Department of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Waehringer Strasse 38, 1090 Vienna, Austria.
Analyst. 2021 Apr 26;146(8):2591-2599. doi: 10.1039/d0an02443k.
We propose a fully automated novel workflow for lipidomics based on flow injection, followed by liquid chromatography-high-resolution mass spectrometry (FI/LC-HRMS). The workflow combined in-depth characterization of the lipidome achieved via reversed-phase LC-HRMS with absolute quantification by using a large number of lipid species-specific and/or retention time (RT)-matched/class-specific calibrants. The lipidome of 13C-labelled yeast (LILY) provided a large panel of cost-effective internal standards (ISTDs) covering triacylglycerols (TG), steryl esters (SE), free fatty acids (FA), diacylglycerols (DG), sterols (ST), ceramides (Cer), hexosyl ceramides (HexCer), phosphatidylglycerols (PG), phosphatidylethanolamines (PE), phosphatidic acids (PA), cardiolipins (CL), phosphatidylinositols (PI), phosphatidylserines (PS), phosphatidylcholines (PC), lysophosphatidylcholines (LPC) and lysophosphatidylethanolamines (LPE). The workflow in combination with the LILY lipid panel enables simultaneous quantification via (1) external multi-point calibration with internal standardization and (2) internal one-point calibration with LILY as a surrogate ISTD, increasing the coverage while keeping the accuracy and throughput high. Extensive measures on quality control allowed us to rank the calibration strategies and to automatically select the calibration strategy of the highest metrological order for the respective lipid species. Overall, the workflow enabled a streamlined analysis, with a limit of detection in the low femtomolar range, and provided validation tools together with absolute concentration values for >350 lipids in human plasma on a species level. Based on the selected standard panel, lipids from 7 classes (LPC, LPE, PC, PE, PI, DG, TG) passed stringent quality filters, which included QC accuracy, a precision and recovery bias of <30% and concentrations within the 99% confidence interval of the international laboratory comparison of SRM 1950, NIST, USA. The quantitative values are independent of common deuterated or non-endogenous ISTDs, thus offering cross-validation of different lipid methods and further standardizing lipidomics.
我们提出了一种基于流动注射的新型脂质组学全自动工作流程，随后是液相色谱-高分辨率质谱（FI/LC-HRMS）。该工作流程将通过反相 LC-HRMS 实现的脂质组深入特征与使用大量脂质特异性和/或保留时间（RT）匹配/分类特异性校准物进行的绝对定量相结合。13C 标记酵母（LILY）的脂质组提供了一组大型的、经济有效的内标（ISTD），涵盖了三酰基甘油（TG）、甾醇酯（SE）、游离脂肪酸（FA）、二酰基甘油（DG）、甾醇（ST）、神经酰胺（Cer）、己糖神经酰胺（HexCer）、磷脂酰甘油（PG）、磷脂酰乙醇胺（PE）、磷脂酸（PA）、心磷脂（CL）、磷脂酰肌醇（PI）、磷脂丝氨酸（PS）、磷脂胆碱（PC）、溶血磷脂酰胆碱（LPC）和溶血磷脂酰乙醇胺（LPE）。该工作流程与 LILY 脂质组结合使用，可通过（1）使用内部标准化的外部多点校准和（2）使用 LILY 作为替代 ISTD 的内部单点校准来同时进行定量，在保持准确性和高通量的同时增加覆盖率。广泛的质量控制措施使我们能够对校准策略进行排名，并自动为各自的脂质种类选择最高计量学等级的校准策略。总体而言，该工作流程实现了简化的分析，具有低至皮摩尔级的检测限，并提供了验证工具以及人血浆中超过 350 种脂质的绝对浓度值，达到物种水平。基于选定的标准面板，7 个类别（LPC、LPE、PC、PE、PI、DG、TG）的脂质通过了严格的质量过滤，其中包括 QC 准确性、精度和回收率偏差<30%，以及国际实验室比较 SRM 1950、NIST、美国的 99%置信区间内的浓度。定量值与常见氘代或非内源性 ISTDs 无关，因此提供了不同脂质方法的交叉验证，并进一步实现了脂质组学的标准化。
