Exo/Metabolomics via LC-UV/RI-MSn
Exo/metabolomics strategies to investigate the metabolic network of microorganism using LC-UV/RI-MSnCopyright: © iAMB
Exometabolomics: Examine extracellular metabolites in cell culture media utilizing metabolic footprinting approach. In the field of microbiology, substrates and products (mainly sugars, organic acids and alcohols) usually occur in various range of concentrations, which makes the traditional ion-exclusion chromatography (IEC) with UV/RI detectors popular for quantification purposes. However, it is usually very difficult to recognize overlapped peaks or to identify unknown substances with this method. The fact that sulphuric acid is mainly utilized as mobile phase additive makes it even harder to adapt the IEC method to any sensitive equipment for peak identification. On the other hand, mass spectrometry is optimal for peak identification, though it has limitation on dynamic range and the choices of mobile phase. Here, we aim to develop a LC-UV/RI-MS/MS method that could generate comparable peaks from UV/RI and MS. With this configuration, we can take advantage of all detectors to identify and quantify extracellular metabolites.
Metabolomics: Investigate intracellular metabolites using metabolic profiling or metabolic fingerprinting approaches. Nowadays, LC-ESI-MS is becoming the method of choice for profiling metabolites in complex biological samples. As most of metabolites related to central metabolic pathways are small molecular weight hydrophilic compounds, one powerful method to analyze them is reverse phase liquid chromatography (RP-LC) with ion-paring reagents. However, these reagents can cause permanent contaminations in the system, which limits the usage of an analytical device to only one method. In this study, an LC-ESI-MS method will be optimized to target a wide range of intracellular metabolites without using any ion-paring reagents.
In combination, we will be able to analyze both intracellular and extracellular metabolites with a single configuration, the LC-UV/RI-MS/MS. The information on exo/metabolome will provide new insights on how metabolites could be taken up, metabolized and/or excreted by cells under certain conditions. The results can be used to alter the metabolic flux distribution by changing endogenous genes or introduction of foreign genes into the host to maximize the production of target compounds. The contributions of this method will be beyond basic research as it can also be applied in both academia and industry engaged in metabolic engineering of microorganism.