Ceramides Analysis
Creative Proteomics provides comprehensive ceramide analysis services tailored to meet diverse research needs. Utilizing advanced technologies like the Agilent 1290 Infinity II LC System and the Agilent 6470 Triple Quadrupole mass spectrometer, the platform ensures unparalleled sensitivity, precision, and resolution. Services include detailed ceramide profiling, structural characterization, pathway analysis, and biomarker discovery. With capabilities to analyze over 200 ceramide species across various biological matrices, the company supports robust lipidomic research and clinical applications with high reproducibility and actionable insights.
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- Define
- List of Ceramides
- Technology Platform
- Advantages
- Sample Requirements
- Applications
- Demo
- FAQs
- Case
- Publications
What are Ceramides?
Ceramides are a group of lipids made up of a fatty acid and sphingosine. Ceramides are component of sphingomyelin lipids, which is one component of lipid bilayer. Therefore, ceramides are highly abundant in cell membranes. In the past, it is assumed that the sole role of ceramides in the cell membrane is acting as the supporting structural elements. However, this assumption turns out to be false. It is shown that ceramides takes part in a series of physiological process in the body.
Ceramides are synthesized by de novo synthesis from serine and palmitoyl-CoA. Acting as precursors for many sphingolipids, ceramides play a crucial role in cell differentiation, cell signaling, proliferation and apoptosis. Catalyzed by the neutral Mg2+-dependent sphingomyelynase, spingomyelin in cell membranes hydrolyze and form ceramides. This is the major synthesis pathway of ceramides. Other minor pathways contributing to ceramide homeostasis include acylation of sphingosine with distinct fatty acyl-CoAs catalyzed by ceramidase and the hydrolysis of ceramide metabolites such as galactosylceramide and glycosylceramide.
What Do Ceramides Do?
Up till now, it's still unclear how the structure of individual ceramide species is related to their physiological functions. However, it is shown in some reports that specific fatty acids produced in response to some stimuli are components of ceramides. This might gives a clue to the relationship between the structure and function for different ceramide species. It is known that C18 ceramide suppress cell growth, while C16 and C24 ceramide species are associated with cell death. In one in vitro study, it is indicated that by inhibiting Akt, a serine protein kinase involved in insulin action, ceramides suppress glucose uptake. Besides, in insulin resistant animal models, the concentration of ceramides increases in muscle, liver and adipose tissue. The elevation of total ceramide concentrations in human muscle is associated with peripheral insulin resistance. Recently, in obese subjects with type 2 diabetes, it is demonstrated that increased plasma ceramides are related to reduced insulin sensitivity.
Ceramides Analysis Offered by Creative Proteomics
Quantitative Ceramides Profiling: High-sensitivity and high-resolution analysis of ceramides across diverse biological matrices. Absolute and relative quantification of individual ceramide species.
Ceramides Structural Characterization: Identification of ceramide backbone structures, fatty acid chain length, and degrees of saturation. Advanced characterization of isomers and related derivatives.
Pathway Analysis and Metabolic Flux: Comprehensive insights into ceramide metabolic pathways. Tracking metabolic intermediates and dynamic flux analysis.
Biomarker Discovery: Identification of ceramide-based biomarkers for health and disease research. Support for clinical and translational research applications.
