In-Depth Structural Characterization and Quantification of Cerebrosides and Glycosphingosines with Gas-Phase Ion Chemistry

Abstract

Cerebrosides (n-HexCer) and glycosphingosines (n-HexSph) constitute two sphingolipid subclasses. Both are comprised of a monosaccharide headgroup (glucose or galactose in mammalian cells) linked via either an α- or β-glycosidic linkage to the sphingoid backbone (n = α or β, depending upon the nature of the linkage to the anomeric carbon of the sugar). Cerebrosides have an additional amide-bonded fatty acyl chain linked to the sphingoid backbone. While differentiating the multiple isomers (i.e. glucose vs galactose, α- vs β-linkage) is difficult, it is crucial for understanding their specific biological roles in health and disease states. Shotgun tandem mass spectrometry has been a powerful tool in both lipidomics and glycomics analysis but is often limited in its ability to distinguish isomeric species. This work describes a new strategy combining shotgun tandem mass spectrometry with gas-phase ion chemistry to achieve both differentiation and quantification of isomeric cerebrosides and glycosphingosines. Briefly, deprotonated cerebrosides, [n-HexCer-H]^-, or glycosphingosines, [n-HexSph-H]^-, are reacted with terpyridine (Terpy) magnesium complex dications, [Mg(Terpy)₂]²⁺, in the gas phase to produce a charge-inverted complex cation, [n-HexCer-H+MgTerpy]⁺ or [n-HexSph-H+MgTerpy]⁺. The collision-induced dissociation (CID) of the charge-inverted complex cations leads to significant spectral differences between the two groups of isomers, α-GalCer, β-GlcCer, and β-GalCer for cerebrosides and α-GlcSph, α-GalSph, β-GlcSph, and β-GalSph for glycosphingosines, which allows for isomer distinction. Moreover, we describe a quantification strategy with the normalized percent area extracted from selected diagnostic ions that quantify either three isomeric cerebroside or four isomeric glycosphingosine mixtures. The analytical performance was also evaluated in terms of accuracy, repeatability, and interday precision. Furthermore, CID of the product ions resulting from 443 Da loss from the charge-inverted complex cations ([n-HexCer-H+MgTerpy]⁺) has been performed and demonstrated for localization of the double-bond position on the amide-bonded monounsaturated fatty acyl chain in the cerebroside structure. The proposed strategy was successfully applied to the analysis of total cerebroside extracts from the porcine brain, providing in-depth structural information on cerebrosides from a biological mixture.

Figure 1.

The general structures of glycosphingosines and cerebrosides with the possible isomeric positions within the structure.

Figure 2.

The comparison of the CID spectra among cerebrosides after gas-phase ion/ion reaction. (a) The post-ion/ion reaction spectrum of cerebroside anion with [Mg(Terpy)₂]²⁺ cation. (b) The CID spectrum of the [α-GalCer–H+MgTerpy]⁺ (m/z 955.6). (c) The CID spectrum of the [β-GlcCer–H+MgTerpy]⁺ (m/z 955.6). (d) The CID spectrum of the [β-GalCer–H+MgTerpy]⁺ (m/z 955.6). The values inside the parenthesis indicate the neutral loss. The lightning bolt (🗲) signifies the collisionally activated precursor ion. The solid circle (●) indicates the mass selection in the negative ion mode analysis and the black and white squares (■/□) indicate the positive ion mode analysis with and without mass selection, respectively.

Figure 3.

The dissociation kinetic plot of isomeric charge-inverted galactosylceramide complex cations. Error bars are express with standard deviation (n=3). The p-value between the two slope is < 0.01 indicating the significantly different rate constant between the two complex cations. (The procedure for the dissociation rate measurement is provided in Supporting Information.)

Figure 4.

The comparison of the CID spectra among glycosphingosines after gas-phase ion/ion reaction. (a) The CID spectrum of the [α-GlcSph–H + MgTerpy]⁺ (m/z 717.4). (b) The CID spectrum of the [α-GalSph–H + MgTerpy]⁺ (m/z 717.4). (c) The CID spectrum of the [β-GlcSph–H + MgTerpy]⁺ (m/z 717.4). (d) The CID spectrum of the [β-GaSph–H + MgTerpy]⁺ (m/z 717.4). The values inside the parenthesis indicate the neutral loss. The symbols represent as same as those in Figure 2.

Figure 5.

The identification of double bond position from the monounsaturated fatty acyl side chain on cerebrosides. (a) The CID spectrum of 443 Da loss ion from [β-GlcCer(d18:1/18:0)−H + MgTerpy]⁺. (b) The CID spectrum of 443 Da loss ion from [β-GlcCer(d18:1/18:1 (n-9))−H + MgTerpy]⁺. The inserts are the zoom-in spectra of m/z region ranged from 350 to 500. The red dashed line signifies the special spectral gap pointing the double bond position. The symbols represent as same as those in Figure 2.