One challenge associated with the discovery and development
of
monoclonal antibody (mAb) therapeutics is the determination of heavy
chain and light chain pairing. Advances in MS instrumentation and
MS/MS methods have greatly enhanced capabilities for the analysis
of large intact proteins yielding much more detailed and accurate
proteoform characterization. Consequently, direct interrogation of
intact antibodies or F(ab′)2 and Fab fragments has the potential
to significantly streamline therapeutic mAb discovery processes. Here,
we demonstrate for the first time the ability to efficiently cleave
disulfide bonds linking heavy and light chains of mAbs using electron
capture dissociation (ECD) and 157 nm ultraviolet photodissociation
(UVPD). The combination of intact mAb, Fab, or F(ab′)2 mass,
intact LC and Fd masses, and CDR3 sequence coverage enabled determination
of heavy chain and light chain pairing from a single experiment and
experimental condition. These results demonstrate the potential of
top-down and middle-down proteomics to significantly streamline therapeutic
antibody discovery.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.