Abstract
Metabolomics studies based on Gas chromatography–Mass spectrometry (GC-MS) are well established and typically employ electron impact (EI) ionisation. Target compounds of interest can be identified by comparison to commercial or public databases. Unfortunately, many possible biomarkers detected in metabolic profiling experiments cannot be identified due to the lack of reference spectra for a majority of biologically relevant compounds. Therefore, many possible biomarkers remain “unknowns” up till now. Hyphenating gas chromatography with high resolution TOF-MS technology with soft atmospheric pressure ionisation (APCI) can preserve the molecular ion information and delivers accurate mass and isotopic pattern information. This data enables a sum formula generation for known and unknown target compounds. Additionally, optionally acquired MS/ MS data can extend the capabilities for structural elucidation. Mass accuracy, resolution and isotopic fidelity are independent of the TOFMS acquisition rate. Therefore, these instruments can be coupled to gas chromatography, which typically delivers narrow peak width requiring fast MS scan speeds. Corynebactrium glutamicum, a gram positive, nontoxic bacterium, is used in the industrial production of amino acids like lysine and glutamate. C. glutamicum can be grown on different carbon sources. Glucose is metabolised via glycolysis and the tricarboxylic acid (TCA) cycle, whereas propionate is catabolised through the methylcitric acid pathway. The prpD2 gene encodes a 2-methylcitrate dehydratase which is involved in the degradation of propionate. Metabolic profiling of Corynebacterium glutamicum delta-prpD2 extracts of cells grown on glucose or glucose and propionate analyzed by GC-APCI-TOF-MS revealed several compounds elevated in cells grown on propionate. Identification of 2-methylcitric acid and alanine using accurate mass and isotopic pattern information in MS and MS/MS spectra provided a proof of concept for the identification of target compounds using high resolution MS technology.