Metabolomics FAQ Summary
If the specific type of "detection" is not clearly mentioned (such as LC-MS metabolomics, ELISA, Western blot, clinical biochemistry, transcriptomics, etc.), the requirements for serum volume and sample size vary significantly across different platforms and analysis content. However, based on routine experimental experience, the following are the basic requirements for mouse serum volume and sample quantity in several typical tests for reference: 1. Required serum volume per tube (estimated based on routine experiments): Experimental type Required volume (μL/sample) Remarks LC-MS metabolomics 100–200 μL Consider loss during freeze-drying/pre-treatment ELISA 50–100 μL Depends on kit sensitivity Western blot (WB) 50–100 μL Low recovery rate if extracting proteins from serum Clinical biochemical index determination 100–200 μL Different indices have different requirements Multi-omics integrated analysis Over 200 μL Multiple platforms require samples to be split into multiple tubes 2. Sample size per group (statistical recommendations): 1. Basic scientific research exploratory phase: It is recommended that each group has n ≥ 6 for preliminary trend judgment. 2. Metabolomics, proteomics, and other high-throughput omics studies: It is recommended that each group has n ≥ 8–10 to enhance statistical power after multiple comparisons. 3. Preclinical animal studies requiring publishable data: Each group should have n ≥ 10–12, especially in models with large individual variability (such as high-fat, high-sugar induction models, etc.). Summary recommendations (preliminary): If it is for omics...
• Should S1P focus on metabolomics or lipidomics?
S1P (Sphingosine-1-phosphate) is a sphingolipid metabolite and a key signaling molecule in the sphingolipid metabolic pathway, exhibiting typical lipid characteristics. Therefore, from the perspectives of analytical methods and metabolic pathway classification: categorically, S1P is more suitable to be included in the scope of 'lipidomics' research; methodologically, lipidomics often employs targeted or untargeted LC-MS/MS methods, especially using reverse phase chromatography (RP-LC) + ESI positive ion mode MRM or PRM methods, which can detect S1P with high sensitivity; in terms of data interpretation, if the research focuses on the sphingolipid metabolic pathway (such as the metabolic conversion between S1P, Sphingosine, and Ceramide), lipidomics databases (such as LIPID MAPS) and pathway tools (such as KEGG sphingolipid metabolism) should be used for annotation and pathway enrichment analysis. If the research emphasis is on signaling functions or interactions with non-lipid molecules, it can also be analyzed as a specific metabolite in targeted metabolomics. However, from a methodological and categorical perspective, it is recommended to follow the lipidomics approach for detection and interpretation.
In the experiment of collecting fish intestinal contents for subsequent bacterial screening (isolation and cultivation of microorganisms), the transportation and preservation conditions are crucial for microbial activity and community structure. Here are professional recommendations suitable for most microbiological operations that require the isolation of anaerobic/facultative bacteria and aerobic bacteria: Key principles for preservation during transportation: 1. Maintain low temperature (4°C) during transportation to inhibit metabolism. Immediately place the samples in pre-cooled sterile centrifuge tubes or sample bags, and transport them in ice boxes/ice bags; Do not freeze (-20°C or below) as this may damage bacterial activity, especially in scenarios where live bacteria are required; 2. Avoid prolonged exposure to air (if screening anaerobic bacteria). If the target includes anaerobic bacteria, it is recommended to use anaerobic sampling tubes or sealed containers filled with nitrogen gas; Anaerobic bags/cans can be used for packaging; 3. It is advisable to add protective agents (optional). If inoculation cannot be performed within a short time, the contents can be mixed with sterile glycerol protective solution (10–20% glycerol) and temporarily stored in refrigeration (4°C, <12 hours); Alternatively, samples can be aliquoted and stored at -80°C, but this method is suitable for preserving microbial DNA/RNA and is not recommended for live bacteria screening; 4. Try to control transportation time within 24 hours. The faster the transportation, the better; it is recommended to transfer to the laboratory or cultivation base immediately after sampling; If the distance is far, dry ice can be used for transportation (for DNA extraction...)
