Structural Analysis of Unsaturated Glycosphingolipids Using Shotgun Ozone-Induced Dissociation Mass Spectrometry

Abstract

Glycosphingolipids are essential biomolecules widely distributed across biological kingdoms yet remain relatively underexplored owing to both compositional and structural complexity. While the glycan head group has been the subject of most studies, there is paucity of reports on the lipid moiety, particularly the location of unsaturation. In this paper, ozone-induced dissociation mass spectrometry (OzID-MS) implemented in a traveling wave-based quadrupole time-of-flight (Q-ToF) mass spectrometer was applied to study unsaturated glycosphingolipids using shotgun approach. Resulting high resolution mass spectra facilitated the unambiguous identification of diagnostic OzID product ions. Using [M+Na]+ adducts of authentic standards, we observed that the long chain base and fatty acyl unsaturation had distinct reactivity with ozone. The reactivity of unsaturation in the fatty acyl chain was about eight-fold higher than that in the long chain base, which enables their straightforward differentiation. Influence of the head group, fatty acyl hydroxylation and length of fatty acyl chain on the oxidative cleavage of double bonds was also observed. Application of this technique to bovine brain galactocerebrosides revealed co-isolated isobaric and regioisomeric species which otherwise would be incompletely identified using contemporary collision-induced dissociation (CID) alone. These results highlight the potential of OzID-MS in glycosphingolipids research, which not only provides complementary structural information to existing CID technique but also facilitates de novo structural determination of these complex biomolecules.

Reaction of ionised steryl esters with ozone in the gas phase

“…While ozonolysis of ionised cholestanyl oleate was dominated by oxidative cleavage of the fatty acyl moiety, cholesteryl stearate ions were found to undergo addition of one or three oxygens to form putative epoxides and lactones (and/or carboxylic acids), respectively. Interestingly, when compared to previous data, ( Barrientos et al, 2017;Ellis et al, 2012;Pham et al, 2013;Poad et al, 2010;Thomas et al, 2008Thomas et al, , 2007 there is some evidence that the ratio of [M+Na+O] + to [M+Na+O 3 ] + is higher for endocyclic double bonds than acyclic olefins, suggesting that this ratio could be used analytically as an indicator of unsaturated ring systems. Nevertheless, further investigation is required to characterise this effect as the data presented here indicate that this ratio can also be influenced by other variables including the identity of the cation (cf.…”

Online Ozonolysis Combined with Ion Mobility-Mass Spectrometry Provides a New Platform for Lipid Isomer Analyses

“…Interestingly, oxidative cleavage of the n -14 double bond in the sphingadiene SM d18:2/22:0 was not observed, which is consistent with previous reports of the sluggish reaction rate of the allylic alcohol motif in sphingolipids. 22,34 …”

An On‐Tissue Paternò–Büchi Reaction for Localization of Carbon–Carbon Double Bonds in Phospholipids and Glycolipids by Matrix‐Assisted Laser‐Desorption–Ionization Mass‐Spectrometry Imaging

“…In combination with electrospray ionization (ESI‐)MS, several techniques have recently emerged that exploit chemical derivatization of C=C bonds and subsequent generation of diagnostic fragments in a CID experiment. These include ozone‐induced dissociation (OzID) of selected ions within a collision cell, epoxidation reaction with acetone enabled by a cold plasma, and on‐line Paternò–Büchi (PB) photo‐derivatization . Very recently, a combination of MALDI‐MSI and CID/OzID, performed in the linear ion trap of a modified MALDI‐LTQ Orbitrap Elite mass spectrometer, was used to visualize the distributions of db‐ and sn ‐positional isomers of several phosphatidylcholines (PC) in mouse brain .…”