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 8-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. Graphical Abstract ᅟ.

Keywords: Accurate mass measurement; Cerebrosides; Globosides; Glycosphingolipids; Ozone-induced dissociation.

Figure 1

High resolution OzID-MS spectrum of (a) GalCer d18:1/18:0(2OH), and (b) GlcCer d18:2/16:0(2OH) standards obtained in Synapt G2™ HDMS. Criegee and aldehyde product ions are depicted as (●) and (■), respectively. Open square (□) indicates ions generated through elimination of H₂C=O from the aldehyde ion.

Figure 2

Comparison of OzID-MS spectra of (a) LacCer d18:1/18:1(9Z) and (b) LacCer d18:1/18:0. Criegee and aldehyde product ions are depicted as (●) and (■), respectively. Open square (□) and circle (○) indicate ions generated through elimination of H₂C=O from the aldehyde ion and Criegee ion, respectively. Asterisk (*) indicates ions generated through secondary oxidation from [M+Na+O₃]⁺. Colors represent different double bond locations.

Figure 3

OzID-MS of (a) GalCer d18:1/18:1(9Z), (b) GalCer d18:1/24:1(15Z), and (c) Gb4Cer d18:1/24:1(15Z)(2OH). (Explanation of symbolic representations is described in Figs. 1 and 2).

Figure 4

Application of high resolution OzID-MS to major isomeric galactocerebroside species in bovine brain. (a) Full scan MS. (b) OzID-MS spectrum of GalCer d18:1/24:1, m/z 832.6644 with n-7/n-14, and n-9/n-14 double bonds (fatty acyl chain/long chain base). (c) OzID-MS of m/z 848.6597 GalCer d18:1/24:1(2OH), with n-7/n-14, and n-9/n-14 double bonds. (Explanation of symbolic representations is described in Figs. 1 and 2).

Figure 5

Application of high resolution OzID-MS to major isomeric and isobaric galactocerebroside species in bovine brain. (a) OzID-MS of m/z 834.6771 with n-7, n-9 (GalCer d18:1/23:1(2OH)), and n-14 double bonds (GalCer d18:1/24:0) (b). (c) OzID-MS spectrum of m/z 850.6727 with n-7, n-9 (GalCer d18:0/24:1(2OH)), and n-14 (GalCer d18:1/24:0(2OH)) double bonds (d). (Explanation of symbolic representations is described in Figs. 1 and 2).

Scheme I

Proposed reaction pathway for the OzID-MS of the [M+Na]⁺ of GalCer d18:1/18:0(2OH) (m/z 766.5814).

Scheme II

Proposed reaction pathway for the OzID-MS of the [M+Na]⁺ of GlcCer d18:2/16:0(2OH) (m/z 736.5330).