Metabolite identification in Synechococcus sp. PCC 7002 using untargeted stable isotope assisted metabolite profiling

Metabolite profiling using mass spectrometry provides an attractive approach for the interrogation of cellular metabolic capabilities. Untargeted metabolite profiling has the potential to identify numerous novel metabolites; however, de novo identification of metabolites from spectral features remains a challenge. Here we present an integrated workflow for metabolite identification using uniform stable isotope labeling. Metabolite profiling of cell and growth media extracts of unlabeled control, (15)N, and (13)C-labeled cultures of the cyanobacterium, Synechococcus sp. PCC 7002 was performed using normal phase liquid chromatography coupled to mass spectrometry (LC-MS). Visualization of three-way comparisons of raw data sets highlighted characteristic labeling patterns for metabolites of biological origin allowing exhaustive identification of corresponding spectral features. Additionally, unambiguous assignment of chemical formulas was greatly facilitated by the use of stable isotope labeling. Chemical formulas of metabolites responsible for redundant spectral features were determined and fragmentation (MS/MS) spectra for these metabolites were collected. Analysis of acquired MS/MS spectra against spectral database records led to the identification of a number of metabolites absent not only from the reconstructed draft metabolic network of Synechococcus sp. PCC 7002 but not included in databases of metabolism (MetaCyc or KEGG).