Abstract

In this work, a lipidomics workflow based on offline semi-preparative lipid class-specific fractionation by supercritical fluid chromatography (SFC) followed by high-resolution mass spectrometry was introduced. The powerful SFC approach offered separation of a wide polarity range for lipids, enabled enrichment (up to 3 orders of magnitude) of lipids, selective fractionation of 14 lipid classes/subclasses, and increased dynamic range enabling in-depth characterization. A significantly increased coverage of low abundant lipids improving lipid identification by numbers and degree (species and molecular level) was obtained in Pichia pastoris when comparing high-resolution mass spectrometry based lipidomics with and without prior fractionation. Proof-of-principle experiments using a standard reference material (SRM 1950, NIST) for human plasma showed that the proposed strategy enabled quantitative lipidomics. Indeed, for 70 lipids, the consensus values available for this sample could be met. Thus, the novel workflow is ideally suited for lipid class-specific purification/isolation from milligram amounts of sample while not compromising on omics type of analysis (identification and quantification). Finally, compared with established fractionation/pre-concentration approaches, semi-preparative SFC is superior in terms of versatility, as it involved only volatile modifiers and salt additives facilitating any follow-up use such as qualitative or quantitate analysis or further purification down to the single lipid species level.

Graphical Abstract

中文翻译：

---

用于脂质类分级的制备型超临界流体色谱—基于高分辨率质谱的脂质组学的新策略

摘要

在这项工作中，介绍了一种脂质组学工作流程，该流程基于通过超临界流体色谱（SFC）进行的离线半制备脂质类别特异性分馏，然后进行高分辨率质谱分析。强大的SFC方法可分离脂质的宽极性范围，实现脂质的富集（最多3个数量级），14种脂质类/亚类的选择性分级分离以及动态范围扩大，从而可进行深入表征。巴斯德毕赤酵母中低丰度脂质的覆盖率显着增加，从而通过数量和程度（种类和分子水平）改善了脂质的鉴定比较使用和不使用事先分级分离的基于高分辨率质谱的脂质组学。使用用于人类血浆的标准参考物质（SRM 1950，NIST）进行的原理验证实验表明，所提出的策略可实现定量脂质组学。实际上，对于70种脂质，可以满足该样品的共识值。因此，新颖的工作流程非常适合从毫克量的样品中进行脂质类特定的纯化/分离，同时又不影响组学类型的分析（鉴定和定量）。最后，与已建立的分馏/预浓缩方法相比，半制备SFC的通用性更高，