Top-Down Deep Sequencing of Ubiquitin Using Two-Dimensional Mass Spectrometry
Top-Down Deep Sequencing of Ubiquitin Using Two-Dimensional Mass Spectrometry
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DOI：
10.1021/acs.analchem.8b00500
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发表时间：
2018-06-19
影响因子：
7.4
通讯作者：
O'Connor, Peter B.
中科院分区：
文献类型：
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作者：
Floris, Federico;Chiron, Lionel;O'Connor, Peter B.
二维质谱 (2DMS) 允许对样品中的所有离子进行数据独立的碎裂，并将碎片离子与其前体相关联，而无需在碎裂之前进行分离。过去十年来，计算机功能和傅里叶变换离子回旋共振 (FTICR) MS 实施的发展使该技术成为自下而上蛋白质组学 (BUP) 以及最近自上而下蛋白质分析 (TDP) 的有用分析工具。在这项工作中，使用 2D FTICR MS 开发了一种新的 TDP 方法，称为 MS/2D FTICR MS 或 MS/2DMS。在 MS/2DMS 中，整个蛋白质最初在六极碰撞池中断裂，例如通过碰撞激活解离 (CAD)。然后将初级碎片发送到 ICR 单元，在其中通过红外多光子解离 (IRMPD) 或电子捕获解离 (ECD) 执行 2DMS。生成的 2D 质谱保留的信息相当于对所选蛋白质进行的一组 TDP MS3 实验。只要感兴趣的离子可以在 ICR 池之前明确裂解，就可以在该过程中添加最多 n - 1 个裂解步骤，从而进行 MSn/2DMS 实验，其输出是保留与 MSn 等效信息的 2D 质谱。本工作中使用 MS/2DMS 和 MS/MS/2DMS 进行泛素 (Ubi) 的结构分析，注意到几个有助于片段识别的独特特征。分别使用源内解离 (ISD)、CAD 和 ECD-2DMS 的 CAD-MS/IRMPD-2DMS、CAD-MS/ECD-2DMS 和 MS2/2DMS 使 Ubi 的裂解覆盖率达到 97%。
Two-dimensional mass spectrometry (2DMS) allows data independent fragmentation of all ions in a sample and correlation of fragment ions to their precursors without isolation prior to fragmentation. Developments in computer capabilities and implementations in Fourier transform ion cyclotron resonance (FTICR) MS over the past decade have allowed the technique to become a useful analytical tool for bottom-up proteomics (BUP) and, more recently, in top-down protein analysis (TDP). In this work, a new method of TDP is developed using 2D FTICR MS, called MS/2D FTICR MS or MS/2DMS. In MS/2DMS, an entire protein is initially fragmented in a hexapole collision cell, e.g., with collisionally activated dissociation (CAD). The primary fragments are then sent to the ICR cell, where 2DMS is performed with infrared multiphoton dissociation (IRMPD) or electron-capture dissociation (ECD). The resulting 2D mass spectra retain information equivalent to a set of TDP MS3 experiments on the selected protein. Up to n - 1 fragmentation steps can be added to the process, as long as an ion of interest can be unambiguously fragmented before the ICR cell, leading to an MSn/2DMS experiment whose output is a 2D mass spectrum retaining information equivalent to MSn. MS/2DMS and MS/MS/2DMS are used in this work for the structural analysis of ubiquitin (Ubi), noting several unique features which aid fragment identification. The use of CAD-MS/IRMPD-2DMS, CAD-MS/ECD-2DMS, and MS2/2DMS using, respectively, in-source dissociation (ISD), CAD, and ECD-2DMS led to 97% cleavage coverage for Ubi.