Oral Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Application of a novel electron capture dissociation (ECD) mass spectrometry to top-down and bottom-up proteoform characterization on a QTOF. (#155)

Blaine R Roberts 1 , Eugene Kapp 1 , Soumya Mukherjee 1 , Yury V Vasil'ev 2 , Valery G Voinov 2 , Joseph S Beckman 2
  1. Florey Institute of Neuroscience and Mental Health, Parkville, VIC, Australia
  2. eMSion, Corvallis, Oregon, USA

Bottom-up approaches using collision induced dissociation for characterizing target proteins yield incomplete information, particularly concerning the colocalization of post translational modifications. Here we described the implementation of a device that yields efficient ECD of proteins that can be reversibly retrofitted into Q-ToFs without diminishing performance. The ECD device does not require trapping ions as needed for ETD and thus is compatible linear designed instruments.  We demonstrate nearly complete sequence coverage of “native”-folded proteins such as the 5+and 6+charge states of ubiquitin and Cu, Zn superoxide dismutase.   Due to the high sequence covnerage (>80%) the localization of non-covalent cofactors like Cu and Zn could be determined from the top-down spectra of SOD. We also benchmarked the molecular weight range accessible for top-down protoemics on our QTOF system. Sequence coverage of 80-95% was obtained for small proteins like ubiquitin, amyloid beta and alpha-synuclein (14 kDa). Sequence coverage was 93% for carbonic anhydrase (29kDa) and similarly for green fluorescent protein (27kDa). Half of the human proteome is smaller than 30kDa making this system a viable option for top-down proteomics. The protein spectra consisted primarily of cand zions, though the ECD cell also produced a substantial number of dand wsidechain fragments. These side-chain fragments allow leucine/isoleucine or lysine/glutamine pairs to be distinguished, facilitating de novosequencing. We then applied this technology to protein extracts from human brain to show that we can conduct top-down protein identification on LC time scales. The simpler fragmentation patterns made possible with the ECD device allows existing mass spectrometers to be able to characterize mid-sized proteins even using fast front-end separations such as ion mobility.