Yes, it is recommended to immediately freeze the leaves in liquid nitrogen after collection and store them at -80°C to maximize the stability of the content of secondary metabolites such as flavonoids, polysaccharides, and triterpenes. The specific explanations are as follows: 1. The necessity of liquid nitrogen freezing (1) Flavonoids and triterpenes are small molecular metabolites, while polysaccharides are high molecular products; all three are susceptible to degradation or transformation due to enzymatic activity, light, temperature, etc. (2) The leaves still have metabolic activity after collection; if the metabolic process is not immediately halted, it will lead to changes in the content of these target substances before analysis, affecting the comparability and accuracy of subsequent data. (3) Liquid nitrogen freezing can quickly inactivate enzymes within the tissue, fixing its metabolic state, especially suitable for complex field sampling conditions that are not easy to process immediately. 2. Recommended storage conditions (1) After liquid nitrogen freezing, transfer to a -80°C freezer, which can stably preserve for several weeks to months. (2) If it is inconvenient to use liquid nitrogen in the field, at least temporarily freeze the samples in a portable liquid nitrogen tank or dry ice and bring them back to the laboratory for processing as soon as possible. 3. Precautions (1) Avoid direct sunlight and mechanical damage during collection, and try to process quickly; (2) Each plant and sampling point should be numbered separately, and information such as time and location should be recorded; (3) If only qualitative screening is to be performed (such as preliminary detection of the presence of certain types of metabolites), refrigerated transport can also be considered, but quantitative studies must use liquid nitrogen freezing for preservation.
• How to calculate relative content in gas chromatography?
The calculation of "relative content" in gas chromatography is typically used for qualitative or semi-quantitative analysis, and essentially uses a certain signal intensity (usually peak area) as an approximate indicator of compound content. Common methods for calculating relative content are as follows: 1. Conventional relative content formula (uncorrected) Figure 1 2. Relative content formula using response factors (corrected) Figure 2 Baitai Parker Biotechnology -- a high-quality service provider for biological product characterization and multi-group biological mass spectrometry testing Related services: Metabolomics
To qualitatively and quantitatively analyze the bile acid profile in feces of C57 neonatal mice, the following key technical points need to be addressed: complex sample matrix, a wide variety of bile acids with significant polarity differences, and potentially low concentrations. The following is a recommended experimental workflow and methods: 1. Sample preprocessing 1. Feces collection Collection time: Ensure timely collection after the first bowel movement of the neonatal mice to avoid contamination from maternal sources. Preservation method: Immediately snap freeze in liquid nitrogen and store at -80°C for long-term preservation. 2. Bile acid extraction Weighing samples: Accurately weigh 10–50 mg of lyophilized feces. Extraction solvent: 70% methanol (containing 0.1% formic acid) or 75% acetonitrile/water, volume ratio 1:20~1:40. Internal standard addition: Add stable isotope-labeled bile acid internal standards (such as d4-CA, d4-CDCA, d4-DCA, etc.) for subsequent quantification. Ultrasonic shaking + vortex mixing: Ice bath sonicate for 15–30 minutes and centrifuge at 4°C. Filtration of supernatant: Treat with a 0.22 μm filter membrane and transfer to the injection vial. 2. Chromatography-mass spectrometry platform selection It is recommended to use the UPLC-MS/MS platform, combined with the MRM mode for highly sensitive quantitative analysis. 1. Chromatographic conditions (recommended) (1) Chromatography column: C18 reverse phase column (e.g., Waters Acquity UPLC BEH C18, 1.7 μm, 2.1×100 mm) (2) Mobile phase: A: 0.1% formic acid aqueous solution B: 0.1% formic acid acetonitrile (3) Gradient elution: Set a gradient suitable for bile acid separation (for example, from 20% within 30 min...
• What is the key rate-limiting enzyme for diacylglycerol synthesis?
The rate-limiting enzyme for diacylglycerol (DAG, diacylglycerol) synthesis depends on the synthesis pathway: 1. In the "phosphatidic acid pathway (Kennedy pathway)," DAG is generated from phosphatidic acid (PA) through dephosphorylation, and the rate-limiting step is usually: phosphatidic acid phosphatase (PAP), especially the Lipin protein family, which catalyzes PA → DAG and is considered the rate-limiting step in this pathway. 2. In the "glycerol-3-phosphate pathway (de novo synthesis)," the precursor for DAG synthesis is phosphatidic acid (PA), and the key rate-limiting enzyme is glycerol-3-phosphate acyltransferase (GPAT), particularly mitochondrial GPAT1. This enzyme acylates glycerol-3-phosphate to LPA, which is the initial rate-limiting step. In summary, if the question focuses on the most direct generation step of DAG, then PAP (such as Lipin) is the key rate-limiting enzyme; if considering the de novo synthesis perspective, then GPAT is the starting rate-limiting enzyme. Further clarification is needed based on the pathway (such as triglyceride synthesis, signal transduction, or membrane lipid synthesis).
