Metabolomics Service

How many biological replicates are generally required in metabolomics studies?

In metabolomic research, there are certain requirements for the number of biological repeat samples. Recommended standards are as follows: not less than 30 clinical samples, not less than 10 animal samples, not less than 8 cell samples, not less than 8 plant samples, and not less than 8 microbial samples. Metabolomics is downstream of systems biology and the amplification of biological processes may lead to amplified individual differences. Influencing factors are numerous, such as age, gender, and the physicochemical property differences of metabolites. To ensure analytical result accuracy and minimize individualized spectral error introduced by sample variability, the sample size should be maximized. Too few biological replicates may led to poor model building in later stages and possibly make statistical analysis impossible, thereby limiting the discovery of differential metabolites.

Why is High Resolution Mass Spectrometry Essential for LC-MS Metabolomics?

In present-day metabolomics research, LC-MS plays a vital role with the primary method of metabolite annotation involving a database search based on accurate molecular weight and secondary fragment information. Accurate molecular weight serves as an indispensable parameter in this procedure. To acquire the accurate molecular weight of substances, the application of high-resolution mass spectrometry is vital. Conventional high-resolution mass spectrometers can ensure the precise mass error of substances is controlled within 5 ppm, and even as low as 3 ppm, thereby significantly enhancing the accuracy of annotation.

What Are TOF, QQQ, QTRAP, and Orbitrap in Mass Spectrometry?

Mass spectrometers can be classified based on their mass analyzers, with TOF, QQQ, QTRAP, and Orbitrap representing different types of such analyzers: TOF (Time-of-Flight); QQQ (Triple Quadrupole); QTRAP (Quadrupole TRAP); Orbitrap (Orbitrap Analyzer).

Serum samples or plasma samples?

Extant research has demonstrated different metabolic varieties and abundances between the two. However, in terms of research objectives, no specific sample type has been definitively proved as superior. Consequently, when deciding to use either serum or plasma, the primary concern should be to ensure consistency in the collection process. In selecting blood samples, plasma anticoagulated with EDTA or heparin is preferable. During the collection process, it is vital to minimize hemolysis. After collection, samples should be stored at -80℃ to prevent any degradation and repeated freeze-thaw cycles should be strictly avoided to maintain sample integrity.

Are the metabolites detected by mass spectrometry all truly existing metabolites?

Qualitative assessment in untargeted metabolomics necessitates aligning the metabolite's tandem mass spectrometry (MS/MS) fragmentation information with database fragment data. Given the variance in fragment patterns for the same substance across different instruments and conditions, a nuanced matching discrepancy arises during data correlation. To mitigate the risk of false positives in substance identification, Creative Proteomics consistently advocates for a stringent matching criterion, emphasizing high concordance in the matching process. This approach underscores the commitment to meticulous data analysis and strives to uphold the integrity of metabolite identification in the pursuit of scientific rigor.

Why are some commonly metabolites sometimes undetectable?

The limitations of untargeted metabolite detection stem from its inherent lack of specificity. For metabolites with low signal intensity, untargeted metabolomics techniques may introduce interference, potentially masking signals and impeding accurate identification. In situations where research objectives are clearly defined, investigators often opt for targeted metabolomics, a method tailored to the specific detection of chosen metabolites, yielding more desirable outcomes. However, it is important to note that, currently, no single technique exists capable of simultaneously identifying all metabolites.

In metabolomics, the query emerging frequently is why, despite detecting thousands of metabolic feature peaks, does the final qualification of compounds remain relatively low?

In instances of insufficient fecal sample collection, is it permissible to supplement with intestinal contents?

The answer is negative. Research indicates significant disparities between metabolites found in intestinal contents and those in feces, with notable variations even among different segments of the intestine. In the context of metabolomic investigations, only samples collected beyond the colon can be considered representative of fecal material. This distinction underscores the importance of precision in sample selection for metabolomics examinations and highlights the necessity of drawing distinct boundaries when characterizing samples in scientific studies.

Does a peak in the TIC/BPC diagram represent a distinct compound?

In TIC/BPC diagrams, a single peak does not always denote a unique compound. In fact, a single peak could potentially be composed of multiple substances.

Is it feasible to submit samples individually for separate testing and subsequently amalgamate them for joint analysis?

What iS QC?

In the process of metabolomics research based on mass spectrometry techniques, the implementation of quality control (QC) is usually necessary to ensure the acquisition of reliable and high-quality metabolomics data. A QC sample, composed by mixing 20 μL from each test sample, principally serves to rectify deviations in the analysis results of mixed samples and to correct errors caused by the analytical instrument itself.

What are primary mass spectrometry (MS) and secondary mass spectrometry (MS/MS)?

During the LC-MS detection process, why are there positive and negative ion modes?

How to assess the stability of the instrument system during the detection process?

In the course of detection, evaluating the stability of the instrument system involves several key considerations. Firstly, prior to sample analysis, meticulous calibration and correction of the instrument are essential to ensure that its response and quality accuracy meet the required standards. Secondly, during the injection process, validation of the analytical system can be achieved by employing Quality Control (QC) samples. Lastly, after obtaining data, assessing the stability of the analytical system during the analysis process can be done by examining the clustering pattern of QC samples in the Principal Component Analysis (PCA) score plot.

When dealing with a large volume of samples, how can the stability of the entire analysis process be ensured, and the impact of instrument fluctuations minimized?

To address this question, the initial step involves the incorporation of Quality Control (QC) samples. Subsequently, depending on the quantity of samples, the detection process can be divided into several batches. During the data processing stage, adopting batch normalization methods becomes crucial to reduce instrument errors and diminish differences between batches. This meticulous approach ensures the stability of the overall analysis process and mitigates the influence of instrument fluctuations.

What types of substances can be detected by LC-MS?

LC-MS technology is versatile in detecting various substance types in the analysis of biological samples. Utilizing the ESI source, as favored by Creative Proteomics, it is particularly suitable for the detection of moderately polar and highly polar compounds. Specific substance categories encompass fatty acids, alcohols, phenols, vitamins, organic acids, polyamines, nucleotides, polyphenols, terpenes, flavones, lipids, and more.

What types of substances can be primarily detected by GC-MS?

Gas Chromatography-Mass Spectrometry (GC-MS) is predominantly applicable for the detection of a spectrum of chemical substances, including short-chain fatty acids, organic acids, fatty acids, sugars, polyols, amines, and sugar phosphates. These substances are chiefly associated with amino acid metabolism, sugar metabolism, the tricarboxylic acid cycle (TCA), and other biological metabolic pathways. It is noteworthy that for water-soluble substances, GC-MS detection requires derivatization treatment. Direct GC-MS analysis is only feasible when the sample possesses volatility and thermal stability.