| Autor: |
Campos CG; Brazilian Agricultural Research Corporation, Embrapa Agroenergy, Brasília, DF, Brazil.; Institute of Chemistry, Federal University of Goiás, Goiânia, GO, Brazil., de Aquino Ribeiro JA; Brazilian Agricultural Research Corporation, Embrapa Agroenergy, Brasília, DF, Brazil., de Almeida JRM; Brazilian Agricultural Research Corporation, Embrapa Agroenergy, Brasília, DF, Brazil.; Graduate Program on Chemical and Biological Technologies, Institute of Chemistry, University of Brasília, Brasília, DF, Brazil., Quirino BF; Brazilian Agricultural Research Corporation, Embrapa Agroenergy, Brasília, DF, Brazil.; Graduate Program on Chemical and Biological Technologies, Institute of Chemistry, University of Brasília, Brasília, DF, Brazil.; Genomic Sciences and Biotechnology Program, Universidade Católica de Brasilia, Brasília, DF, Brazil., Abdelnur PV; Brazilian Agricultural Research Corporation, Embrapa Agroenergy, Brasília, DF, Brazil. patricia.abdelnur@embrapa.br.; Institute of Chemistry, Federal University of Goiás, Goiânia, GO, Brazil. patricia.abdelnur@embrapa.br. |
| Abstrakt: |
Mass spectrometry is a sensitive and selective analytical technique that enables detection and quantitation of low abundance compounds in a complex sample matrix. Targeted metabolomics allows for quantitative analysis of metabolites, providing kinetic information of production and consumption rates, an essential step to investigate microbial metabolism. Here, we describe a targeted metabolomics protocol for yeast samples, from sample preparation to mass spectrometry analysis, which enables the identification of metabolic fluxes after xylose consumption. Sample preparation methods were optimized for quenching of yeast metabolism followed by intracellular metabolite extraction, using cold methanol and boiling ethanol protocols. Ultrahigh performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) methods using ion pair chromatography (IPC) and hydrophilic interaction liquid chromatography (HILIC) allowed for the quantitation of 18 metabolites involved in central carbon metabolism (glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle). The protocol here described was successfully applied to quantify metabolites in Scheffersomyces stipitis, Spathaspora passalidarum, Spathaspora arborariae, and Candida tenuis samples after xylose consumption. |
