PT  - JOURNAL ARTICLE
AU  - Weiwei Peng
AU  - Matti F. Pronker
AU  - Joost Snijder
TI  - Mass spectrometry-based <em>de novo</em> sequencing of the anti-FLAG-M2 antibody using multiple proteases and a dual fragmentation scheme
AID  - 10.1101/2021.01.07.425675
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.01.07.425675
4099  - http://biorxiv.org/content/early/2021/01/12/2021.01.07.425675.short
4100  - http://biorxiv.org/content/early/2021/01/12/2021.01.07.425675.full
AB  - 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 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.Competing Interest StatementThe authors have declared no competing interest.