Metabolomics Service for Glutamine Metabolism Analysis
Creative Proteomics employs advanced metabolomic techniques to analyze alterations in glutamine and its metabolic pathways, providing a scientific foundation for the exploration of disease mechanisms and drug target identification strategies.
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- Background
- Service Offering
- Advantage
- Analytical Method
- Sample Requirements
- Case Study
- FAQ
- Publications
Glutamine represents the most abundant free amino acid within mammalian systems, playing a pivotal role in cellular energy metabolism, nitrogen transport, nucleotide biosynthesis, and the modulation of oxidative stress. Recent investigations have demonstrated a significant association between the reprogramming of glutamine metabolism and various pathological conditions, including tumor proliferation, immune modulation, neurodegenerative disorders, and metabolic syndromes.
Metabolomics has emerged as a robust tool for in-depth exploration of glutamine metabolism, enabling a comprehensive analysis of metabolites within biological systems to elucidate the dynamic changes and regulatory mechanisms associated with glutamine metabolism. This approach allows for precise detection of glutamine and its metabolic derivatives, thereby offering a detailed depiction of its metabolic landscape.
The Significance of Glutamine Analysis
The analysis of glutamine holds significant importance within the realm of scientific research. In the study of disease mechanisms, aberrations in glutamine metabolism are closely linked to the pathogenesis and progression of various diseases. A comprehensive analysis of glutamine metabolism can uncover underlying mechanisms of disease development, providing critical insights for early diagnosis and therapeutic intervention. For instance, in certain neoplastic conditions, tumor cells exhibit increased uptake and metabolism of glutamine. Investigating alterations in glutamine metabolism may elucidate the mechanisms underlying the growth and proliferation of tumor cells, thereby laying a foundation for the development of targeted anticancer therapeutics.
In the pharmaceutical research domain, glutamine analysis serves as a crucial parameter for assessing the efficacy and safety of drugs. Monitoring changes in glutamine metabolism under pharmacological influence enables the determination of whether a drug achieves its intended effect and whether it provokes adverse reactions. This information is vital for optimizing the drug development process and enhancing the success rate of novel therapeutics. Furthermore, glutamine analysis plays an integral role in fields such as nutritional science and physiology, providing robust support for the exploration of life's mysteries.
Schematic summary of intracellular glutamine metabolism. (Zohreh Mirveis et al., 2023)
Service Offerings
Precision Qualitative and Quantitative Analysis of Glutamine
- Accurately measure both the absolute concentration and relative abundance of glutamine within samples.
- Monitor the dynamic changes in related metabolites, such as glutamic acid, α-ketoglutarate, and ammonia.
For qualitative analysis, high-resolution analytical instruments in conjunction with specialized databases are utilized to accurately identify glutamine in samples. The reliability of identification is ensured through precise recognition of its chemical structure and physical properties. Quantitative analysis employs high-precision detection methods to measure glutamine content accurately. Using the standard curve method, known concentration glutamine standards are analyzed alongside samples, allowing precise determination of sample glutamine content through comparative signal strength analysis. To ensure the accuracy and reproducibility of analytical results, strict control over experimental conditions is maintained, alongside regular calibration and maintenance of instruments, thereby guaranteeing precise and reliable data with each analysis.
Elucidation of Glutamine Metabolic Pathways
The metabolic pathway of glutamine involves several critical stages. Initially, glutamine is transported into cells via membrane transport proteins. Within the cell, glutamine undergoes conversion to glutamic acid catalyzed by glutaminase, marking a crucial initiation step in the metabolic pathway. Glutamic acid can then participate in diverse metabolic processes, such as the tricarboxylic acid (TCA) cycle to provide cellular energy, or as a precursor in the synthesis of other biomolecules. Regulatory mechanisms of the glutamine metabolic pathway are influenced by various factors; for instance, intracellular energy status and amino acid concentrations can adjust enzyme activity through feedback mechanisms, modulating the rate of glutamine metabolism.
Recent studies have continuously uncovered novel insights, including the observation that in tumor cells, there is an upregulation of key enzyme expressions within the glutamine metabolic pathway. This enhancement in glutamine metabolism fuels the rapid growth and proliferation of tumor cells by providing essential energy and substrates.
- Integrate multi-omics data, such as transcriptomics and proteomics, to analyze the activity and regulatory networks of critical glutamine metabolic enzymes, including glutaminase and glutamate dehydrogenase.
- Construct metabolic pathway maps to reveal abnormal regulatory mechanisms within metabolic fluxes, such as in the TCA cycle and glutathione synthesis.
Service Advantages
- Integration of Multiple Technologies: The complementary use of HPLC and LC-MS ensures both high throughput and precision in analysis.
- Data Reliability: We provide raw data, standardized reports, and bioinformatics interpretations to ensure the reliability and comprehensiveness of results.
- Personalized Analysis: We offer customized metabolic pathway modeling, including specialized analyses such as interactions between glutaminolysis and mTOR signaling pathways.
Analytical Methods & Platform
High-Performance Liquid Chromatography (HPLC): Employed for the quantitative analysis of glutamine with high sensitivity and stability, making it suitable for the testing of large sample batches.
