Institute of Analytical Chemistry, Department of Chemistry, University of Natural Resources and Life Sciences, Vienna (BOKU), Muthgasse 18, 1190 Vienna, Austria.
Agilent Technologies, Santa Clara, 95051, CA, USA.
Anal Chim Acta. 2021 Jun 8;1163:338508. doi: 10.1016/j.aca.2021.338508. Epub 2021 Apr 12.
The focus of this work was the implementation of ion mobility (IM) and a prototype quadrupole driver within data independent acquisition (DIA) using a drift tube IM-QTOFMS aiming to improve the level of confidence in identity confirmation workflows for non-targeted metabolomics. In addition to conventional all ions (IM-AI) acquisition, quadrupole resolved all ions (IM-QRAI) acquisition allows a drift time-directed precursor ion isolation in DIA using sequential isolation of precursor ions using mass windows of up to 100 Da which can be rapidly ramped across single ion mobility transients (i.e., <100 ms) according to the arrival times of precursor ions. Both IM-AI and IM-QRAI approaches were used for identity confirmation and relative quantification of metabolites in cellular extracts of the cell factory host Pichia pastoris. Samples were spiked with a uniformly C-labeled (UC) internal standard and LC with low-field drift tube IM separation was used in combination with IM-AI and IM-QRAI. Combining excellent hardware performance and correlation of IM arrival times of natural (C) and UC metabolites enabled alignment of signals in the arrival time domain (CCS differences ≤0.3%), and, in the case of IM-QRAI operation, maintenance of quantitative signals in comparison to IM-AI. The combination of tailored IM-QRAI methods for precursor ion isolation and IM separation also minimized the occurrence of spectral interferences in complex DIA datasets. Combined use of the software tools MS-DIAL, MS-Finder and Skyline for peak picking, feature alignment, reconciliation of C and UC isotopologue pairs, deconvolution of fragment spectra from DIA data, identity confirmation (including CCS) and targeted re-extraction of datafiles were employed for the data processing workflow. Overall, the combined new acquisition and data processing approaches enabled 87 metabolites to be identified between Level 1 (identified by standard compound) and Level 3.2 (accurate mass spectrum and number of carbons confirmed). The developed methods constitute promising metabolomics discovery tools and can be used to elucidate the number of carbon atoms present in unknown metabolites in stable isotope-supported metabolomics.
这项工作的重点是在使用 drift tube IM-QTOFMS 的数据非依赖性采集 (DIA) 中实现离子淌度 (IM) 和四极杆驱动器的原型，旨在提高非靶向代谢组学中身份确认工作流程的置信度。除了常规的所有离子 (IM-AI) 采集外，四极杆分辨所有离子 (IM-QRAI) 采集允许在 DIA 中使用质量窗口（最大可达 100 Da）进行导向漂移时间的前体离子隔离，该质量窗口可以根据前体离子的到达时间快速跨越单个离子淌度瞬变（即，<100 ms）。两种 IM-AI 和 IM-QRAI 方法都用于鉴定和相对定量细胞工厂宿主毕赤酵母细胞提取物中的代谢物。在 LC 与低场 drift tube IM 分离联用的基础上，样品中加入了均匀 C 标记 (UC) 的内标，并使用 IM-AI 和 IM-QRAI 进行鉴定和相对定量。将出色的硬件性能与天然 (C) 和 UC 代谢物的 IM 到达时间的相关性相结合，实现了在到达时间域中的信号对齐（CCS 差异≤0.3%），并且在 IM-QRAI 操作的情况下，与 IM-AI 相比，保持定量信号。用于前体离子隔离和 IM 分离的定制 IM-QRAI 方法的组合也最大限度地减少了复杂 DIA 数据集中的光谱干扰的发生。MS-DIAL、MS-Finder 和 Skyline 等软件工具的组合用于峰提取、特征对齐、C 和 UC 同位素对的协调、从 DIA 数据中解卷积碎片光谱、身份确认（包括 CCS）和数据文件的靶向再提取，用于数据处理工作流程。总的来说，结合新的采集和数据处理方法，在 Level 1（通过标准化合物鉴定）和 Level 3.2（准确质量谱和确认的碳原子数）之间鉴定了 87 种代谢物。所开发的方法构成了有前途的代谢组学发现工具，可以用于阐明稳定同位素支持的代谢组学中未知代谢物中存在的碳原子数。
