Electron Capture Dissociation for Discovery Top-Down Proteomics of Peptides and Small Proteins on Chromatographic Time Scales

IF 2.7 2区 化学 Q2 BIOCHEMICAL RESEARCH METHODS
Lester S. Manly, , , Anne M. Roberts, , , Joseph S. Beckman, , and , Blaine R. Roberts*, 
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Abstract

Bottom-up proteomics introduces proteoform ambiguity due to the loss of connectivity between peptides and their original proteoforms. Top-down proteomics (TDP) removes the ambiguity through the direct identification and characterization of intact proteoforms and their respective post-translational modifications (PTM). Electron capture dissociation (ECD) is an efficient and gentle peptide and protein fragmentation strategy that can be used for both bottom-up and top-down approaches. Here, we used an Agilent 6550 Q-TOF mass spectrometer retrofitted with an e-MSion ECD cell. Top-down sequencing capabilities of the cell were evaluated by sequencing of intact peptides and proteins on high-performance liquid chromatography (HPLC) time scales. Amyloid beta 1-40 (Aβ40) was first tested due to its pathophysiological role in Alzheimer’s disease and served as our large peptide standard. We sequenced Aβ40 via reverse-phase HPLC-MS and achieved 95% sequence coverage on chromatographic time scales utilizing a data-dependent acquisition (DDA)-based method. Acetone-precipitated protein extracts from human brain were then separated by HPLC and analyzed with a DDA method, which identified 16 proteoforms between 2 and 17 kDa with sequence coverage ranging from 7 to 90% based on proteoform size and composition. In addition to proteoform identification, ECD fragmentation distinguished multiple isoaspartate modifications from aspartate, as well as accurately differentiating leucine from isoleucine residues directly from the human brain sample. Here, we observed isoaspartate within a thymosin beta-4 proteoform. Additionally, we demonstrated the differentiation of leucine and isoleucine within a subunit of ubiquitin. This study advances the application of LC-Q-TOF instrumentation for discovery-based top-down proteomics utilizing ECD as enabled by the e-MSion ECD cell.

电子捕获解离在色谱时间尺度上发现多肽和小蛋白自上而下的蛋白质组学。
自底向上的蛋白质组学引入了由于肽和其原始蛋白质形态之间的连接丧失的蛋白质形态的模糊性。自上而下的蛋白质组学(TDP)通过直接鉴定和表征完整的蛋白质形态及其相应的翻译后修饰(PTM)来消除这种模糊性。电子捕获解离(ECD)是一种高效且温和的肽和蛋白质碎片化策略,可用于自下而上和自上而下的方法。在这里,我们使用了安捷伦6550 Q-TOF质谱仪,并安装了e-MSion ECD电池。通过在高效液相色谱(HPLC)时间尺度上对完整肽和蛋白质进行测序,评估细胞的自上而下测序能力。淀粉样蛋白β 1-40 (Aβ40)因其在阿尔茨海默病中的病理生理作用而首次进行测试,并作为我们的大肽标准。我们通过反相HPLC-MS对a β40进行了测序,并利用基于数据依赖采集(DDA)的方法在色谱时间尺度上实现了95%的序列覆盖率。利用高效液相色谱法对人脑丙酮沉淀蛋白提取物进行分离,并采用DDA方法进行分析,鉴定出16种2 ~ 17 kDa的蛋白质形态,根据蛋白质形态的大小和组成,序列覆盖率为7% ~ 90%。除了鉴定蛋白质形态外,ECD片段还区分了多种异天冬氨酸修饰和天冬氨酸,以及直接从人脑样本中准确区分亮氨酸和异亮氨酸残基。在这里,我们观察到异天冬氨酸在胸腺蛋白酶-4蛋白形式。此外,我们证明了在泛素的一个亚基内亮氨酸和异亮氨酸的分化。本研究推进了LC-Q-TOF仪器在基于发现的自上而下蛋白质组学中的应用,利用e-MSion ECD细胞实现了ECD。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CiteScore
5.50
自引率
9.40%
发文量
257
审稿时长
1 months
期刊介绍: The Journal of the American Society for Mass Spectrometry presents research papers covering all aspects of mass spectrometry, incorporating coverage of fields of scientific inquiry in which mass spectrometry can play a role. Comprehensive in scope, the journal publishes papers on both fundamentals and applications of mass spectrometry. Fundamental subjects include instrumentation principles, design, and demonstration, structures and chemical properties of gas-phase ions, studies of thermodynamic properties, ion spectroscopy, chemical kinetics, mechanisms of ionization, theories of ion fragmentation, cluster ions, and potential energy surfaces. In addition to full papers, the journal offers Communications, Application Notes, and Accounts and Perspectives
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