An Optimized Liquid Chromatography-Mass Spectrometry Method for Ganglioside Analysis in Cell Lines

Abstract

Gangliosides are glycosphingolipids composed of a sialylated glycan head group and a ceramide backbone. These anionic lipids form lipid rafts and play crucial roles in regulating various proteins involved in signal transduction, adhesion, and cell-cell recognition. Neuroblastoma, a pediatric cancer of the sympathetic nervous system, is treated with intensive chemotherapy, radiation, and an antibody targeting the GD2 ganglioside. Gangliosides are critical in neuroblastoma development and serve as therapeutic targets, making it essential to establish a reliable, rapid, and cost-effective method for profiling gangliosides, particularly one capable of isomeric separation of intact species. In this study, liquid chromatography-mass spectrometry (LC-MS) was optimized using standard gangliosides, followed by the optimization of sphingolipid extraction methods from cell lines by comparing Folch and absolute methanol extraction techniques. Percent recovery and the number of identified sphingolipids were used to evaluate the analytical merits of these methods. A standard gangliosides calibration curve demonstrated excellent linearity (R² = 0.9961-0.9975). The ZIC-HILIC column provided the best separation of ganglioside GD1 isomers with a 25 min runtime. GD1a elutes before GD1b on the ZIC-HILIC column. Absolute methanol yielded better percent recovery (96 ± 7) and identified 121 different sphingolipids, the highest number between the two extraction methods. The optimized method was applied to profile gangliosides in neuroblastoma (COG-N-683), pancreatic cancer (PSN1), breast cancer (MDA-MB-231BR), and brain tumor (CRL-1620) cell lines. The ganglioside profile of the neuroblastoma cell line COG-N-683 showed an inverse relationship between GD1 and GD2. Ceramide, Hex1Cer, GM1, and GM3 were highly abundant in CRL-1620, PSN1, and MDA-MB-231BR, respectively. These results suggest that our method provides a sensitive, reliable, and high-throughput workflow for ganglioside profiling across different cell types.

Keywords: LC-MS; ZIC-HILIC; gangliosides; neuroblastoma.

Figure 1

Experimental workflow for ganglioside extraction and analysis, including sample preparation, LC-MS, and data analysis. Cells were lysed using zirconium beads and sonicated on ice for 60 min. The protein concentration of each cell line was determined. Homogenates were then centrifuged, and the supernatant was collected for sphingolipid extraction. The extracted gangliosides were subsequently analyzed using LC-MS with negative mode detection.

Figure 2

EIC traces for GM3, GD2, GD1a, and GD1b, including their structural derivatives. The structures of each ganglioside are shown in the inset, with the designations of each abbreviated functional group provided as a legend within the inset.

Figure 3

The (A) 3:1 and (B) 2:3 mixtures of GD1a and GD1b standards with isomeric chromatographic separation. This provides verification for the or der of elution of GD1a and GD1b. (B) Side-by-side comparison of Folch and absolute methanol extraction methods. Monosaccharide symbol legends: glucose (blue circle), galactose (yellow circle), N-acetylgalactosamine (yellow square), and N-acetylneuraminic acid (magenta diamond).

Figure 4

(A) Extracted ion chromatogram for GD1a and GD1b isomers in biological sample at different m/z, (B) because of varying ceramide moiety in each GD1 structures, which shows the complexity of gangliosides. (C) MS traces corresponding to each GD1 ion chromatogram.

Figure 5

(A) Ganglioside biosynthesis pathway and (B) their MS traces in biological samples. Isomers are indicated by an asterisk (*) next to the name. The MS trace corresponding to each ganglioside in the pathway are highlighted in a similar colored textbox.

Figure 6

Pie charts of the average relative abundance of gangliosides in (A) PSN1, (B) CRL-1620, (C) MDA-MB-231BR, and (D) COG-N-683.

Figure 7

Boxplot of the relative abundance of all sphingolipids identified and quantified in cell lines from metastatic breast cancer (MDA-MB-231BR), neuroblastoma (COG-N-683), brain cancer cell line (CRL-1620), and pancreatic cancer cell line (PSN1). The total relative abundance for sphingolipids in each cell line was summed for each replicate (n = 3).