A prototype timsOmni platform enables confident annotation of key hypervariable 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 and applied to antibody analysis. A modified electrode design for the electron-based fragmentation (ExD) section of the Omnitrap platform is introduced, enhancing both the robustness and performance of the method. 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 selected regions for fragmentation such as some of the hypervariable complementarity determining regions (CDRs) that are unique for each clone and necessary for target recognition. Using electron-based fragmentation methods, we employed the prototype timsOmni mass spectrometer to obtain full CDR3 sequences for paired heavy and light chains with high confidence. Optimal results were obtained 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) as well as (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, demonstrating exceptional sequencing power for comprehensive protein characterization.