A Prototype timsOmni Platform Enables Confident Annotation of the Key Hypervariable CDR3 Regions of IgG Immunoglobulins Using Low- and High-Energy Electron-Based Fragmentation

The configuration of the first prototype timsOmni instrument, which integrates an Omnitrap linear ion trap into a timsTOF platform, is presented. A modified electrode design for the electron-based fragmentation (ExD) section of the Omnitrap is described, enhancing both the robustness and performance. Optimal characterization of antibodies requires characterizing light and heavy chains as pairs in addition to sequencing their variable domains and identifying any modifications. This is best addressed using protein-centric proteomics, as heterogeneity information such as the specific clonal origin of each identified fragment can be retained. Furthermore, by acting on intact proteins that retain part of their structure, such as disulfide bonds, it is possible to target key regions for fragmentation such as the hypervariable complementarity determining regions (CDR3) that are unique for each clone and necessary for target recognition. Therefore, as a proof of concept, we used the prototype timsOmni mass spectrometer for antibody analysis. Using solely electron-based fragmentation methods, we obtained full CDR3 sequences for paired heavy and light chains. Optimal results were achieved by performing Electron Induced Dissociation (EID) at ∼35 eV electron energy on native-like fragment antigen-binding (Fab) precursor ions. This approach yields both (a, x) and (c, z) fragment ion pairs with the potential to enhance both sequence coverage and annotation confidence. Overall, the timsOmni mass spectrometer presented here serves as an advanced and versatile platform for protein-centric proteomics.