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.
High throughput native mass spectrometry analysis of proteins and protein complexes has been enabled by recent development of infusion and liquid chromatography (LC) systems, which often include complete LC pumps without fully utilizing their gradient flows. We demonstrated a lower-cost infusion cart for native mass spectrometry applications using a single isocratic solvent pump that can operate at both nano-and high-flow configurations (0.05−150 μL/min) for both infusion and online buffer exchange experiments. The platform is controlled via opensource software and can potentially be expanded for customized experimental designs, offering a lower cost alternative to laboratories with limited budgets and/or needs in student training.
High throughput native mass spectrometry analysis of proteins and protein complexes has been enabled by recent development of infusion and liquid chromatography (LC) systems, which often include complete LC pumps without fully utilizing their gra-dient flows. We demonstrated a lower-cost infusion cart for native mass spectrometry applications using a single isocratic solvent pump that can operate at both nano- and high-flow configurations (0.05-150 µL/min) for both infusion and online buffer exchange experiments. The platform is controlled via open-source software and can potentially be expanded for customized experimental designs, offering a lower cost alternative to labs with limited budget and/or needs in student training.
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