Unknown Metabolite Identification

Metabolomics generally categorizes the confidence level of metabolite identification based on the following four dimensions: compounds identified with confidence (Level 1), compounds identified through presumptive annotation (Level 2), classification of compounds through presumptive annotation (Level 3), and unknown compounds (Level 4). Level 1 identification requires comparing two or more orthogonal characteristics (such as retention time, m/z, and fragment mass spectra) of the target metabolite with the same characteristics observed in authentic chemical standards under identical analytical conditions. Presumed (Level 2 or Level 3) annotations essentially rely on one or two characteristics and are often based on comparisons of data collected by different labs and obtained through different analytical methods, rather than direct comparison with authentic chemical standards under the same analytical conditions.

Unknown metabolites refer to small molecules that can be repeatedly detected but whose chemical identities remain undetermined. Although these compounds have not yet been identified and classified, they can still be distinguished and quantified based on spectral data in metabolomics experiments. In LC-MS experiments, an unknown compound is defined by a unique retention time, one or more masses, or a specific primary ion fragmentation pattern. In NMR experiments, an unknown compound is defined by a pattern of chemical shifts. Unknowns may represent previously unrecognized small molecules, such as secondary products of metabolism or rare exogenous substances, or they may be molecular constituents of established pathways that cannot be assigned to existing NMR reference libraries or MS fragmentation patterns.

The identification of unknown compounds is very time-consuming and costly, often requiring preparative-scale separations for NMR studies or extensive chemical synthesis for structural comparisons using MS/MS. Therefore, most reports focus on detecting metabolites with available authentic commercial standards or those present in metabolite databases, categorized as Level 1, Level 2, and Level 3 metabolites. Due to the inherent instability of many metabolites and the lack of demand, only a few thousand commercial analytical standards are available. However, the metabolites present in these standards and databases represent only a small portion of endogenous metabolites. Therefore, effective methods for prioritizing, studying, and ultimately identifying uncharacterized metabolites are crucial for metabolomics research. The successful identification of unknown metabolites will have a significant impact on biomarker discovery and omics studies.

Identification of Unknown Metabolites

To identify unknown compounds in metabolomic samples, different categories of metabolites should be distinguished. Those with the same distinguishable nominal mass, those with the same distinguishable nominal mass but different molecular formulas and isotopic masses, and those with the same distinguishable nominal mass and isotopic mass but different chemical structures should all be differentiated. For example, leucine and isoleucine are structural isomers with the same distinguishable nominal and isotopic mass. Additionally, since a single metabolite can be detected in a mass spectrometer with different derivative species, it is important to correctly assign different derivative species to the parent metabolite. For example, in amino acid analysis, trimethylsilylation (TMS) chemically derivatizes amino acids to generate amino acids containing one, two, or three TMS groups, all of which should belong to the same parent amino acid.

BiotechPack combines NMR, LC-MS/MS, and bioinformatics analysis to provide mass spectrometry-based analysis services for identifying unknown metabolites, enabling accurate identification of metabolites in complex biological samples. BiotechPack continuously develops tools and databases to achieve true and complete identification of all metabolites in an automated manner. Welcome to inquire!

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