Profiling Intact Glycosphingolipids with Automated Structural Annotation and Quantitation from Human Samples with Nanoflow Liquid Chromatography Mass Spectrometry

Abstract

Sphingolipids are an essential subset of bioactive lipids found in most eukaryotic cells that contribute to membrane biophysical properties and are involved in cellular differentiation, recognition, and mediating interactions. The described nanoHPLC-ESI-Q/ToF methodology utilizes known biosynthetic pathways, accurate mass detection, optimized collision-induced disassociation, and a robust nanoflow chromatographic separation for the analysis of intact sphingolipids found in human tissue, cells, and serum. The methodology was developed and validated with an emphasis on addressing the common issues experienced in profiling these amphipathic lipids, which are part of the glycocalyx and lipidome. The high sensitivity obtained using nanorange flow rates with robust chromatographic reproducibility over a wide range of concentrations and injection volumes results in confident identifications for profiling these low-abundant biomolecules.

Figure 1

Molecular structure of GM1_a and summary of the structural diversity of human sphingolipids. The head groups are drawn in blue, and the sphingoid base is in black.

Figure 2

Example chromatogram of human brain tissue sphingolipid profile annotating 22 of 118 compounds found. Inset structures were assigned based on the methods described.

Figure 3

Heatmap summarizing the relative intensities of sphingolipids (≥0.01% relative abundance) in human brain tissue. The major products correspond to GM1, GD1, and SM.

Figure 4

Typical fragmentations generated by CID MS/MS for compounds with various GSL headgroups including (A) Glucose-, (B) SHex-, (C) OAc-GT1_b-, and (D) Fuc-GM0-Cer. Dominant dissociation products correspond to cleavages of glycan linkages.

Figure 5

(A) Elution profile of representative LCBs. The MS/MS spectra of GM1-LCB/C18 with (B) d18:2, (C) t18:0, and (D) d18:0.

Figure 6

Heatmap summarizing observed sphingolipids (≥0.01% relative abundances) in human serum.