Time-related multivariate strategy for the comprehensive evaluation of microbial chemical data.

INTRODUCTION: In microbial metabolomics, the use of multivariate data analysis (MDVA) has not been comprehensively explored regarding the different techniques available and the information that each gives about the metabolome. To overcome these limitations, here we show the use of Fusarium oxysporum cultured in the presence of exogenous alkaloids as a model system to demonstrate a comprehensive strategy for metabolic profiling. MATHERIALS AND METHODS: F. oxysporum was harvested on different days of incubation after alkaloidal addition, and the chemical profiles were compared using LC-MS data and MDVA. We show significant innovation to evaluate the chemical production of microbes during their life cycle by utilizing the full capabilities of Partial Least Square (PLS) with microbial-specific modeling that considers incubation days, media culture availability, and growth rate in solid media. RESULTS AND DISCUSSCION: Results showed that the treatment of the Y-data and the use of both PLS regression and discrimination (PLSr and PLS-DA) inferred complemental chemical information. PLSr revealed the metabolites that are produced/consumed during fungal growth, whereas PLS-DA focused on metabolites that are only consumed/produced at a specific period. Both regression and classificatory analysis were equally important to identify compounds that are regulated and/or selectively produced as a response to the presence of the alkaloids. Lastly, we report the annotation of analogs from the piperidine alkaloids biotransformed by F. oxysporum as a defense response to the toxic plant metabolites. These molecules do not show the antimicrobial potential of their precursors in the fungal extracts and were rapidly produced and consumed within 4 days of microbial growth.

Mass spectrometry for characterization of homologous piperidine alkaloids and their activity as acetylcholinesterase inhibitors.

RATIONALE: Piperidine alkaloids from Senna spectabilis constitute a rare class of natural products with several biological activities. However, the absence of chromophores makes their structural elucidation by conventional methods a great challenge. In this context, mass spectrometry emerges as a powerful tool for metabolomics studies. METHODS: The piperidine alkaloids (-)-cassine and (-)-spectaline and the semisynthetic derivatives (-)-3-O-acetylcassine and (-)-3-O-acetylspectaline were investigated by electrospray ionization tandem mass spectrometry (ESI-MS/MS) in the positive mode and electron ionization mass spectrometry (EI-MS). ESI fragmentation studies were performed with a quadrupole time-of-flight instrument; N2 was used as collision gas. The acetylcholinesterase inhibitory activity of the investigated compounds was evaluated by bioautography and microplate screening assays. RESULTS: ESI-MS/MS and EI-MS provided valuable and complementary information about the structure of the piperidine compounds. Collision-induced dissociation experiments (MS/MS) revealed that neutral elimination of water or acetic acid is the major fragmentation pathway, which agrees with the stereochemistry proposed for (-)-cassine and (-)-spectaline and the semisynthetic derivatives (-)-3-O-acetylcassine and (-)-3-O-acetylspectaline. CONCLUSIONS: The ESI-MS/MS and EI-MS studies allowed us to propose fragmentation mechanisms for piperidine alkaloids and derivatives. Therefore, mass spectrometry is an important tool for characterizing the structure of these compounds and for supporting further metabolomics studies.