Top-Down Mass Spectrometry of a Clinical Antibody Light Chain Using the Omnitrap-Orbitrap-Booster Platform

The Omnitrap-Orbitrap-Booster (OOB) mass spectrometry (MS) platform was developed to advance the top-down (TD) MS analysis of proteins. It integrates a multimodal tandem mass spectrometry (MS/MS) ion trap system (Omnitrap), a high-resolution Orbitrap Fourier transform mass spectrometer (FTMS), and a high-performance data acquisition system (FTMS Booster) to improve fragmentation efficiency and spectral quality by increasing the signal-to-noise (S/N) ratio of product ions. In this study, we evaluate the OOB platform for the electron capture dissociation (ECD)-based TD MS analysis of a P15 multiple myeloma antibody light chain. Single precursor charge state analysis of P15 23+ yielded relatively high sequence coverage of 68%, albeit indicating a limitation caused by the overlap of certain product ions with the charge reduced precursors. The corresponding method development, leveraging consecutive analysis of multiple precursor charge states (15+ to 19+) across triplicate LC-MS/MS runs on the OOB platform, enhanced P15 sequence coverage to 93%, demonstrating its capacity for comprehensive protein characterization. In addition, we demonstrate that the obtained ECD-based TD MS performance on the OOB platform for P15 light chain is comparable to the “gold-standard” electron transfer/higher-energy collision dissociation (EThcD)-based TD MS on an Orbitrap Eclipse. Serendipitously, ECD exhibits a lower spectral peak density (i.e., reduced spectral congestion) due to reduced redundancy of product ions. These results establish the OOB platform as a powerful and efficient tool for TD MS of proteins.