Farwanah Hany, Wirtz Jennifer, Kolter Thomas, Raith Klaus, Neubert Reinhard H H, Sandhoff Konrad
LiMES - Life and Medical Sciences Institute, Membrane Biology and Lipid Biochemistry Unit, c/o Kekulé-Institut für Organische Chemie und Biochemie, University of Bonn, Gerhard-Domagk Str. 1, D-53121 Bonn, Germany.
J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Oct 1;877(27):2976-82. doi: 10.1016/j.jchromb.2009.07.008. Epub 2009 Jul 14.
Many lipidomic approaches focus on investigating aspects of sphingolipid metabolism. Special emphasis is put on neutral sphingolipids and cholesterol and their interaction. Such an interest is attributed to the fact that those lipids are altered in a series of serious disorders including various sphingolipidoses. High performance thin-layer chromatography (HPTLC) has become a widely used technique for lipid analysis. However, mass spectrometric profiling is irreplaceable for gaining an overview about the various molecular species within a lipid class. In this work we have developed a sensitive method based on a gradient normal phase high performance liquid chromatography (HPLC) coupled to quadrupole time of flight (QTOF) atmospheric pressure chemical ionization mass spectrometry (APCI-MS) in positive mode, which for the first time enables separation, on-line detection, and mass spectrometric profiling of multiple neutral sphingolipids including ceramide, glucosylceramide, lactosylceramide, globotriaosylceramide, globotetraosylceramide, sphingomyelin as well as cholesterol within less than 15min. An important advantage of the presented HPLC/APCI-MS approach is that the separation pattern emulates the one obtained by an optimized HPTLC method with a multiple stage development. Thus, the lipid classes previously separated and quantified by HPTLC can be easily screened regarding their mass spectrometric profiles by HPLC/APCI-MS. In addition, the selected ionization conditions enable in-source fragmentation providing useful structural information. The methods (HPLC/APCI-MS and the optimized HPTLC) were applied for the analysis of the mentioned lipids in human fibroblasts. This approach is aimed basically at investigators who perform studies based on genetic modifications or treatment with pharmacological agents leading to changes in the biochemical pathways of neutral sphingolipids and cholesterol. In addition, it can be of interest for research on disorders related to impairments of sphingolipid metabolism.
许多脂质组学方法专注于研究鞘脂代谢的各个方面。特别强调中性鞘脂和胆固醇及其相互作用。这种兴趣归因于这样一个事实，即这些脂质在一系列严重疾病中发生改变，包括各种鞘脂贮积症。高效薄层色谱法（HPTLC）已成为一种广泛应用于脂质分析的技术。然而，质谱分析对于全面了解脂质类别中的各种分子种类是不可替代的。在这项工作中，我们开发了一种灵敏的方法，该方法基于梯度正相高效液相色谱（HPLC）与四极杆飞行时间（QTOF）大气压化学电离质谱（APCI-MS）联用，采用正模式，首次能够在不到15分钟的时间内分离、在线检测和质谱分析多种中性鞘脂，包括神经酰胺、葡萄糖神经酰胺、乳糖神经酰胺、球三糖神经酰胺、球四糖神经酰胺、鞘磷脂以及胆固醇。所提出的HPLC/APCI-MS方法的一个重要优点是，其分离模式类似于通过优化的多阶段展开HPTLC方法获得的分离模式。因此，先前通过HPTLC分离和定量的脂质类别可以通过HPLC/APCI-MS轻松地根据其质谱图进行筛选。此外，选定的电离条件能够实现源内裂解，提供有用的结构信息。这些方法（HPLC/APCI-MS和优化的HPTLC）被应用于分析人类成纤维细胞中的上述脂质。这种方法主要针对那些基于基因修饰或用药物处理导致中性鞘脂和胆固醇生化途径变化进行研究的研究人员。此外，它对于与鞘脂代谢受损相关疾病的研究可能也有意义。