List of Ceramides We Can Analyze
| Category | Subtype | Examples | Description |
|---|---|---|---|
| Ceramides (CER) | Ceramide NS | CER (C16:0), CER (C18:1), CER (C24:1) | Non-hydroxy sphingosine ceramides. Found in skin and tissues. |
| Ceramide NP | CER NP (C16:0), CER NP (C24:0) | Non-hydroxy phytosphingosine ceramides. Essential for skin. | |
| Ceramide AS | CER AS (C18:0), CER AS (C20:0) | α-hydroxy sphingosine ceramides. Skin barrier-related. | |
| Ceramide AP | CER AP (C24:1), CER AP (C26:0) | α-hydroxy phytosphingosine ceramides. Specialized roles in lipid rafts. | |
| Ceramide EOS | CER EOS (C24:0), CER EOS (C26:1) | ω-hydroxy ceramides esterified with fatty acids. Critical for skin. | |
| Glycosylated Ceramides | Glucosylceramides | GlcCER (C16:0), GlcCER (C24:1) | Ceramides with a glucose moiety. Biomarkers in metabolic disorders. |
| Lactosylceramides | LacCER (C16:0), LacCER (C24:0) | Ceramides with a lactose group. Key in cell signaling and inflammation. | |
| Gangliosides | GM3, GM1, GD3 | Ceramide-based glycosphingolipids with sialic acid residues. | |
| Phosphorylated Ceramides | Ceramide-1-Phosphate | CER-1P (C16:0), CER-1P (C24:1) | Phosphorylated ceramides involved in apoptosis and inflammation. |
| Ceramide-2-Phosphate | CER-2P (C18:1), CER-2P (C24:0) | Rare phosphorylated ceramides with specific cellular functions. | |
| Dihydroceramides | Dihydroceramides | dhCER (C16:0), dhCER (C24:1) | Reduced ceramides (no double bonds). Precursor to bioactive ceramides. |
| Other Derivatives | Ceramide Sulfates | CER-S (C18:1), CER-S (C24:0) | Sulfated ceramides found in the skin and central nervous system. |
| Hexosylceramides | HexCER (C16:0), HexCER (C24:0) | Hexose-modified ceramides. Important for glycosphingolipid metabolism. |
Technology Platforms for Ceramides Analysis
To ensure the highest quality of data and insights, Creative Proteomics employs the Agilent 1290 Infinity II LC System coupled with the Agilent 6470 Triple Quadrupole mass spectrometer.
Features of Benefits of the Agilent 6470 Triple Quadrupole:
- Unmatched Resolution: Delivers exceptional chromatographic resolution for separating complex ceramide mixtures.
- Flexibility: Compatible with a wide range of columns and solvents for versatile applications.
- High Throughput: Fast analysis without compromising data quality.
Benefits of the Agilent 6470 Triple Quadrupole:
- High Sensitivity: Achieves femtomole-level detection limits, ideal for low-abundance ceramides.
- Exceptional Quantification: Provides accurate and reproducible quantitative analysis across a broad dynamic range.
- Advanced Multiple Reaction Monitoring (MRM): Enables targeted detection of specific ceramide species with high specificity.
Agilent 1290 Infinity II LC System coupled with Agilent 6470 Triple Quadrupole (Figure from Agilent)
Advantages of Ceramides Assay
- Unparalleled Sensitivity and Precision: Using the Agilent 1290 Infinity II LC System coupled with Agilent 6470 Triple Quadrupole, our ceramide assay achieves detection limits as low as 1 femtomole, ensuring the quantification of even trace-level ceramides.
- Comprehensive Coverage: Capable of analyzing over 200 ceramide species, including ceramides (CER), glycosylated ceramides, dihydroceramides, and phosphorylated ceramides. This enables the most extensive lipidomic profiling available in the market.
- High Reproducibility: Analytical precision is ensured with a coefficient of variation (CV) below 5% for repeated measurements, critical for robust biomarker discovery and clinical studies.
- Dynamic Quantification Range: Our assays provide a dynamic range of 5 orders of magnitude, allowing accurate quantification of ceramides in diverse biological matrices, from plasma to tissues.
- Advanced Data Analysis: Deliverables include detailed statistical data, with correlation coefficients (R²) exceeding 0.99 for calibration curves. Advanced bioinformatic tools are utilized for pathway mapping and cluster analysis, ensuring actionable insights.
Sample Requirements for Ceramides Analysis
| Sample Type | Minimum Sample Amount | Notes |
|---|---|---|
| Plasma/Serum | 100 µL | Use EDTA- or heparin-treated tubes; avoid hemolysis. |
| Tissue Samples | ≥ 20 mg | Snap-frozen in liquid nitrogen; store at -80°C. |
| Cell Pellets | ≥ 5 × 10⁶ cells | Washed with PBS and flash-frozen; store at -80°C. |
| Urine | 200 µL | Centrifuge to remove debris; store at -80°C. |
| CSF (Cerebrospinal Fluid) | 50 µL | Collect in sterile tubes; store at -80°C. |
| Purified Lipids | ≥ 100 µg | Solubilized in chloroform or methanol. |
| Milk | 500 µL | Centrifuge and collect lipid-rich layer; store at -80°C. |
Applications of Ceramides Assay Service
Disease Biomarker Discovery
- Metabolic Disorders: Quantify ceramides such as Ceramide NS (C16:0) and Glucosylceramides to identify biomarkers linked to insulin resistance, obesity, and diabetes.
