University of Münster, Institute of Inorganic and Analytical Chemistry, Corrensstraße 30, 48149 Münster, Germany.
Food Chemistry, Faculty of Mathematics and Natural Sciences, University of Wuppertal, Gaußstr. 20, 42119, Wuppertal, Germany.
J Chromatogr A. 2021 Oct 11;1655:462481. doi: 10.1016/j.chroma.2021.462481. Epub 2021 Aug 18.
Sphingolipids exert important functions in cells, ranging from stabilising the cell membrane to bioactive signalling in signal transduction pathways. Changed concentrations of sphingolipids are associated with, among others, neurodegenerative and cardiovascular diseases. In this work, we present a novel two-dimensional liquid chromatography method (2D-LC) coupled to tandem mass spectrometry (MS/MS) for the identification of ceramides, hexosylceramides and sphingomyelins in the model organism Caenorhabditis elegans (C. elegans). The method utilises a multiple heart-cut approach with a hydrophilic interaction liquid chromatography (HILIC) separation in the first dimension. The fractions of the sphingolipid classes were cut out and thereby separated from the abundant glycerolipids, which offers a simplified sample preparation and a high degree of automation as it compensates the alkaline depletion step usually conducted prior to the chromatographic analysis. The fractions were stored in a sample loop and transferred onto the second column with the combination of two six port valves. A reversed phase liquid chromatography was performed as the second dimension and allowed for a separation of the species within a sphingolipid class and according to the fatty acid moiety of the sphingolipid. The segregation of the abundant glycerolipids and the reduced matrix effects allowed for better identification of low abundant species, especially dihydro-sphingolipids with a saturated sphingoid base. In addition, the separation of the three fractions was carried out parallel to the separation and equilibration in the first dimension, which leads to no extension of the analysis time for the 2D-LC compared to the one-dimensional HILIC method. In total 45 sphingolipids were detected in the C. elegans lipid extract and identified via accurate mass and MS/MS fragments.
神经酰胺、己糖神经酰胺和神经鞘磷脂在细胞中发挥着重要的功能，从稳定细胞膜到信号转导通路中的生物活性信号传递。鞘脂浓度的改变与神经退行性和心血管疾病等有关。在这项工作中，我们提出了一种新的二维液相色谱法（2D-LC）与串联质谱（MS/MS）相结合，用于鉴定模式生物秀丽隐杆线虫（C. elegans）中的神经酰胺、己糖神经酰胺和神经鞘磷脂。该方法利用亲水相互作用液相色谱（HILIC）在第一维中的多次心脏切割方法。将鞘脂类的馏分切出，从而与丰富的甘油脂质分离，这提供了简化的样品制备和高度的自动化，因为它补偿了通常在色谱分析之前进行的碱性消耗步骤。馏分储存在样品环中，并通过两个六通阀转移到第二根柱上。第二维进行反相液相色谱，允许在鞘脂类内和根据鞘脂的脂肪酸部分分离物种。丰富的甘油脂质的分离和基质效应的减少允许更好地鉴定低丰度的物种，特别是具有饱和鞘氨醇碱基的二氢鞘脂。此外，三个馏分的分离与第一维中的分离和平衡平行进行，与一维 HILIC 方法相比，2D-LC 的分析时间没有延长。在秀丽隐杆线虫脂质提取物中检测到 45 种鞘脂，并通过精确质量和 MS/MS 片段进行鉴定。
