Chemistry and Biochemistry Department, Texas Tech University, Lubbock, TX 79409, USA.
Cancer Center, School of Medicine, Texas Tech University Health Sciences Center (TTUHSC), Lubbock, TX 79416, USA.
Cells. 2024 Oct 2;13(19):1640. doi: 10.3390/cells13191640.
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 (R = 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.
神经节苷脂是由唾液酸化糖脂头基团和神经酰胺骨架组成的糖脂。这些阴离子脂质形成脂筏，在调节参与信号转导、黏附和细胞-细胞识别的各种蛋白质方面发挥着关键作用。神经母细胞瘤是一种儿童期交感神经系统的癌症，采用强化化疗、放疗和针对 GD2 神经节苷脂的抗体进行治疗。神经节苷脂在神经母细胞瘤的发展中起着至关重要的作用，并作为治疗靶点，因此建立一种可靠、快速且具有成本效益的神经节苷脂分析方法至关重要，特别是一种能够对完整物种进行异构体分离的方法。在这项研究中，使用标准神经节苷脂对液相色谱-质谱（LC-MS）进行了优化，然后通过比较 Folch 和绝对甲醇提取技术，对细胞系中的神经节苷脂提取方法进行了优化。用回收率百分比和鉴定的神经节苷脂数量来评估这些方法的分析优点。标准神经节苷脂校准曲线表现出极好的线性（R = 0.9961-0.9975）。ZIC-HILIC 柱提供了 GD1 异构体的最佳分离效果，运行时间为 25 分钟。ZIC-HILIC 柱上 GD1a 在 GD1b 之前洗脱。绝对甲醇的回收率百分比更高（96 ± 7），鉴定出的神经节苷脂数量为 121 种，在两种提取方法中数量最多。优化后的方法应用于神经母细胞瘤（COG-N-683）、胰腺癌（PSN1）、乳腺癌（MDA-MB-231BR）和脑肿瘤（CRL-1620）细胞系中的神经节苷脂分析。神经母细胞瘤细胞系 COG-N-683 的神经节苷脂图谱显示 GD1 和 GD2 之间呈反比关系。神经酰胺、Hex1Cer、GM1 和 GM3 在 CRL-1620、PSN1 和 MDA-MB-231BR 中含量丰富。这些结果表明，我们的方法为不同细胞类型的神经节苷脂分析提供了一种敏感、可靠和高通量的工作流程。
