Abstract
Ceramides are a lipid subclass of the sphingolipids that show large structural diversity. Structural characterization of the ceramides (CERs) can lead to better understanding of their role and function in the biological system. Here we investigated representatives of NP (CER III, CER IIIB) and AP ceramide classes (CER VI) that contain phytosphingosine (P) backbone. Ceramides were characterized in positive ionization mode by hydrogen-deuterium exchange mass spectrometry (HDX-MS). Fragmentation in positive ionization mode of the CER III and CER VI resulted in abundant ions assigned to phytosphingosine moiety at m/z 282, 300 and 318. HDX-MS of fragments showed increase in m/z of corresponding ions confirming the exchange of deuterium. In negative ionisation spectra multiple fragment ions were assigned to fatty acyl (RCOO–) moiety. Presence of RCOO– allowed unambiguous identification of CER III and CER IIIB which were distinguished by the presence of double bond on fatty acyl chain.
Highlights
S PHINGOLIPIDS represent an important group of molecules involved in key metabolic and structural functions in organism.[1]
We have focused on structural analysis of synthetic CER III, CER IIIB, CER VI and P to get better insight into the CER structure in this work
Similar fragmentation pattern was showed for CER VI-Hydrogen-deuterium exchange (HDX) disolved in EtOH-d6 (Figure 4)
Summary
S PHINGOLIPIDS represent an important group of molecules involved in key metabolic and structural functions in organism.[1]. CER present in SC were analyzed by thin layer chromatography (TLC),[10,11,12] traditionally; TLC does not provide structural information of CERs which is neccessary in some studies. Capability of MS to provide structural information of CER is considered as advantage over other techniques since CERs might be detected as positively or negatively charged species.[15] CERs are most commonly analyzed by ESI and atmospheric pressure chemical ionisation (APCI), eventhough shotgun and matrix assisted laser desorption/ionisation (MALDI) techniques are reported.[16] Scherer et al used hydrophilic interaction chromatography-MS (HILIC-MS) for direct profiling of hexosylceramide, lactosylceramide, sphingosine, sphinganine, phytoSPH, di- and trimethyl-SPH, sphingosylphosphorylcholine, ceramide-1-phosphate, and dihydroceramide-1-phosphate and achieved base separation of all sphingolipid classes within 2 min.[17] Using reversed phase liquid chromatography (RP-LC) t'Kindt et al.[4] established analytical
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