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173 Ashley Avenue Building CRI Room 505c Charleston, SC, 29425 United StatesMUSC Lipidomics Shared Resource The Lipidomics Shared Resource at MUSC represents expertise specializing in sphingolipid biology, providing services to the Medical University of South Carolina and institutions or industries throughout the world. The Lipidomics Shared Resource is composed of analytical and synthetic units providing expertise, synthetic compounds/standards, and analytical methodology (LC-MS/MS and SFC-MS/MS) to enhance an understanding of the role of bioactive lipids in disease pathology. Overview: The Lipidomics Shared Resource builds on the unique expertise of key personnel in sphingolipid chemistry, analysis, and biology providing intellectual and physical resources to enhance the understanding of bioactive sphingolipids and lipids in signal transduction and cell regulation. Sphingolipids are recognized as key components regulating fundamental cell biology processes regulating transmembrane signaling. Diversity of bioactive sphingolipids and their interconnected metabolism provide a network of pathways. Key sphingolipid metabolites are: ceramides (Cer), sphingosine (Sph), sphingosine 1-phosphate (S1P), and ceramide 1-phosphate (Cer1P). Services & Expertise: The Lipidomics Shared Resource is designed to support research in lipidomics by providing advanced methodology and expertise for both synthesis and analysis of bioactive lipids in a cost-effective manner, with a focus on several key areas: - Providing qualitative and quantitative analysis of >300 sphingolipids using high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) technologies. - Providing analytical method development of new lipid components. - Providing and developing synthetic lipid tools/standards, including functionalized and fluorescent ceramides, 17C-sphingolipids; or synthetic lipid analogs, including organelle-targeted sphingolipids. - Mentoring investigators in the understanding of lipid biology through lipid chemistry, experimental design, selection of lipids of interest, and interpretation of the analytical data. Services: The Analytical Unit provides sample preparation/lipid extraction, data normalization measurements (levels of inorganic phosphate/sample), and lipid analysis based on High-Performance Liquid Chromatography-tandem Mass Spectrometry (HPLC-MS/MS) or Supercritical Fluid Chromatography-tandem Mass Spectrometry (SFC-MS/MS) approaches. The core provides both qualitative (compound identification) and quantitative mass spectral analysis of key lipids from various biological materials (cells, tissues, serum, blood, etc) with assays to measure over 300 different sphingolipid, DAG components, and Free Fatty Acids at a basic metabolic level. Analytical Units: 1. Sph/S1P/Cer Sphingoid bases (Sphingosine [Sph]& Dihydro-Sphingosine [dhSph]) Sphingoid base-1- Phosphates (S1P & dhS1P), Ceramide molecular species (Cn-Cer) This is the most basic analysis, covering 18 different molecular species 2. dhCer Dihydro-Ceramide molecular species 3. Hexosyl-Cer Hexosylceramide (Glucosyl/Galactosyl-Ceramide) molecular species 4. Lactosyl-Cer Lactosyl-Ceramide molecular species 5. SM Sphingomyelin molecular species 6. DAG Diacyl-Glycerol molecular species 7. α-OH-Cers (2’OH-Cer) Alpha-Hydroxy-Ceramide molecular species 8. Cer-1P Ceramide 1-Phosphate molecular species 9. PhytoSph/PhytoCer Phytosphinghosine, Phytosphingosine-1-Phosphate and Phytoceramide molecular species 10. α-OH-PhytoCer Alpha-Hydroxy-Phytoceramide molecular species 11. 17CSph/S1P/Cer 17C-Sphingoid bases, 17C-Sphingoid base-1- Phosphates and 17C-Ceramide molecular species 12. dh17CSph/S1P/Cer 17C-Dihydro-Sphingoid bases, 17C-Dihydro-Sphingoid base-1- Phosphates and 17C-Dihydro-Ceramide molecular species 13. Glu/Gal-Cer by SFC Glucosylceramide and Galactosylceramide species separated by SFC-MS/MS 14. 1-Deoxy-Sph/Cer 1-Deoxy-Sphingoid bases, 1-Deoxy-Ceramide molecular species 15. 1-Deoxy-dhCer 1-Deoxy-dihydro-Ceramide molecular species 16. Free Fatty Acids FFA molecular species C12- to C26- saturated and monounsaturated species 17. Special Request available upon consultation Analytical Approach: Quantitative analyses of sphingolipids, are based on eight-point calibration curves generated for each target analyte. The synthetic standards along with a set of internal standards are spiked into an artificial matrix, they are then subjected to an identical extraction procedure as the biological samples. These extracted standards are then analyzed by the HPLC/MS/MS or SFC/MS/MS system operating in positive multiple reaction-monitoring (MRM) mode employing a gradient elution. Plotting the analyte/internal standard peak area ratios against analyte concentrations generates the analyte specific calibration curves. Any lipid with no authentic standards is quantitated using the calibration curve of its closest counterpart. Publication Acknowledgement: “Supported in part by the Lipidomics Shared Resource, Hollings Cancer Center, Medical University of South Carolina (P30 CA138313 and P30 GM103339)”.
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