• What methods are currently used for the characterization of yak milk oligosaccharides?
Milk oligosaccharides, especially yak milk oligosaccharides, are mainly identified and quantified through the combination of chromatography and mass spectrometry techniques. The specific methods include the following categories: 1. Sample extraction and purification: After protein precipitation and fat removal, methods such as solid phase extraction (SPE) are used to separate lactose, oligosaccharides, and other components, cleaning up background interference for subsequent analysis. 2. Liquid chromatography–electrospray ionization–tandem mass spectrometry (LC‑ESI‑MS/MS): (1) Commonly used for qualitative/quantitative analysis of free and conjugated oligosaccharides, such as simultaneously detecting neutral and sialic acid-type glycosides. (2) Combining high-performance liquid chromatography (such as reverse phase or porous graphitized carbon chromatography PGC) with ESI‑MS/MS to obtain parent ions and fragmentation spectra for structural inference and isomer differentiation. (3) Treating sugar chains with permethylation helps enhance the clarity of MS/MS fragmentation and signal intensity, improving structural elucidation capabilities. 3. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI‑TOF‑MS): (1) Used for rapid and intuitive analysis of oligosaccharide population characteristics (m/z distribution), especially with good coverage for sialic acid and neutral oligosaccharides. (2) Often combined with high-performance liquid chromatography or front-end fractionation to improve component clarity and identify novel structures. 4. Nuclear magnetic resonance spectroscopy (NMR) —^1H/^13C and 2D experiments: When the purification level is sufficient (≥10 mg), through........
• What are the unique metabolites of gut microbiota?
The unique metabolites of gut microbiota mainly refer to the metabolic products synthesized by gut microorganisms that the host itself cannot directly synthesize, or metabolic products that significantly depend on microbial participation in the host's metabolic pathways. They can be summarized into several major categories, with representative examples as follows: 1. Short-Chain Fatty Acids (SCFAs) mainly include: 1. Acetate 2. Propionate 3. Butyrate Source: Generated by microbial fermentation of dietary fibers (such as resistant starch, fructooligosaccharides, etc.) Function: Regulate energy metabolism, maintain gut barrier, modulate immunity, and influence the nervous system (gut-brain axis) 2. Metabolites of aromatic amino acids 1. Indole derivatives: such as Indole-3-acetic acid (IAA), Indole propionic acid (IPA), Indole lactic acid (ILA) 2. Phenylpyruvic acid, p-Hydroxyphenylacetic acid, etc. Source: Aromatic amino acids such as tryptophan, tyrosine, phenylalanine are metabolized by microorganisms. Function: Regulate neural signaling, immune modulation, activate AhR and other nuclear receptors 3. Bile acid metabolites (Secondary Bile Acids) 1. Deoxycholic acid (DCA) 2. Lithocholic acid (LCA) 3. Ursodeoxycholic acid.......
Under the condition of having only the retention time and peak area of the samples, and lacking standard concentration gradient data, absolute quantitative analysis cannot be performed, meaning the quality or concentration of each component cannot be accurately calculated. However, with a reasonable experimental design and clear peak identification, the following meaningful analytical results can still be obtained from these data: 1. Component identification (qualitative) 1. Retention Time (RT) is the most commonly used qualitative indicator in gas chromatography. 2. If the chromatographic column model, carrier gas flow rate, temperature control program, and other conditions remain unchanged, the target components in unknown samples can be preliminarily identified based on the retention time of standard samples or historical data. 3. It can be used for sample component identification, trend tracking, impurity analysis, etc. 2. Relative content analysis (semi-quantitative) 1. Peak area can be regarded as an indicator of the relative abundance of each component in the sample, calculated as follows: 2. It can be used for (1) relative comparison of component composition in multiple batches of samples; (2) analysis of trends in the same sample under different treatment conditions (such as temperature, time, storage); (3) construction of composition profiles of natural products (such as volatile oils, fragrances); (4) impurity tracking or main component monitoring. 3. Retention behavior analysis (for process or mechanism research) Comparing the retention time shifts of different samples can be used to analyze: 1. Whether isomers or derivatives exist; 2. Whether chromatographic conditions (such as column efficiency, active sites) change; 3. In some mechanistic studies (such as reaction kinetics, metabolic pathways, etc.).
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