
• Total number of polymer chains: 2
• Total formula weight: 48499.23

Authors

Pronker, M.F.,Snijder, J. (deposition date: 2021-01-05, release date: 2021-07-21, Last modification date: 2024-10-23)

Primary citation

Peng, W.,Pronker, M.F.,Snijder, J.
Mass Spectrometry-Based De Novo Sequencing of Monoclonal Antibodies Using Multiple Proteases and a Dual Fragmentation Scheme.
J.Proteome Res., 20:3559-3566, 2021

PubMed Abstract: 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 sequencing of monoclonal IgG from the purified antibody products. The method uses a panel of multiple complementary proteases to generate suitable peptides for 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.

PubMed: 34121409
DOI: 10.1021/acs.jproteome.1c00169

Experimental method

X-RAY DIFFRACTION (1.86 Å)