Top-down proteomics by mass spectrometry (MS) involves the mass measurement of an intact protein followed by subsequent activation of the protein to generate product ions. Electron-based fragmentation methods like electron capture dissociation and electron transfer dissociation are widely used for these types of analyses. Recently, electron ionization dissociation (EID), which utilizes higher energy electrons (>20 eV) has been suggested to be more efficient for top-down protein fragmentation compared to other electron-based dissociation methods. Here, we demonstrate that the use of EID enhances protein fragmentation and subsequent detection of protein fragments. Protein product ions can form by either single cleavage events, resulting in terminal fragments containing the C-terminus or N-terminus of the protein, or by multiple cleavage events to give rise to internal fragments that include neither the C-terminus nor the N-terminus of the protein. Conventionally, internal fragments have been disregarded, as reliable assignments of these fragments were limited. Here, we demonstrate that internal fragments generated by EID can account for ∼20-40% of the mass spectral signals detected by top-down EID-MS experiments. By including internal fragments, the extent of the protein sequence that can be explained from a single tandem mass spectrum increases from ∼50 to ∼99% for 29 kDa carbonic anhydrase II and 8.6 kDa ubiquitin. When searching for internal fragments during data analysis, previously unassigned peaks can be readily and accurately assigned to confirm a given protein sequence and to enhance the utility of top-down protein sequencing experiments.

中文翻译：

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电子电离解离产生的内部碎片增强了蛋白质自上而下的质谱分析。

通过质谱法 (MS) 自上而下的蛋白质组学涉及对完整蛋白质的质量测量，然后激活蛋白质以生成产物离子。电子捕获解离和电子转移解离等基于电子的碎裂方法被广泛用于这些类型的分析。最近，与其他基于电子的解离方法相比，使用更高能量电子 (>20 eV) 的电子电离解离 (EID) 被认为对于自上而下的蛋白质碎裂更有效。在这里，我们证明了 EID 的使用增强了蛋白质片段化和随后的蛋白质片段检测。蛋白质产物离子可以通过单一裂解事件形成，导致含有蛋白质 C 端或 N 端的末端片段，或通过多次切割事件产生既不包括蛋白质的 C 端也不包括 N 端的内部片段。通常，内部碎片已被忽略，因为这些碎片的可靠分配是有限的。在这里，我们证明了 EID 产生的内部碎片可以占自上而下 EID-MS 实验检测到的质谱信号的 20-40%。通过包括内部片段，对于 29 kDa 碳酸酐酶 II 和 8.6 kDa 泛素，可以从单个串联质谱解释的蛋白质序列的范围从~50% 增加到~99%。在数据分析期间搜索内部片段时，可以轻松准确地分配以前未指定的峰，以确认给定的蛋白质序列并增强自上而下蛋白质测序实验的效用。