- Neurodegenerative Diseases: Assess altered ceramide levels in cerebrospinal fluid (e.g., Ceramide NP) associated with Alzheimer's and Parkinson's diseases.
- Cardiovascular Research: Detect ceramide ratios (e.g., CER C24:1/CER C16:0) predictive of cardiovascular risk.
Dermatological Research
- Skin Barrier Function: Measure ceramides (e.g., Ceramide EOS, Ceramide AS) critical for stratum corneum integrity in studies on atopic dermatitis, psoriasis, and aging skin.
- Cosmetic Development: Evaluate ceramide content in skincare formulations to ensure efficacy in hydration and barrier repair.
Drug Development and Toxicology
- Therapeutic Targets: Screen ceramide-modulating drugs, such as inhibitors of ceramide synthase, for efficacy in metabolic and inflammatory diseases.
- Toxicological Studies: Assess ceramide dysregulation as a marker of cell apoptosis in response to pharmaceutical agents.
Nutritional Research
Dietary Lipid Studies: Analyze ceramide changes induced by diets rich in sphingolipids (e.g., milk, soy) to understand their impact on metabolism and health.
Environmental and Plant Research
- Plant Stress Responses: Study ceramides in plant extracts (e.g., Ceramide AS and hexosylceramides) to understand their role in drought and pathogen resistance.
- Ecotoxicology: Monitor ceramide metabolism disruptions in organisms exposed to environmental toxins.
Demo
MS/MS spectra of synthetic CERs using an ion trap (IT) system. (van Smeden, et al., Journal of lipid research, 2011).
FAQ of Ceramides Analysis
What is the impact of hemolysis or contamination on ceramide analysis?
Hemolysis or contamination can interfere with ceramide profiling by introducing unwanted lipids or altering lipid composition. We recommend using non-hemolyzed samples and clean collection methods. For plasma or serum, gentle handling during collection and centrifugation can help avoid hemolysis.
How does the sample's lipid content affect analysis?
Samples with extremely high or low lipid content may require adjusted extraction methods. For example, lipid-rich samples might need additional cleanup steps to prevent ion suppression during mass spectrometry. Low-lipid samples can still be analyzed using enhanced sensitivity settings.
Can your analysis differentiate between ceramide isomers?
Yes, we use high-resolution mass spectrometry and specialized chromatographic techniques to separate and quantify ceramide isomers. This allows us to provide detailed information on the structural diversity of ceramide species in your samples.
Can I combine ceramide analysis with other lipidomics studies?
Yes, ceramide profiling can be integrated into a broader lipidomics workflow to study sphingolipids, glycosphingolipids, or other lipid classes. This provides a comprehensive understanding of lipid metabolism and interactions within your samples.
How do you handle variability between biological replicates?
We minimize variability by standardizing sample preparation and extraction methods. For biological replicates, we recommend submitting samples from consistent sources and preparation conditions. Statistical tools are used during data analysis to account for biological and technical variation.
Learn about other Q&A.
Ceramides Analysis Case Study
Publications
Here are some publications in proteomics research from our clients:
- Nutritional analysis of commercially available, complete plant-and meat-based dry dog foods in the UK. 2024. https://doi.org/10.1101/2024.09.11.612409
- Mechanisms underlying neonate-specific metabolic effects of volatile anesthetics. 2021. https://doi.org/10.7554/eLife.65400
- Lipin-1 regulates lipid catabolism in pro-resolving macrophages. 2020. https://doi.org/10.1101/2020.06.03.121293
- Pan-lysyl oxidase inhibition disrupts fibroinflammatory tumor stroma, rendering cholangiocarcinoma susceptible to chemotherapy. 2024. https://doi.org/10.1097/HC9.0000000000000502
- Polyamine metabolism impacts T cell dysfunction in the oral mucosa of people living with HIV. 2023. https://doi.org/10.1038/s41467-023-36163-2
