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

The impact of supercharging on the fragmentation patterns of six proteins, ubiquitin, cytochrome c, staph nuclease, myoglobin, dihydrofolate reductase, and carbonic anhydrase, was investigated for five activation methods, HCD, ETD, EThcD, 213 nm UVPD, and 193 nm UVPD under denaturing conditions. Changes in sequence coverage, alterations in the number and abundance of preferential cleavages (N-terminal to proline, C-terminal to aspartic or glutamic acid, adjacent to aromatic residues), and changes in individual fragment ion abundances were evaluated. Large decreases in sequence coverage were observed upon supercharging of proteins activated by HCD, whereas modest gains were observed for ETD. Minimal changes in sequence coverage were observed when using EThcD, 213 nm UVPD, and 193 nm UVPD, all of which tended to display the highest sequence coverages of the activation methods. Specific preferential backbone cleavage sites were increasingly enhanced for all proteins in supercharged states for all activation methods, particularly for HCD, 213 nm UVPD, and 193 nm UVPD. Even if large gains in sequence coverages were not apparent for the highest charge states, supercharging consistently led to at least a few new backbone cleavage sites for ETD, EThcD, 213 nm UVPD, and 193 nm UVPD for all proteins.