Liquid Chromatography-Mass Spectrometry (LC-MS): Utilizes both targeted and untargeted metabolomics techniques to achieve comprehensive qualitative and quantitative analysis of glutamine and its metabolites.
Instrumentation: Agilent 1260 HPLC System and Thermo Scientific Q Exactive HF-X High-Resolution Mass Spectrometer.
Chromatographic Conditions: Utilizes a Hydrophilic Interaction Liquid Chromatography (HILIC) column, which is optimal for the separation of polar metabolites, enhanced by gradient elution to achieve improved separation performance.
Workflow of Metabolomics Service
The general workflow of metabolomics analysis. (Jiajia Yang et al., 2023)
Sample Requirements
| Sample Type | Minimum Sample Size | Sample Preparation Requirements | Recommended Storage Conditions |
|---|---|---|---|
| Cultured Cells | (1 \times 10^6) cells | After collection, wash cells with phosphate-buffered saline (PBS) and lyse to release metabolites. | Long-term storage at -80°C |
| Primary Mouse Cells (Cultured/Uncultured) | (1 \times 10^6) cells | Follow the same procedure as cultured cells: collect, wash with PBS, and lyse. | Long-term storage at -80°C |
| Tissue (e.g., liver, heart, brain) | 50-100 mg | Homogenize tissue using an appropriate buffer, followed by metabolite extraction. | Long-term storage at -80°C |
| Serum/Plasma | 200 µL | Centrifuge at 3000 RPM for 10 minutes to separate serum/plasma, avoiding the use of anticoagulants. | Long-term storage at -80°C |
| Biological Fluids (e.g., urine, bile) | 500 µL | After collection, centrifuge to remove particulates. | Long-term storage at -80°C |
| Thioglycollate-Induced Macrophages | (1 \times 10^6) cells | Collect cells post-thioglycollate injection; follow cultured cell procedure (PBS wash and lysis). | Long-term storage at -80°C |
Case Study
Guo et al. (2023). "SLC38A2 and glutamine signalling in cDC1s dictate anti-tumour immunity". Nature, 621(7977), 132-141.
DOI: 10.1038/s41586-023-06299-8
- Background
- Methods
- Conclusions
- Case Analysis and Significance
This study examines the competition for glutamine between cancer cells and immune cells, particularly type 1 conventional dendritic cells (cDC1), within the tumor microenvironment (TME), and its impact on antitumor immune responses. The research aims to elucidate whether glutamine metabolism indirectly enhances CD8+ T cell activity by modulating dendritic cell function.
Metabolomics Analysis: Using LC-MS, the researchers quantified glutamine levels and its metabolites (such as α-ketoglutarate and glutamic acid) within tumor tissues and cDC1 cells.
Gene Knockout Model: Guo et al,. generated SLC38A2 (glutamine transporter) deficient mice to assess the antigen presentation function of cDC1 and activation markers of CD8+ T cells (e.g., IFN-γ, Granzyme B) via flow cytometry.
Metabolic Flux Tracing: Employing stable isotope-labeled glutamine (¹³C₅-glutamine), we traced the carbon and nitrogen flux within cDC1 and its contribution to the TCA cycle.
Therapeutic Intervention Experiments: In a melanoma mouse model, we evaluated the synergistic antitumor effects of local glutamine supplementation combined with PD-1 immune checkpoint inhibitors.
Tumor cells, through the high expression of SLC38A2, compete with cDC1 for glutamine uptake, leading to impaired cDC1 function and insufficient CD8+ T cell activation.
Supplementing exogenous glutamine restores the metabolic activity of cDC1, enhances antigen presentation capability via the FLCN-TFEB signaling axis, and significantly boosts the tumor-killing function of CD8+ T cells.
Co-deletion of TFEB restores the priming effect of FLCN-deficient or glutamine-deprived cDC1s.
The intervention with glutamine has been shown to more than double the response rate to immune checkpoint blockade therapy, specifically anti-PD-1, providing a novel strategy for "metabolism-immunity" combination therapy.
This pioneering study illuminates the role of glutamine as an "immune-metabolic checkpoint" within the tumor microenvironment. Metabolomics analyses distinctly demonstrated the dependency of cDC1 on glutamine, thereby offering a theoretical foundation for targeting the metabolic microenvironment to enhance immunotherapy. The employment of LC-MS and isotope tracing techniques exemplifies the application of metabolomics services in immuno-oncology, aiding researchers in accurately elucidating the relationship between metabolite dynamics and cellular functions.
FAQ
What are the differences between HPLC and LC-MS technologies for glutamine analysis?
HPLC: Ideal for high-sensitivity and high-throughput absolute quantification of glutamine in large sample volumes, such as culture media or plasma samples. It is cost-effective but does not allow for full-spectrum detection of all metabolites.
LC-MS: Combines targeted quantification (precise analysis of glutamine, glutamic acid, etc.) and untargeted screening (discovery of unknown metabolites). It facilitates comprehensive analyses of metabolic pathways, with sensitivity down to the picomole level, suitable for complex samples like tissues or tumor microenvironments.
