Top-Down Analysis of Supercharged Proteins Using Collision-, Electron-, and Photon-Based Activation Methods

The impact of supercharging on the fragmentation patternsof sixproteins, ubiquitin, cytochrome c, staph nuclease, myoglobin, dihydrofolatereductase, and carbonic anhydrase, was investigated for five activationmethods, HCD, ETD, EThcD, 213 nm UVPD, and 193 nm UVPD under denaturingconditions. Changes in sequence coverage, alterations in the numberand abundance of preferential cleavages (N-terminal to proline, C-terminalto aspartic or glutamic acid, adjacent to aromatic residues), andchanges in individual fragment ion abundances were evaluated. Largedecreases in sequence coverage were observed upon supercharging ofproteins activated by HCD, whereas modest gains were observed forETD. Minimal changes in sequence coverage were observed when usingEThcD, 213 nm UVPD, and 193 nm UVPD, all of which tended to displaythe highest sequence coverages of the activation methods. Specificpreferential backbone cleavage sites were increasingly enhanced forall proteins in supercharged states for all activation methods, particularlyfor HCD, 213 nm UVPD, and 193 nm UVPD. Even if large gains in sequencecoverages were not apparent for the highest charge states, superchargingconsistently led to at least a few new backbone cleavage sites forETD, EThcD, 213 nm UVPD, and 193 nm UVPD for all proteins.

Automated summary

The impact of supercharging on the fragmentation patterns of six proteins under denaturing conditions was investigated using five activation methods. Changes in sequence coverage, preferential cleavage sites, and fragment ion abundances were evaluated for each method. The results showed that supercharging had different effects on sequence coverage and preferential cleavage sites depending on the activation method and the protein.