Capillary Zone Electrophoresis-Tandem Mass Spectrometry with Activated Ion Electron Transfer Dissociation for Large-scale Top-down Proteomics
Capillary Zone Electrophoresis-Tandem Mass Spectrometry with Activated Ion Electron Transfer Dissociation for Large-scale Top-down Proteomics
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DOI：
10.1007/s13361-019-02206-6
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发表时间：
2019-12-01
影响因子：
3.2
通讯作者：
Sun, Liangliang
中科院分区：
文献类型：
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作者：
McCool, Elijah N.;Lodge, Jean M.;Sun, Liangliang
毛细管区带电泳(CZE)串联质谱仪(MS/MS)以其高效的分离能力和对蛋白质组的高灵敏度检测，近年来被认为是一种有效的自上而下蛋白质组学研究方法。然而，常用的基于碰撞的解离方法往往不能提供广泛的蛋白质形式的碎片以进行彻底的表征。与高能碰撞解离(HCD)和标准电子转移解离(ETD)相比，活化离子电子转移解离(AIETD)结合了红外光活化和ETD，对蛋白质形式的碎解表现出更好的性能。在这里，我们首次提出了CZE-AI-ETD在Orbitrap融合光质谱计上的应用，用于大规模自上而下的大肠杆菌细胞蛋白质组学研究。在蛋白质形态和蛋白质识别(ID)方面，CZE-AI-ETD优于CZE-ETD。CZE-AI-ETD达到了与CZE-HCD相当的蛋白质形态和蛋白质ID。CZE-AI-ETD往往比CZE-HCD和CZE-ETD产生更好的蛋白质组分期望值(E值)，表明AI-ETD产生的序列信息片段离子的数量具有更高质量的MS/MS谱。CZE-AI-ETD具有良好的重复性，相对标准偏差分别小于4%和2%(n=3)。将CZE-AI-ETD与尺寸排除层析(SEC)联用，从大肠杆菌细胞中鉴定出3028种蛋白质形式和387种蛋白质，光谱水平为1%，蛋白质形式水平的错误发现率为5%。这些数据是迄今为止使用AI-ETD方法的最大的自上而下的蛋白质组学数据集。一个SEC组分的单次CZE-AI-ETD鉴定了957种蛋白质形式和253种蛋白质。检测到N-末端截断、信号肽切割、N-末端蛋氨酸去除以及各种翻译后修饰，包括蛋白质N-末端乙酰化、甲基化、S-硫代化、二硫键和赖氨酸琥珀酸化。
Capillary zone electrophoresis (CZE)tandem mass spectrometry (MS/MS) has been recognized as an efficient approach for topdown proteomics recently for its high-capacity separation and highly sensitive detection of proteoforms. However, the commonly used collision-based dissociation methods often cannot provide extensive fragmentation of proteoforms for thorough characterization. Activated ion electron transfer dissociation (AIETD), that combines infrared photoactivation concurrent with ETD, has shown better performance for proteoform fragmentation than higher energy-collisional dissociation (HCD) and standard ETD. Here, we present the first application of CZE-AI-ETD on an Orbitrap Fusion Lumosmass spectrometer for large-scale top-down proteomics of Escherichia coli (E. coli) cells. CZE-AI-ETD outperformed CZE-ETD regarding proteoform and protein identifications (IDs). CZE-AI-ETD reached comparable proteoform and protein IDs with CZE-HCD. CZE-AI-ETD tended to generate better expectation values (E values) of proteoforms than CZE-HCD and CZE-ETD, indicating a higher quality of MS/MS spectra from AI-ETD respecting the number of sequence-informative fragment ions generated. CZE-AI-ETD showed great reproducibility regarding the proteoform and protein IDs with relative standard deviations less than 4% and 2% (n = 3). Coupling size exclusion chromatography (SEC) to CZE-AI-ETD identified 3028 proteoforms and 387 proteins from E. coli cells with 1% spectrum level and 5% proteoform-level false discovery rates. The data represents the largest top-down proteomics dataset using the AI-ETD method so far. Single-shot CZE-AI-ETD of one SEC fraction identified 957 proteoforms and 253 proteins. N-terminal truncations, signal peptide cleavage, N-terminal methionine removal, and various post-translational modifications including protein N-terminal acetylation, methylation, S-thiolation, disulfide bonds, and lysine succinylation were detected.