Department of Analytical Chemistry, University of Vienna, 1090 Vienna, Austria.
Vienna Doctoral School in Chemistry, University of Vienna, 1090 Vienna, Austria.
Anal Chem. 2024 Jun 11;96(23):9643-9652. doi: 10.1021/acs.analchem.4c01388. Epub 2024 May 25.
Signaling lipids are key players in cellular processes. Despite their importance, no method currently allows their comprehensive monitoring in one analytical run. Challenges include a wide dynamic range, isomeric and isobaric species, and unwanted interaction along the separation path. Herein, we present a sensitive and robust targeted liquid chromatography-mass spectrometry (LC-MS/MS) approach to overcome these challenges, covering a broad panel of 17 different signaling lipid classes. It involves a simple one-phase sample extraction and lipid analysis using bioinert reversed-phase liquid chromatography coupled to targeted mass spectrometry. The workflow shows excellent sensitivity and repeatability in different biological matrices, enabling the sensitive and robust monitoring of 388 lipids in a single run of only 20 min. To benchmark our workflow, we characterized the human plasma signaling lipidome, quantifying 307 endogenous molecular lipid species. Furthermore, we investigated the signaling lipidome during platelet activation, identifying numerous regulations along important lipid signaling pathways. This highlights the potential of the presented method to investigate signaling lipids in complex biological systems, enabling unprecedentedly comprehensive analysis and direct insight into signaling pathways.
信号脂质是细胞过程中的关键参与者。尽管它们很重要，但目前没有一种方法可以在一次分析运行中全面监测它们。挑战包括广泛的动态范围、异构体和等摩尔物质，以及沿分离路径的不想要的相互作用。在此，我们提出了一种灵敏而稳健的靶向液相色谱-质谱联用 (LC-MS/MS) 方法来克服这些挑战，涵盖了广泛的 17 种不同的信号脂质类别。它涉及使用生物惰性反相液相色谱结合靶向质谱进行单相样品提取和脂质分析。该工作流程在不同的生物基质中表现出出色的灵敏度和重现性，能够在单次 20 分钟的运行中灵敏而稳健地监测 388 种脂质。为了基准测试我们的工作流程，我们对人血浆信号脂质组进行了表征，定量了 307 种内源性分子脂质物种。此外，我们还研究了血小板激活过程中的信号脂质组，确定了沿重要脂质信号通路的许多调节。这突显了所提出方法在复杂生物系统中研究信号脂质的潜力，能够进行前所未有的全面分析，并直接洞察信号通路。
