Mass Spectrometry-BasedDe NovoSequencing of Monoclonal Antibodies Using Multiple Proteases and a Dual Fragmentation Scheme

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 directde novosequencing of monoclonal IgG from the purified antibody products. The method uses a panel of multiple complementary proteases to generate suitable peptides forde novosequencing 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.