What is the detection sensitivity of glutamine analysis?
Under our optimized conditions, we achieved a limit of detection for glutamine as low as 0.1 nM using LC-MS, and a limit of quantitation of 1 nM using HPLC, enabling precise analysis of low-abundance samples.
Does your service support the analysis of small sample sizes?
Yes, we offer an ultra-sensitive LC-MS approach, reducing the minimum sample requirements to as low as (10^5) cells or 20 mg of tissue.
How should cell or tissue samples be preserved post-collection? Are there special handling requirements?
Cell Samples: Wash with pre-cooled PBS, instantly snap-freeze in liquid nitrogen to halt metabolic activity, and store at -80°C. For intracellular metabolite extraction, add metabolite quenching agents (e.g., 80% methanol-water solution).
Tissue Samples: Rapidly dissect and freeze in liquid nitrogen, avoiding freeze-thaw cycles. Use dry ice for cold chain maintenance during transport.
Blood/Plasma: Within 30 minutes of collection, centrifuge to separate plasma, with no anticoagulants like EDTA or heparin, and store at -80°C.
Comprehensive Analysis and Reporting
Does the metabolomics data come with extensive bioinformatics analysis?
Yes, apart from standardized reports (metabolite concentrations, pathway enrichment analysis), we provide the following advanced analytic options:
Metabolic Flux Modeling: Uses 13C-labeled glutamine to trace carbon-nitrogen flow, calculating TCA cycle fluxes.
Multi-omics Integration: Combines transcriptomics/proteomics data to construct metabolic-gene regulatory networks (e.g., correlation between mTORC1 and glutaminase expression).
Machine Learning Prediction: Employs metabolite profiling for disease biomarker discovery (e.g., tumor glutamine metabolism scoring models).
What does data analysis include?
The analysis encompasses differential metabolite screening, pathway enrichment analysis, metabolic network visualization, and statistical reporting.
Can I obtain raw mass spectrometry data for further research?
Yes, we provide:
Raw mass spectrometry files (.raw format).
Peak identification and integration results (including retention times, mass-to-charge ratios, peak areas).
How does glutamine metabolism analysis integrate with immunotherapy research?
Typical applications include:
Evaluating glutamine-dependent metabolic reprogramming of tumor-infiltrating lymphocytes (TILs).
Analyzing the impact of immune checkpoint inhibitors (e.g., anti-PD-1) on glutamine metabolic pathways.
Identifying metabolic intervention targets to enhance CAR-T cell persistence (e.g., glutaminase inhibition to alleviate exhaustion).
Species-Specific and Integrative Analysis
Can you customize glutamine metabolism analysis for specific species (e.g., zebrafish)?
Cross-species analysis is supported. We have established metabolite databases for zebrafish, mice, humans, and plant samples, offering species-specific metabolic pathway annotation (e.g., glutamine-uric acid axis in insects).
Is it possible to integrate other metabolic pathways (e.g., glycolysis) into the analysis?
We support the integrated analysis of multiple pathways. Please specify the requirement ahead of time.
Learn about other Q&A.
Publications
Here are some publications from our clients:
- Inflammation primes the kidney for recovery by activating AZIN1 A-to-I editing. 2023. https://doi.org/10.1101/2023.11.09.566426
- Neddylation is required for perinatal cardiac development through stimulation of metabolic maturation. 2023. https://doi.org/10.1016/j.celrep.2023.112018
- Quantifying forms and functions of intestinal bile acid pools in mice. 2024. https://doi.org/10.1101/2024.02.16.580658
- Teriflunomide/leflunomide synergize with chemotherapeutics by decreasing mitochondrial fragmentation via DRP1 in SCLC. 2024. https://doi.org/10.1016/j.isci.2024.110132
- Glycine supplementation can partially restore oxidative stress-associated glutathione deficiency in ageing cats. 2024. https://doi.org/10.1017/S0007114524000370 [Opens in a new window]
References
- DeBerardinis RJ, Cheng T. Q's next: the diverse functions of glutamine in metabolism, cell biology and cancer. Oncogene. 2010;29(3):313-324. DOI: 10.1038/onc.2009.358
- Wise DR, Thompson CB. Glutamine addiction: a new therapeutic target in cancer. Trends Biochem Sci. 2010;35(8):427-433. DOI: 10.1016/j.tibs.2010.05.003
- Chen L, Cui H (2015) Targeting Glutamine Induces Apoptosis: A Cancer Therapy Approach. Int J Mol Sci 16(9), 22830–55. DOI: 10.3390/ijms160922830
- Hensley CT, Wasti AT, DeBerardinis RJ (2013) Glutamine and cancer: cell biology, physiology, and clinical opportunities. J. Clin. Invest. 123(9), 3678–84. DOI: 10.1172/JCI69600
- Jin L, Alesi GN, Kang S (2016) Glutaminolysis as a target for cancer therapy. Oncogene 35(28), 3619–25. https://doi.org/10.1038/onc.2015.447
