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. 2021 Feb 22;12(1):1201.
doi: 10.1038/s41467-021-21480-1.

Unravelling the structural complexity of glycolipids with cryogenic infrared spectroscopy

Carla Kirschbaum  1   2 , Kim Greis  1   2 , Eike Mucha  2 , Lisa Kain  3 , Shenglou Deng  4 , Andreas Zappe  1 , Sandy Gewinner  2 , Wieland Schöllkopf  2 , Gert von Helden  2 , Gerard Meijer  2 , Paul B Savage  4 , Mateusz Marianski  5   6 , Luc Teyton  3 , Kevin Pagel  7   8
Affiliations

Unravelling the structural complexity of glycolipids with cryogenic infrared spectroscopy

Carla Kirschbaum et al. Nat Commun. .

Abstract

Glycolipids are complex glycoconjugates composed of a glycan headgroup and a lipid moiety. Their modular biosynthesis creates a vast amount of diverse and often isomeric structures, which fulfill highly specific biological functions. To date, no gold-standard analytical technique can provide a comprehensive structural elucidation of complex glycolipids, and insufficient tools for isomer distinction can lead to wrong assignments. Herein we use cryogenic gas-phase infrared spectroscopy to systematically investigate different kinds of isomerism in immunologically relevant glycolipids. We show that all structural features, including isomeric glycan headgroups, anomeric configurations and different lipid moieties, can be unambiguously resolved by diagnostic spectroscopic fingerprints in a narrow spectral range. The results allow for the characterization of isomeric glycolipid mixtures and biological applications.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Structures and IR spectra of α-GalCer and β-GalCer (d18:1/24:1(15Z)).
a The stereoisomers α-GalCer and β-GalCer differ by the configuration of the glycosidic bond. b Sodium adducts of α-GalCer and β-GalCer yield unique fingerprints in the 1000–1150 cm−1 region. c The assignment of vibrational bands is based on computed vibrational spectra of the lowest-energy conformer of [α-GalCer+Na]+ with the lipid chains trimmed to four heavy atoms. The anharmonic spectrum (orange) provides a clear match with the experimental fingerprint. Source data are provided as a Source Data file.
Fig. 2
Fig. 2. Spectroscopic fingerprints of protonated isomeric Gal- and Glc sphingosines.
The highly resolved absorption patterns of each permutation are diagnostic for both the monosaccharide and the configuration of the glycosidic bond. Source data are provided as a Source Data file.
Fig. 3
Fig. 3. IR spectra of isomeric Gb3-sphingosine and iGb3-sphingosine.
The regioisomeric trisaccharides yield distinguishable and well-resolved absorption patterns in the fingerprint region. Source data are provided as a Source Data file.
Fig. 4
Fig. 4. Influence of different lipid moieties on the IR spectra of α-Gal lipids.
The exchange of sphingosine for phytosphingosine leads to subtle differences in the spectral fingerprint, whereas ceramide and diacylglycerol considerably alter the fingerprint region. The amide- and ester groups yield additional bands beyond 1450 cm−1. Source data are provided as a Source Data file.
Fig. 5
Fig. 5. IR spectra of sodiated glycosylceramides (m/z 832.7) from biological lipid extracts and from synthetic α-Glc/GalCer and β-Glc/GalCer (d18:1/24:1(15Z)) standards.
The five main absorption bands found in both spectra of biological glycolipids are equally present in the spectrum of β-GlcCer. In addition, α-GlcCer contributes to the spectrum of Folch extract 2 isolated from spleen of GAA knockout mice. The isomer contributions obtained from spectral deconvoluted by NMF are depicted in the left panel. Source data are provided as a Source Data file.
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