School of Chemistry, University of Melbourne, Parkville, Victoria, 3010, Australia.
Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, 3010, Australia.
J Am Soc Mass Spectrom. 2017 Jul;28(7):1406-1419. doi: 10.1007/s13361-017-1668-1. Epub 2017 Apr 28.
Sphingolipids serve not only as components of cellular membranes but also as bioactive mediators of numerous cellular functions. As the biological activities of these lipids are dependent on their structures, and due to the limitations of conventional ion activation methods employed during tandem mass spectrometry (MS/MS), there is a recognized need for the development of improved structure-specific methods for their comprehensive identification and characterization. Here, positive-ionization mode 193 nm ultraviolet photodissociation (UVPD)-MS/MS has been implemented for the detailed structural characterization of lipid species from a range of sphingolipid classes introduced to the mass spectrometer via electrospray ionization as their lithiated or protonated adducts. These include sphingosine d18:1(4E), dihydrosphingosine (sphinganine) d18:0, sphingadiene d18:2(4E,11Z), the isomeric sphingolipids ceramide d18:1(4E)/18:0 and dihydroceramide d18:0/18:1(9Z), ceramide-1-phosphate d18:1(4Z)/16:0, sphingomyelin d18:1(4E)/18:1(9Z) the glycosphingolipids galactosyl ceramide d18:1(4E)/24:1(15Z) and lactosyl ceramide d18:1(4E)/24:0, and several endogenous lipids present within a porcine brain total lipid extract. In addition to the product ions formed by higher energy collision dissociation (HCD), UVPD is shown to yield a series of novel structurally diagnostic product ions resulting from cleavage of both sphingosine carbon-carbon and acyl chain carbon-carbon double bonds for direct localization of site(s) of unsaturation, as well as via diagnostic cleavages of the sphingosine backbone and N-C amide bond linkages. With activation timescales and dissociation efficiencies similar to those found in conventional MS/MS strategies, this approach is therefore a promising new tool in the arsenal of ion activation techniques toward providing complete structural elucidation in automated, high-throughput lipid analysis workflows. Graphical Abstract ᅟ.
鞘脂不仅作为细胞膜的组成部分，而且作为许多细胞功能的生物活性介质。由于这些脂质的生物学活性取决于它们的结构，并且由于在串联质谱（MS/MS）期间采用的传统离子活化方法的限制，因此需要开发改进的结构特异性方法来对它们进行全面鉴定和表征。在这里，采用正离子模式 193nm 紫外光解（UVPD）-MS/MS 对通过电喷雾电离引入质谱仪的一系列鞘脂类脂质物种进行详细的结构表征，这些脂质以其锂化或质子化加合物的形式存在。这些包括鞘氨醇 d18:1(4E)、二氢鞘氨醇（神经酰胺）d18:0、鞘二烯 d18:2(4E,11Z)、异构鞘脂类神经酰胺 d18:1(4E)/18:0 和二氢神经酰胺 d18:0/18:1(9Z)、神经酰胺-1-磷酸 d18:1(4Z)/16:0、鞘磷脂 d18:1(4E)/18:1(9Z)、糖鞘脂类半乳糖基神经酰胺 d18:1(4E)/24:1(15Z)和乳糖基神经酰胺 d18:1(4E)/24:0，以及来自猪脑总脂质提取物中的几种内源性脂质。除了由高能碰撞解离（HCD）形成的产物离子之外，UVPD 还显示出一系列新型结构诊断产物离子，这些产物离子是由鞘氨醇碳-碳和酰链碳-碳双键的裂解产生的，用于直接定位不饱和位点，以及通过鞘氨醇骨架和 N-C 酰胺键键合的诊断裂解。由于具有与在传统 MS/MS 策略中发现的相似的激活时间尺度和解离效率，因此该方法是离子激活技术的一种有前途的新工具，可在自动化、高通量脂质分析工作流程中提供完整的结构阐明。
