使用循环离子淌度光谱仪进行串联离子淌度实验
Summary
离子淌度光谱法（IMS）是质谱法表征生物分子的有趣补充，特别是因为它对异构体敏感。该协议描述了串联IMS（IMS / IMS）实验，该实验允许分离分子并生成其片段的迁移率曲线。
Abstract
准确表征化学结构对于了解其潜在的生物学机制和功能特性非常重要。质谱（MS）是一种流行的工具，但并不总是足以完全揭示所有结构特征。例如，尽管碳水化合物具有生物学相关性，但它们的表征因许多级别的异构体而变得复杂。离子淌度光谱（IMS）是一个有趣的补充，因为它对离子构象敏感，因此对异构体敏感。
此外，最近的进展显着改进了该技术：与线性IMS仪器相比，上一代Cyclic IMS仪器提供了额外的功能，例如增加的分辨能力或执行串联离子迁移率（IMS / IMS）实验的可能性。在IMS/IMS期间，根据其离子迁移率选择离子，碎片化并重新分析，以获得有关其片段的离子迁移率信息。最近的研究表明，这种IMS / IMS数据中包含的片段的迁移率曲线可以充当特定聚糖的指纹，并且可以用于分子网络策略中，以结构相关的方式组织糖组学数据集。
因此，该协议的目标是描述如何生成IMS / IMS数据，从样品制备到离子迁移率维度的最终碰撞截面（CCS）校准，从而产生可重复的光谱。以一种代表性聚糖为例，该协议将展示如何在循环IMS仪器上构建IMS/IMS控制序列，如何考虑该控制序列以将IMS到达时间转换为漂移时间（即应用于离子的有效分离时间），以及如何从原始数据中提取相关的迁移率信息。该协议旨在清楚地解释IMS / IMS实验的关键点，从而帮助新的Cyclic IMS用户执行直接且可重复的采集。
Introduction
生物分子的完整化学表征是了解其潜在生物学和功能特性的关键。为此，近年来，“组学”科学不断发展，旨在大规模表征生物浓度下的化学结构。在蛋白质组学和代谢组学中，MS已成为揭示生物介质中发现的结构异质性的核心工具 – 特别是由于其灵敏度和通过串联MS（MS / MS）提供结构信息的能力。在MS / MS策略中，根据其质量选择离子，然后片段化，最后获取其片段的质量以建立分子的指纹。MS/MS光谱尤其可用于匹配光谱数据库1，2，或试探性地重建父结构3，4。在相似光谱属于相似化合物的假设下，MS/ MS数据也可用于构建通过相似性评分连接相关物种的分子网络（MNs）5，6。
然而，由于MS检测离子的质荷比（m / z）的固有特性，该技术对落在（立体）异构体范围内的许多结构特征视而不见。例如，碳水化合物由几种单糖亚基组成，其中许多是立体异构体甚至异构体（例如，Glc与Gal或Glc与人）。这些亚基由糖苷键连接，糖苷键可以通过键的位置（区域异构体）和无构体碳的空间位阻构型（非对称性）而不同。这些特性使得独立的MS难以区分碳水化合物异构体7，并且只能使用高能活化方法解决区域异构体8，9，10。虽然衍生化是破坏立体异构群等价性的一种选择11，但它需要大量的样品制备。另一个更直接的选择是将MS与对异构体敏感的分析维度（如IMS）耦合。
由于此协议是为已经熟悉 IMS 基本概念的用户设计的，并且由于其他地方提供了详细的评论12，13，因此此处仅简要概述了 IMS 的原理。IMS是一种气相分离方法，它依赖于离子与缓冲气体和电场的相互作用，最终根据其气相构象分离离子。在商用仪器上可以找到耦合到MS的IMS的不同原理：一些在高低电场（场不对称IMS，FAIMS）的交替下工作，而大多数在低场限值内工作 – 特别是漂移管IMS（DTIMS，线性递减电场），行波IMS（TWIMS，对称电位波）和捕获IMS（TIMS，高流量缓冲气体捕获离子对抗电场）13.低场方法允许访问所谓的CCS，这是离子 – 气体对的一种性质，代表离子的表面（在Å2 或nm2中），在分离过程中与缓冲气体相互作用。CCS在理论上与仪器无关，因此可用于生成可在不同实验室之间再现的数据14。离子淌度分离可能受到各种参数的影响，特别是受迁移池中气体压力和气体温度波动的影响。CCS校准是解决这个问题的一种方法，因为校准剂和感兴趣的物种都会受到类似的影响13。但是，必须将仪器安装在温度受控的房间内，并具有可靠的气体压力控制系统。
IMS的一个有趣的演变是IMS / IMS，它于2006年由Clemmer的小组首次引入，作为MS / MS15，16的类似物。在IMS / IMS中，根据其离子迁移率选择性地分离感兴趣的离子;然后它被激活（直到可能的碎片），并对激活的离子或片段进行新的IMS分析。在第一个仪器设计中，两个IMS细胞串联放置，由激活所在的离子漏斗隔开。从那时起，尽管提出了许多IMS / IMS设置（有关审查，请参阅Eldrid和Thalassinos17），但第一台具有IMS / IMS功能的商用质谱仪直到2019年才上市18。该仪器通过将它与另一项技术突破相结合，大大改进了最初的概念：IMS单元的循环设计。
理论上，循环IMS单元允许近乎无限地增加漂移路径长度，从而增加仪器的分辨能力19。这是通过特定的仪器几何形状实现的，其中循环TWIMS单元正交放置在主离子光轴上。IMS单元入口处的多功能阵列区域允许控制离子路径的方向：（i）将离子侧向发送以进行IMS分离，（ii）向前用于MS检测，或（iii）从IMS单元向后存储到预阵列单元中。从这个预阵列存储池中，离子可以被激活，片段在IMS细胞中重新注入以进行离子迁移率测量，这种方法已成功用于表征立体异构体20。最终，收集的数据包含前体及其片段的离子迁移率和 m / z 信息。
在最近一篇使用这种循环设计进行聚糖分析的出版物（Ollivier等人21）中，我们表明，这种IMS / IMS数据中包含的片段的迁移率曲线充当生物分子的指纹，可用于分子网络策略。由此产生的网络称为IM-MN，导致糖组学数据集以结构相关的方式进行组织，而仅由MS / MS数据（MS-MN）构建的网络几乎没有透露信息。为了补充本出版物并帮助Cyclic IMS用户实现此工作流，此协议提供了用于收集数据的协议的完整描述。该协议仅关注 IMS/IMS 数据的生成，然后用户可以使用这些数据来构建 IM-MN 网络（请参阅 21），或用于他们选择的任何其他应用程序。本文将不考虑IM-MN的构建，因为分子网络的方案已经可用22。强调了为产生有价值和可重复的IMS / IMS收购而必须遵循的关键点。以Ollivier等人研究的寡糖之一 为例。21、以下步骤详述：（i）样品制备，（ii）循环IMS仪器的调谐，（iii）数据的自动峰值拾取，以及（iv）CCS校准。
