A protocol for the investigation of the intracellular Staphylococcus aureus metabolome.

Systems biology studies assume the acquisition of reliable and reproducible data sets. Metabolomics, in particular, requires comprehensive evaluated workflows to enable the analysis of hundreds of different compounds. Therefore, a protocol to elucidate the metabolome of the gram-positive pathogen, Staphylococcus aureus COL strain, grown in a chemically defined medium is introduced here. Different standard operating procedures in the field of metabolome experiments were tested for common pitfalls. These included suitable and fast sampling processes, efficient metabolite extraction, quenching effectiveness (energy charge), and estimation of leakage and recovery of metabolites. Moreover, a cell disruption protocol for S. aureus was developed and optimized for metabolome analyses, for the express purpose of obtaining reproducible data. We used complementary methods (e.g., gas chromatography and/or liquid chromatography coupled with mass spectrometry) to detect the highly chemically diverse groups of metabolites for a global insight into the intracellular metabolism of S. aureus. Copyright (c) 2010 Elsevier Inc. All rights reserved.