Free Radical-based Sequencing for Native Top-Down Mass Spectrometry

Native top-down proteomics allows for both proteoform identification and high-order structure characteri-zation for cellular protein complexes. Unfortunately, tandem MS-based fragmentation efficiencies for such targets are low due to an increase in analyte ion mass and the low ion charge states that characterize native MS data. Multiple fragmenta-tion methods can be integrated in order to increase protein complex sequence coverage, but this typically requires use of specialized hardware and software. Free-radical initiated peptide sequencing (FRIPS) enables access to charge-remote and electron-based fragmentation channels within the context of conventional CID experiments. Here, we optimize FRIPS la-belling for native top-down sequencing experiments. Our labelling approach is able to access intact complexes with TEMPO-based FRIPS reagents without significant protein denaturation or assembly disruption. By combining CID and FRIPS datasets, we observed sequence coverage improvements as large as 50% for protein complexes ranging from 36 kDa to 106 kDa. Fragment ion production in these experiments was increased by as much as 102%. In general, our results indicate that TEMPO-based FRIPS reagents have the potential to dramatically increase sequence coverage obtained in na-tive top-down experiments.