University of Tübingen, Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, Auf der Morgenstelle 8, 72076, Tübingen, Germany.
Department of Cardiology and Cardiovascular Medicine, University Hospital Tübingen, Otfried-Müller-Strasse 10, 72076, Tübingen, Germany.
Anal Chim Acta. 2019 Jan 10;1046:1-15. doi: 10.1016/j.aca.2018.08.060. Epub 2018 Sep 3.
A non-targeted lipidomics workflow based on C8 core-shell particle ultra high-performance liquid chromatography (UHPLC) hyphenated to ESI-QTOF-MS in data-independent acquisition (DIA) mode with sequential window acquisition of all theoretical fragment ion spectra (SWATH) was developed and applied to differential platelet lipidomics profiling of cardiovascular disease patients (stable angina pectoris (n = 10), ST-elevated myocardial infarction (n = 13)) against healthy controls (n = 10). DIA with SWATH generates comprehensive MS and MS/MS data throughout the entire chromatograms and all study samples. Hence, chromatograms can be extracted based on precursors or fragments which provided some benefits in terms of assay specificity in some cases. SWATH acquisition offers flexible experimental design with variable Q1 isolation windows. Liquid chromatography as well as SWATH settings were optimized to cover the lipidome of human platelets. The flexibility of the SWATH experiment design was utilized to implement target SWATH windows with narrow 5 Da Q1 precursor ion selection width (multiple reaction monitoring (MRM)-like SWATH windows) for the detection of low abundant oxidized phospholipids. Data processing was performed with MS-DIAL, and its feasibilities and caveats are discussed by illustrative examples. Thereby, identification of lipids is still a bottleneck in non-targeted lipidomics workflow. MS-DIAL, however, offers automatic identification via spectral matching using an in silico library. In total 1971 molecular features were detected cross the samples of which 611 were identified (total score >70%). The quality of the acquired data was validated with embedded quality control samples (n = 11). 80.3% of all features detected in the QC samples showed a coefficient of variation of below 30%. Multivariate statistics were used to visualize differences in the lipidome of distinct sample groups at a false discovery rate of 5%.
建立了一种基于 C8 核壳粒子超高效液相色谱（UHPLC）与电喷雾-四极杆飞行时间质谱（ESI-QTOF-MS）联用的非靶向脂质组学工作流程，在数据非依赖性采集（DIA）模式下采用顺序窗口采集所有理论碎片离子谱（SWATH），并应用于心血管疾病患者（稳定型心绞痛（n=10）、ST 段抬高型心肌梗死（n=13））与健康对照者（n=10）的血小板脂质组学差异分析。DIA 与 SWATH 在整个色谱图和所有研究样本中生成全面的 MS 和 MS/MS 数据。因此，基于前体或碎片提取色谱图，在某些情况下可提供一些分析物特异性方面的优势。SWATH 采集提供了具有可变 Q1 隔离窗口的灵活实验设计。优化了液相色谱和 SWATH 参数，以涵盖人血小板的脂质组。SWATH 实验设计的灵活性用于实现具有较窄 5 Da Q1 前体离子选择宽度的靶向 SWATH 窗口（类似多重反应监测（MRM）的 SWATH 窗口），用于检测低丰度氧化磷脂。数据处理采用 MS-DIAL 进行，并通过示例讨论了其可行性和注意事项。因此，非靶向脂质组学工作流程中，脂质的鉴定仍然是一个瓶颈。然而，MS-DIAL 通过使用虚拟库进行基于谱图匹配的自动鉴定。共检测到 1971 个分子特征，其中 611 个被鉴定（总得分>70%）。通过嵌入式质量控制样品（n=11）验证了获得数据的质量。在 QC 样品中检测到的所有特征中有 80.3%的变异系数低于 30%。采用多元统计方法在错误发现率为 5%的情况下可视化不同样本组脂质组的差异。
