Analytical platform for metabolome analysis of microbial cells using methyl chloroformate derivatization followed by gas chromatography–mass spectrometry

Abstract

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).

Early pregnancy metabolite profiling discovers a potential biomarker for the subsequent development of gestational diabetes mellitus

“…The samples were centrifuged and the pooled supernatants dried. Following extraction, the samples were derivatised using the modified methyl chloroformate alkylation procedure detailed by Smart et al [6], before being analysed using a GC-MS instrument.…”

Toxin-Antitoxin Systems of Mycobacterium smegmatis Are Essential for Cell Survival

“…For the analysis of intracellular metabolites, the quenched samples were centrifuged at 36,086 ϫ g for 20 min at Ϫ20°C to separate the microbial cells from the extracellular medium. The supernatants were discarded, and the cell pellets were submitted to metabolite extraction as described in Smart et al (45). Before extraction, 20 l of internal standard (L-alanine-2,3,3,3-d 4 10 mM) was added to each sample.…”

“…Samples were then processed as described in Smart et al (45). The samples were quenched by rapidly transferring 50 ml of the microbial culture into Falcon tubes containing 150 ml of a precooled (Ϫ20°C) solution of cold glycerol/saline.…”

“…The column used for all analyses was a ZB-1701 (Phenomenex), 30 ϫ 250 m (internal diameter) ϫ 0.15 (film thickness), with a 5-m guard column. The MS was operated in scan mode (start after 6 min; mass range 38 -650 atomic mass units at 1.47 scans/s (45).…”

Metabolic profiling of mussel larvae: effect of handling and culture conditions

“…Raw spectral data were pre-processed according to Smart et al ( 2010 ), using the R package Metab V1.0 (Aggio et al 2011 ) to facilitate highthroughput processing of the data by the Automated Mass Spectral Deconvolution and Identification System (AMDIS). Twenty seven metabolites from different chemical classes were reliably identified by comparing retention times and m/z ratios against an in-house library of spectra.…”