What categories of metabolites can be detected by GC-MS?
A: GC-MS can detect metabolites such as organic acids, amino acids, monosaccharides, sugar alcohols, amines, fatty acids, indoles, monoglycerides, nucleosides, disaccharides, tocopherols, sterols, etc.
It is said that GC should be derivatized, is this the process to be done in all GC assays?
A: No, it is not. The samples of volatile substances do not need to be derivatized. The data from derivatization analysis can be searched by both fiehn and NIST, but only the NIST library can be searched without derivatization.
What metabolites are suitable for detection by LC-MS?
A: Most substances such as lipids, bile acids, amino acids, and nucleotides can be detected using LC-MS
How to choose positive or negative ion mode when mass spectrometry?
A: The choice of positive or negative ion mode is mainly based on the nature of the compound, and the mobile phase environment affects the sensitivity of the analysis. Some substances tend to be positively charged and some tend to be negatively charged. For example, alkaline compounds tend to be positively charged with additionally charged protons or other positively charged ions. Acidic compounds tend to be negatively charged, losing protons or adding other negatively charged ions.
Substances containing carboxyl and sulfonic acid groups can generally be used in negative ion mode because they can be ionized to R-COO- and R-SO3- under general conditions. In an acidic mobile phase, e.g. below pH 3, carboxylate may not ionize well into negative ions. The sensitivity of negative ion monitoring decreases at this point, while sulfonic acid roots are more acidic and can still be ionized.
At present, the positive ion mode is generally used more often. Because of the nature of the column mobile phase is generally on the acidic side (pH 2-8), and the ESI ion source is commonly used. Under acidic conditions most of the more polar compounds can add and hydrogen ions to form positive ions, such as flavonoids, lipids, sugars, etc. without nitrogen. The negative ion mode is correspondingly generally one order of magnitude smaller than the positive ion mode.
Therefore, in practice, it is necessary to choose according to the actual situation conditions, or both modes can be applied.
How to have more reliable metabolomics results?
A: The TIC of the blank can be used to see if the column has residues from the previous project and if the instrument is clean. QC can be used to measure the stability of the instrument, to do inter-batch correction, and to screen for FEATURES.
Do I need to add internal standards for target assays?
A: The commonly used quantitative methods for target assays are external and internal standards. The external standard method does not add internal standards. The internal standard method requires the addition of an internal standard, and the addition of a stable isotope internal standard is preferable.
What is the difference between a base peak map (BPC) and a total ion flow map (TIC)?
A: The Base Peak is the peak of the most intense ion in the mass spectrogram within a specified mass-to-charge ratio.
Base Peak Chromatogram (BPC): A chromatogram obtained by successively depicting the intensity of the most intense ion in the mass spectrometer at each time point.
Total ion flow chromatogram (TIC): A plot of the sum of the intensities of all ions over a selected mass range against time or number of scans.
Both TIC and BPC are reflections of the overall information of the sample. In general, BPC plots are more beautiful than TIC plots, so they are often used in articles. However, some scholars think that the BPC graph is not a true reflection of the sample, so they do not accept the BPC but only the TIC.
Why is the content of substance A significantly greater than substance B in the experiment, yet the signal intensity of substance B is significantly greater than that of substance A in the metabolomic data?
A: Since different substances have different molecular structures and property groups, the ionization efficiency, signal response intensity and other parameters of different substances in mass spectrometry are different, and the final result is that the mass spectrometry detection efficiency of different substances varies greatly. Therefore, the comparison of quantitative results of metabolomics is only applicable to the comparison between the same substance in different states (experimental/control), not the comparison of different substances in the same experimental state.
After finding the differential metabolite, how do you validate the function of this metabolite?
A: Qualitative and quantitative validation is performed first. We use triple quadruple rods for targeting validation to obtain absolute quantitative information. Then analyze which regulatory pathway the differential metabolite belongs to by metabolic pathway. Functional validation of molecules on that pathway can be performed. In addition, other omics data, such as transcriptome and proteome, can be added to complement each other.