Protocol
Representative Results
Discussion
SELECT系列循环IMS是一个功能强大的工具，允许选择给定的 m / z 和离子迁移率的已定义离子群，而无需上游色谱分离。该仪器提供了生成该离子群的双次碎片图的可能性，可以从中提取MS / MS和IMS / IMS光谱。但是，用户必须注意在实验过程中需要注意的几个关键点。
首先，用户应仔细检查MS隔离窗口是否存在可能的等压污染物。实际上，四极杆的隔离窗口相对较宽，并且?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
S.O.感谢法国国家研究机构资助他的博士学位（授予ANR-18-CE29-0006）。
Materials
| 33-α-L- plus 23-α-L-Arabinofuranosyl-xylotetraose (XA3XX/XA2XX) mixture | Megazyme Ltd., Wicklow, Ireland | O-XAXXMIX | XA2XX + XA3XX mixture |
| 33-α-L-Arabinofuranosyl-xylotetraose (XA3XX) | Megazyme Ltd., Wicklow, Ireland | O-XA3XX | Pure XA3XX standard |
| Eppendorf Safe-Lock Tubes, 1.5 mL, Eppendorf Quality, colorless, 1,000 tubes | Eppendorf, Hamburg, Germany | 0030120086 | Used to prepare the carbohydrate stock solution and dilution |
| FALCON 50 mL Polypropylene Conical Tube 30 x 115 mm | Corning Science México S.A. de C.V., Reynosa, Tamaulipas, Mexico | 352070 | Used to prepare the aqueous stock solution of 100 mM LiCl |
| Lithium Chloride (ACS reagent, ≥99 %) | Sigma-Aldrich Inc., Saint Quentin Fallavier, France | 310468 | Used to dope the sample with lithium |
| Major Mix IMS/Tof Calibration Kit | Waters Corp., Wilmslow, UK | 186008113 | Calibration solution for MS and IMS |
| MassLynx 4.2 SCN1016 Release 6 (Waters Embedded Analyser Platform for Cyclic IMS 2.9.1 Release 9) | Waters Corp., Wilmslow, UK | 721022377 | Cyclic IMS vendor software for instrument control and data processing |
| Methanol for HPLC PLUS Gradient grade | Carlo-Erba Reagents, Val de Reuil, France | 412383 | High-purity solvent |
| MS Leucine Enkephaline Kit | Waters Corp., Wilmslow, UK | 700002456 | Reference compound used for tuning of the mass spectrometer |
| SCHOTT DURAN 100 mL borosilicate glass bottle | VWR INTERNATIONAL, Radnor, Pennsylvania, US | 218012458 | Used to prepare the solution of 500 µM LiCl in 50:50 MeOH/Water |
| SELECT SERIES Cyclic IMS | Waters Corp., Wilmslow, UK | 186009432 | Ion mobility-mass spectrometer equipped with a cylic IMS cell |
| Website: http://mzmine.github.io/ | MZmine Development Team | – | Link to download the MZmine software |
| Website: https://github.com/siollivier/IM-MN | INRAE, UR BIA, BIBS Facility, Nantes, France | – | Link to an in-house R script containing a CCS calibration function |
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