Electron Transfer Dissociation Parameter Optimization Using Design of Experiments Increases Sequence Coverage of...

Middle-down analysis of monoclonal antibodies (mAbs) by tandem mass spectrometry (MS²) can provide detailed insight into their primary structure with minimal sample preparation. The middle-down approach uses an enzyme to cleave mAbs into Fc/2, LC, and Fd subunits that are then analyzed by reversed phase liquid chromatography tandem mass spectrometry (RPLC-MS²). As maximum sequence coverage is desired to obtain meaningful structural information at the subunit level, a host of dissociation methods have been developed, and sometimes combined, to bolster fragmentation and increase the number of identified fragments. Here, we present a design of experiments (DOE) approach to optimize MS² parameters, in particular those that may influence electron transfer dissociation (ETD) efficiency to increase the sequence coverage of antibody subunits. Applying this approach to the NIST monoclonal antibody standard (NISTmAb) using three RPLC-MS² runs resulted in high sequence coverages of 67%, 67%, and 52% for Fc/2, LC, and Fd subunits, respectively. In addition, we apply this DOE strategy to model the parameters required to maximize the number of fragments produced in "low", "medium", and "high" mass ranges, which ultimately resulted in even higher sequence coverages of NISTmAb subunits (75%, 78%, and 64% for Fc/2, LC, and Fd subunits, respectively). The DOE approach provides high sequence coverage percentages utilizing only one fragmentation method, ETD, and could be extended to other state-of-the-art techniques that combine multiple fragmentation mechanisms to increase coverage.