An Optimized Liquid Chromatography–Mass Spectrometry Method for Ganglioside Analysis in Cell Lines
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...
Saved in:
| Main Authors: | , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2024-10-01
|
| Series: | Cells |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2073-4409/13/19/1640 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1832577703840055296 |
|---|---|
| author | Akeem Sanni Andrew I. Bennett Yifan Huang Isabella Gidi Moyinoluwa Adeniyi Judith Nwaiwu Min H. Kang Michelle E. Keyel ChongFeng Gao C. Patrick Reynolds Brian Haab Yehia Mechref |
| author_facet | Akeem Sanni Andrew I. Bennett Yifan Huang Isabella Gidi Moyinoluwa Adeniyi Judith Nwaiwu Min H. Kang Michelle E. Keyel ChongFeng Gao C. Patrick Reynolds Brian Haab Yehia Mechref |
| author_sort | Akeem Sanni |
| collection | DOAJ |
| description | 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<sup>2</sup> = 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. |
| format | Article |
| id | doaj-art-dcbfcea98922479394a4e1d678775c76 |
| institution | Kabale University |
| issn | 2073-4409 |
| language | English |
| publishDate | 2024-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cells |
| spelling | doaj-art-dcbfcea98922479394a4e1d678775c762025-01-30T17:45:36ZengMDPI AGCells2073-44092024-10-011319164010.3390/cells13191640An Optimized Liquid Chromatography–Mass Spectrometry Method for Ganglioside Analysis in Cell LinesAkeem Sanni0Andrew I. Bennett1Yifan Huang2Isabella Gidi3Moyinoluwa Adeniyi4Judith Nwaiwu5Min H. Kang6Michelle E. Keyel7ChongFeng Gao8C. Patrick Reynolds9Brian Haab10Yehia Mechref11Chemistry and Biochemistry Department, Texas Tech University, Lubbock, TX 79409, USAChemistry and Biochemistry Department, Texas Tech University, Lubbock, TX 79409, USAChemistry and Biochemistry Department, Texas Tech University, Lubbock, TX 79409, USAChemistry and Biochemistry Department, Texas Tech University, Lubbock, TX 79409, USAChemistry and Biochemistry Department, Texas Tech University, Lubbock, TX 79409, USAChemistry and Biochemistry Department, Texas Tech University, Lubbock, TX 79409, USACancer Center, School of Medicine, Texas Tech University Health Sciences Center (TTUHSC), Lubbock, TX 79416, USACancer Center, School of Medicine, Texas Tech University Health Sciences Center (TTUHSC), Lubbock, TX 79416, USAVan Andel Institute, Grand Rapids, MI 49503, USACancer Center, School of Medicine, Texas Tech University Health Sciences Center (TTUHSC), Lubbock, TX 79416, USAVan Andel Institute, Grand Rapids, MI 49503, USAChemistry and Biochemistry Department, Texas Tech University, Lubbock, TX 79409, USAGangliosides 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<sup>2</sup> = 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.https://www.mdpi.com/2073-4409/13/19/1640gangliosidesZIC-HILICLC-MSneuroblastoma |
| spellingShingle | Akeem Sanni Andrew I. Bennett Yifan Huang Isabella Gidi Moyinoluwa Adeniyi Judith Nwaiwu Min H. Kang Michelle E. Keyel ChongFeng Gao C. Patrick Reynolds Brian Haab Yehia Mechref An Optimized Liquid Chromatography–Mass Spectrometry Method for Ganglioside Analysis in Cell Lines Cells gangliosides ZIC-HILIC LC-MS neuroblastoma |
| title | An Optimized Liquid Chromatography–Mass Spectrometry Method for Ganglioside Analysis in Cell Lines |
| title_full | An Optimized Liquid Chromatography–Mass Spectrometry Method for Ganglioside Analysis in Cell Lines |
| title_fullStr | An Optimized Liquid Chromatography–Mass Spectrometry Method for Ganglioside Analysis in Cell Lines |
| title_full_unstemmed | An Optimized Liquid Chromatography–Mass Spectrometry Method for Ganglioside Analysis in Cell Lines |
| title_short | An Optimized Liquid Chromatography–Mass Spectrometry Method for Ganglioside Analysis in Cell Lines |
| title_sort | optimized liquid chromatography mass spectrometry method for ganglioside analysis in cell lines |
| topic | gangliosides ZIC-HILIC LC-MS neuroblastoma |
| url | https://www.mdpi.com/2073-4409/13/19/1640 |
| work_keys_str_mv | AT akeemsanni anoptimizedliquidchromatographymassspectrometrymethodforgangliosideanalysisincelllines AT andrewibennett anoptimizedliquidchromatographymassspectrometrymethodforgangliosideanalysisincelllines AT yifanhuang anoptimizedliquidchromatographymassspectrometrymethodforgangliosideanalysisincelllines AT isabellagidi anoptimizedliquidchromatographymassspectrometrymethodforgangliosideanalysisincelllines AT moyinoluwaadeniyi anoptimizedliquidchromatographymassspectrometrymethodforgangliosideanalysisincelllines AT judithnwaiwu anoptimizedliquidchromatographymassspectrometrymethodforgangliosideanalysisincelllines AT minhkang anoptimizedliquidchromatographymassspectrometrymethodforgangliosideanalysisincelllines AT michelleekeyel anoptimizedliquidchromatographymassspectrometrymethodforgangliosideanalysisincelllines AT chongfenggao anoptimizedliquidchromatographymassspectrometrymethodforgangliosideanalysisincelllines AT cpatrickreynolds anoptimizedliquidchromatographymassspectrometrymethodforgangliosideanalysisincelllines AT brianhaab anoptimizedliquidchromatographymassspectrometrymethodforgangliosideanalysisincelllines AT yehiamechref anoptimizedliquidchromatographymassspectrometrymethodforgangliosideanalysisincelllines AT akeemsanni optimizedliquidchromatographymassspectrometrymethodforgangliosideanalysisincelllines AT andrewibennett optimizedliquidchromatographymassspectrometrymethodforgangliosideanalysisincelllines AT yifanhuang optimizedliquidchromatographymassspectrometrymethodforgangliosideanalysisincelllines AT isabellagidi optimizedliquidchromatographymassspectrometrymethodforgangliosideanalysisincelllines AT moyinoluwaadeniyi optimizedliquidchromatographymassspectrometrymethodforgangliosideanalysisincelllines AT judithnwaiwu optimizedliquidchromatographymassspectrometrymethodforgangliosideanalysisincelllines AT minhkang optimizedliquidchromatographymassspectrometrymethodforgangliosideanalysisincelllines AT michelleekeyel optimizedliquidchromatographymassspectrometrymethodforgangliosideanalysisincelllines AT chongfenggao optimizedliquidchromatographymassspectrometrymethodforgangliosideanalysisincelllines AT cpatrickreynolds optimizedliquidchromatographymassspectrometrymethodforgangliosideanalysisincelllines AT brianhaab optimizedliquidchromatographymassspectrometrymethodforgangliosideanalysisincelllines AT yehiamechref optimizedliquidchromatographymassspectrometrymethodforgangliosideanalysisincelllines |