Department of Chemistry, University of Texas at Austin, Austin, Texas 78712, United States.
J Am Soc Mass Spectrom. 2023 Jul 5;34(7):1467-1476. doi: 10.1021/jasms.3c00138. Epub 2023 Jun 26.
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.
研究了在变性条件下，五种激活方法（HCD、ETD、EThcD、213nm UVPD 和 193nm UVPD）对六种蛋白质（泛素、细胞色素 c、枯草杆菌核酸酶、肌红蛋白、二氢叶酸还原酶和碳酸酐酶）的碎片化模式的超荷影响。评估了序列覆盖率的变化、优先切割（脯氨酸 N 端、天冬氨酸或谷氨酸 C 端、芳香族残基附近）数量和丰度的变化以及各个片段离子丰度的变化。在用 HCD 激活的蛋白质中超荷时，序列覆盖率大幅下降，而 ETD 则略有增加。当使用 EThcD、213nm UVPD 和 193nm UVPD 时，序列覆盖率变化很小，所有这些方法的序列覆盖率都倾向于最高。对于所有激活方法，所有蛋白质在超荷状态下的特定优先骨架裂解位点都得到了增强，特别是对于 HCD、213nm UVPD 和 193nm UVPD。即使对于最高电荷状态，序列覆盖率没有明显增加，但超荷总能为所有蛋白质的 ETD、EThcD、213nm UVPD 和 193nm UVPD 产生至少几个新的骨架裂解位点。
