This protocol describes an analytical platform for the analysis of intra- and extracellular metabolites of microbial cells (yeast, filamentous fungi and bacteria) using gas chromatography–mass spectrometry (GC-MS). The protocol is subdivided into sampling, sample preparation, chemical derivatization of metabolites, GC-MS analysis and data processing and analysis. This protocol uses two robust quenching methods for microbial cultures, the first of which, cold glycerol-saline quenching, causes reduced leakage of intracellular metabolites, thus allowing a more reliable separation of intra- and extracellular metabolites with simultaneous stopping of cell metabolism. The second, fast filtration, is specifically designed for quenching filamentous micro-organisms. These sampling techniques are combined with an easy sample-preparation procedure and a fast chemical derivatization reaction using methyl chloroformate. This reaction takes place at room temperature, in aqueous medium, and is less prone to matrix effect compared with other derivatizations. This protocol takes an average of 10 d to complete and enables the simultaneous analysis of hundreds of metabolites from the central carbon metabolism (amino and nonamino organic acids, phosphorylated organic acids and fatty acid intermediates) using an in-house MS library and a data analysis pipeline consisting of two free software programs (Automated Mass Deconvolution and Identification System (AMDIS) and R).
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- Supplementary Data 1 (13K)
MCFMS.CID file from MCF MS Library. Our in house library consists of two files: MCFMS.CID and MCFMS.MSL (supplemented material 2). Box 1 presents all steps and necessary instructions to load our in-house library in AMDIS. Download the files and follow the instruction in Box 1.
- Supplementary Data 2 (141K)
MCFMS.MSL file from MCF MS Library. Our in house library consists of two files: MCFMS.CID (supplemented material 1) and MCFMS.MSL. Box 1 presents all steps and necessary instructions to load our in-house library in AMDIS. Download the files and follow the instruction in Box 1.
- Supplementary Data 3 (47K)
Step 21(B) (i). Our in-house script consists of one R scrip file called ‘mining_script’ and all requirements and instructions to use it are described in the first rows of the script.
- Supplementary Data 4 (15K)
Step 21(B) (i). The reference ion library consists of one comma separated file (.csv) called ‘ref_ion_library.csv’. This file is complementary to the in-house script (mining_script) and both files have to be stored in the same folder.
- Supplementary Video 1 (3M)
Step 2 (A) (i), (ii). Quenching using cold glycerol-saline. Rapid transfer microbial culture suspension to a cold solution of glycerol-saline at −23°C. Note that the volume of sample transferred can be roughly controlled by level marks in the sampling flasks. This is not accurate and, therefore, the quantification of biomass in each sample must be carried out after extraction.
- Supplementary Video 2 (4M)
Step 14 (A) (iii)-(v). MCF reaction after the samples have been resuspended in NaOH and mixed with methanol and pyridine. Although it is not clear in this video, the vortex agitation must be “vigorous”.
- Supplementary Video 3 (10M)
Step 14 (A) (vii). Removal of aqueous phase. The top aqueous layer should be totally removed.
- Supplementary Video 4 (2M)
Step 14 (A) (ix). End of derivatization. Sodium sulphate-dried chloroform solution containing the MCF derivatives is transferred to the GC-MS vial. Avoid transferring granules of sodium sulphate together with the sample.