Liebisch G, Drobnik W, Reil M, Trümbach B, Arnecke R, Olgemöller B, Roscher A, Schmitz G
Institut für Klinische Chemie und Laboratoriumsmedizin, Klinikum der Universität Regensburg, Franz-Josef-Strauss-Allee 11, 93042 Regensburg, Germany.
J Lipid Res. 1999 Aug;40(8):1539-46.
Ceramide (CER) is an important signaling molecule involved in a variety of cellular processes, including differentiation, cell growth, and apoptosis. Currently, different techniques are applied for CER quantitation, some of which are relatively insensitive and/or time consuming. Tandem mass spectrometry with its high selectivity and sensitivity is a very useful technique for detection of low abundant metabolites without prior purification or derivatization. In contrast to existing mass spectrometry methods, the developed electrospray tandem mass spectrometry (ESI-MS/MS) technique is capable of quantifying different CER species from crude cellular lipid extracts. The ESI-MS/MS is performed with a continuous flow injection and the use of an autosampler, resulting in a high throughput capability. The collision-induced fragmentation of CER produced, in addition to others, a characteristic fragment of m/z 264, making a precursor ion scan of 264 well suited for CER quantitation. Quantitation is achieved by use of a constant concentration of a non-naturally occurring internal standard C8-CER, together with a calibration curve established by spiking different concentrations of naturally occurring CER. The calibration curves showed linearity over a wide concentration range and sample volumes equivalent to 10 microg of cell protein corresponding to about 20, 000 fibroblasts were sufficient for CER analysis. Moreover this assay showed a detection limit at the subpicomole level. In summary, this methodology enables accurate and rapid analysis of CER from small samples without prior separation steps, thus providing a useful tool for signal transduction research.
神经酰胺（CER）是一种重要的信号分子，参与多种细胞过程，包括分化、细胞生长和凋亡。目前，用于CER定量的技术各不相同，其中一些技术相对不灵敏和/或耗时。串联质谱具有高选择性和灵敏度，是一种无需事先纯化或衍生化即可检测低丰度代谢物的非常有用的技术。与现有的质谱方法不同，所开发的电喷雾串联质谱（ESI-MS/MS）技术能够从粗制细胞脂质提取物中定量不同种类的CER。ESI-MS/MS采用连续流动注射和自动进样器进行，具有高通量能力。CER的碰撞诱导碎片化除了产生其他碎片外，还产生了一个m/z 264的特征性碎片，使得对264进行前体离子扫描非常适合CER定量。通过使用恒定浓度的非天然存在的内标C8-CER以及通过加入不同浓度的天然存在的CER建立的校准曲线来实现定量。校准曲线在很宽的浓度范围内呈线性，相当于10微克细胞蛋白的样品体积对应约20,000个成纤维细胞，足以进行CER分析。此外，该测定法显示出亚皮摩尔水平的检测限。总之，这种方法能够在无需事先分离步骤的情况下对小样品中的CER进行准确快速的分析，从而为信号转导研究提供了一种有用的工具。
