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Direct Determination of Antibody Chain Pairing by Top-down and Middle-down Mass Spectrometry Using Electron Capture Dissociation and Ultraviolet Photodissociation.

ABSTRACT: 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.

SUBMITTER: Shaw JB

PROVIDER: S-EPMC7819135 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Project description:Fast photochemical oxidation of proteins (FPOP) footprinting is a structural mass spectrometry method that maps proteins by fast and irreversible chemical reactions. The position of oxidative modification reflects solvent accessibility and site reactivity and thus provides information about protein conformation, structural dynamics, and interactions. Bottom-up mass spectrometry is an established standard method to analyze FPOP samples. In the bottom-up approach, all forms of the protein are digested together by a protease of choice, which results in mixture of peptides from various subpopulations of proteins with varying degrees of photochemical oxidation. But, once the protein is already oxidized at least once, the spatial distribution of any consecutive oxidation no longer truly reflects initial protein solvent accessibility and residue reactivity, because the site preference of further oxidation may also be influenced by the changes caused by the prior oxidation. Thus, it would be interesting to obtain an assessment of the protein structure based on the FPOP data, by analyzing only singly oxidized protein populations. This requires utilization of more specific top-down mass spectrometry approaches. The key element of any top-down experiment is selection of a suitable method of ion isolation, excitation and fragmentation. Here we employ and compare collision-induced dissociation (CID), electron-transfer dissociation (ETD), and electron-capture dissociation (ECD) combined with multi continuous accumulation of selected ions (CASI). Singly oxidized subpopulation of FPOP labeled ubiquitin was used to optimize the method. The usefulness was then demonstrated further by using it to visualize structural changes induced by cofactor removal from holo/apo myoglobin system. The top-down data were compared with the literature and with the bottom-up data set obtained on the same samples. The top-down results were found to be in a good agreement, which indicates that monitoring singly FPOP oxidized ion population by top-down is a functional workflow for oxidative protein footprinting.

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Direct Determination of Antibody Chain Pairing by Top-down and Middle-down Mass Spectrometry Using Electron Capture Dissociation and Ultraviolet Photodissociation.

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