Internal Fragments Generated by Electron Ionization Dissociation Enhance Protein Top-Down Mass Spectrometry
Internal Fragments Generated by Electron Ionization Dissociation Enhance Protein Top-Down Mass Spectrometry
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DOI：
10.1021/jasms.0c00160
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发表时间：
2020-09-02
影响因子：
3.2
通讯作者：
Loo, Joseph A.
中科院分区：
文献类型：
--
作者：
Zenaidee, Muhammad A.;Lantz, Carter;Loo, Joseph A.
自上而下的蛋白质组学通过质谱法（MS）包括完整蛋白质的质量测量，随后激活蛋白质以产生产物离子。基于电子的分裂方法，如电子捕获解离和电子转移解离被广泛用于这些类型的分析。近年来，电子电离解离（EID）被认为比其他基于电子的解离方法更有效地用于自上而下的蛋白质断裂，电子电离解离利用的电子能量更高（bbb20 eV）。在这里，我们证明了EID的使用增强了蛋白质片段和随后的蛋白质片段检测。蛋白质产物离子可以通过单次裂解事件形成，产生含有蛋白质c端或n端的末端片段，也可以通过多次裂解事件产生既不包括蛋白质c端也不包括蛋白质n端的内部片段。通常，内部片段被忽略，因为这些片段的可靠分配是有限的。在此，我们证明了EID产生的内部片段可以占到自上而下EID- ms实验检测到的质谱信号的20-40%。通过包含内部片段，对于29 kDa的碳酸酐酶II和8.6 kDa的泛素，可以从单个串联质谱解释的蛋白质序列范围从类似于50%增加到类似于99%。在数据分析过程中寻找内部片段时，可以方便、准确地分配先前未分配的峰，以确认给定的蛋白质序列，提高自上而下的蛋白质测序实验的实用性。
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 similar to 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 similar to 50 to similar 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.