University of Münster, Institute of Inorganic and Analytical Chemistry, Corrensstraße 48, 48149, Münster, Germany.
Food Chemistry, Faculty of Mathematics and Natural Sciences, University of Wuppertal, Gaußstraße 20, 42119, Wuppertal, Germany.
Anal Chim Acta. 2024 Aug 15;1317:342913. doi: 10.1016/j.aca.2024.342913. Epub 2024 Jun 26.
Lipidomics studies require rapid separations with accurate and reliable quantification results to further elucidate the role of lipids in biological processes and their biological functions. Supercritical fluid chromatography (SFC), in particular, can provide this rapid and high-resolution separation. The combination with trapped ion mobility spectrometry (TIMS) has not yet been applied, although the post-ionization separation method in combination with liquid chromatography or imaging techniques has already proven itself in resolving isomeric and isobaric lipids and preventing false identifications. However, a multidimensional separation method should not only allow confident identification but also provide quantitative results to substantiate studies with absolute concentrations.
A SFC method was developed and the hyphenation of SFC and TIMS was further explored towards the separation of different isobaric overlaps. Furthermore, lipid identification was performed using mass spectrometry (MS) and parallel accumulation serial fragmentation (PASEF) MS/MS experiments in addition to retention time and collision cross section (CCS). Quantification was further investigated with short TIMS ramps and performed based on the ion mobility signal of lipids, since TIMS increases the sensitivity by noise filtering. The final method was, as an exemplary study, applied to investigate the function of different ceramide synthases (CerS) in the nematode and model organism Caenorhabditis elegans (C. elegans). Loss of three known CerS hyl-1, hyl-2 and lagr-1 demonstrated different influences on and alterations in the sphingolipidome.
This method describes for the first time the combination of SFC and TIMS-MS/MS, which enables a fast and sensitive quantification of lipids. The results of the application to C. elegans samples prove the functionality of the method and support research on the metabolism of sphingolipids in nematodes.
脂质组学研究需要快速分离，并获得准确可靠的定量结果，以进一步阐明脂质在生物过程中的作用及其生物学功能。超临界流体色谱（SFC）尤其可以提供这种快速和高分辨率的分离。尽管与液相色谱或成像技术相结合的后离子化分离方法已经在解决异构体和等质异位体的脂质以及防止错误鉴定方面得到了证明，但与捕集离子淌度谱（TIMS）相结合的方法尚未得到应用。然而，多维分离方法不仅应该允许进行有信心的鉴定，还应该提供定量结果，以证实具有绝对浓度的研究。
开发了一种 SFC 方法，并进一步探索了 SFC 和 TIMS 的串联，以分离不同的等质异位体重叠。此外，除了保留时间和碰撞截面（CCS）外，还使用质谱（MS）和平行累积串联碎裂（PASEF）MS/MS 实验进行脂质鉴定。定量进一步研究了短 TIMS 斜坡，并基于脂质的离子淌度信号进行，因为 TIMS 通过噪声滤波提高了灵敏度。最终方法作为一个示例研究，应用于研究不同神经酰胺合成酶（CerS）在线虫和模式生物秀丽隐杆线虫（C. elegans）中的功能。已知的三种 CerS hyl-1、hyl-2 和 lagr-1 的缺失对鞘脂组的影响和改变不同。
该方法首次描述了 SFC 和 TIMS-MS/MS 的组合，该组合能够快速灵敏地定量脂质。将该方法应用于 C. elegans 样品的结果证明了该方法的功能，并支持线虫鞘脂代谢的研究。
