Institute of Clinical Chemistry and Laboratory Medicine, Regensburg University Hospital, Regensburg, Germany.
Department of Biochemistry and Molecular Biology, Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense, Denmark; Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
J Lipid Res. 2021;62:100050. doi: 10.1016/j.jlr.2021.100050. Epub 2021 Feb 16.
Lipidomics data require consideration of ions with near-identical masses, which comprises among others the Type-II isotopic overlap. This overlap occurs in series of lipid species differing only by number of double bonds (DBs) mainly because of the natural abundance of C-atoms. High-resolution mass spectrometry, such as Fourier-transform mass spectrometry (FTMS), is capable of resolving Type-II overlap depending on mass resolving power. In this work, we evaluated FTMS quantification accuracy of lipid species affected by Type-II overlap. Spike experiments with lipid species pairs of various lipid classes were analyzed by flow injection analysis-FTMS. Accuracy of quantification was evaluated without and with Type-II correction (using relative isotope abundance) as well as utilizing the first isotopic peak (M+1). Isobaric peaks, which were sufficiently resolved, were most accurate without Type-II correction. In cases of partially resolved peaks, we observed peak interference causing distortions in mass and intensity, which is a well-described phenomenon in FTMS. Concentrations of respective species were more accurate when calculated from M+1. Moreover, some minor species, affected by considerable Type-II overlap, could only be quantified by M+1. Unexpectedly, even completely unresolved peaks were substantially overcorrected by Type-II correction because of peak interference. The described method was validated including intraday and interday precisions for human serum and fibroblast samples. Taken together, our results show that accurate quantification of lipid species by FTMS requires resolution-depended data analysis.
脂质组学数据需要考虑具有几乎相同质量的离子，其中包括 II 型同位素重叠。这种重叠主要发生在仅通过双键数（DB）不同的脂质物种系列中，这是由于 C 原子的自然丰度所致。傅立叶变换质谱（FTMS）等高分辨率质谱能够根据质量分辨率解析 II 型重叠。在这项工作中，我们评估了受 II 型重叠影响的脂质物种的 FTMS 定量准确性。通过流动注射分析-FTMS 分析了各种脂质类别的脂质物种对的加标实验。在没有和有 II 型校正（使用相对同位素丰度）以及利用第一同位素峰（M+1）的情况下评估了定量准确性。足够分辨的等质量峰在没有 II 型校正的情况下最为准确。在部分分辨的峰的情况下，我们观察到峰干扰导致质量和强度的扭曲，这是 FTMS 中描述的一种现象。从 M+1 计算时，各物种的浓度更准确。此外，一些受相当大的 II 型重叠影响的次要物种只能通过 M+1 进行定量。出乎意料的是，即使是完全未分辨的峰也由于峰干扰而被 II 型校正大大过校正。该方法已通过人血清和成纤维细胞样品的日内和日间精密度进行了验证。综上所述，我们的结果表明，FTMS 准确定量脂质物种需要依赖分辨率的数据分析。
