The aim of this experiment was to study the metabolites of various organs of Nymphaea minuta.
Materials and methods
Different from transcriptome and proteomics technology, metabolomics technology is the latest development. There are a wide variety of metabolites studied, and there are a large number of isomers and metabolites with similar molecular weights, resulting in the accurate identification of metabolites is a major difficulty and challenge. Different identification methods directly affect the reliability of metabolite identification grades, and then affect the reliability of experimental results and subsequent functional studies, which has a significant impact. Oliver Fiehn et al., an international expert in metabolomics, suggested that the classification of metabolites be included in the metabolome analysis report (Blazenovic et al., 2018). It is worth noting that there is currently an article in Cell that provides Metabolomics Standards Initiative (MSI) criteria for metabolite identification. Moreover, the definition of MSI metabolite levels is included in the method section (Contrepois et al., 2020; L et al., 2020).
There has always been a clear definition of the identification grade of metabolites in the world. As early as 2007, MSI is Chemical Analysis Working Group defined the reliability levels of four metabolite identification results (Dunn et al., 2012). Later, at the 2017 annual meeting of the Metabolomics Society in Brisbane, Australia, a new reliability level of metabolite identification was redefined (Blazenovic et al., 2018), and a new "level 0" was added, increasing from the original 4 to 5, and the higher the number, the lower the reliability level. As shown in the following picture.
Grade 0, with clear three-dimensional structure and stereochemical information. Class 1, reliable 2-D structural identification, requires at least two or more orthogonal properties (such as MS/MS spectra, retention time RT, or collision cross section (CCS) values) of a real chemical standard to be compared with the same properties of metabolites of interest analyzed under the same analytical conditions. Grades 2 or 3 are assumed annotated results, usually based on only one or two properties and relying on comparisons with data collected by different laboratories or obtained by different analytical methods, rather than direct comparisons with real chemical standards under the same analytical conditions. For example, matching MS/MS spectra from public databases such as HMDB, MoNA, MassBank, METLIN and NIST, the identification Level of metabolites is defined as Level 2. Those that cannot be identified as above are classified as level 4 unknowns.
This project adopted local self-built database and public database search database (Luo et al., 2017; Zhaobing et al., 2018). By matching with the information of metabolite retention time, molecular mass (molecular mass error within <10 ppm), secondary fragmentation spectra, collision energy and other information in the database, the structure of metabolites in biological samples was identified, and the identification results were strictly checked and confirmed manually. The identification Level is Level 2 and above.
