An optimized protocol for metabolome analysis in yeast using direct infusion electrospray mass spectrometry.
A method for the global analysis of yeast intracellular metabolites, based on electrospray mass spectrometry (ES-MS), has been developed. This has involved the optimization of methods for quenching metabolism in Saccharomyces cerevisiae and extracting the metabolites for analysis by positive-ion electrospray mass spectrometry. The influence of cultivation conditions, sampling, quenching and extraction conditions, concentration step, and storage have all been studied and adapted to allow direct infusion of samples into the mass spectrometer and the acquisition of metabolic profiles with simultaneous detection of more than 25 intracellular metabolites. The method, which can be applied to other micro-organisms and biological systems, may be used for comparative analysis and screening of metabolite profiles of yeast strains and mutants under controlled conditions in order to elucidate gene function via metabolomics. Examples of the application of this analytical strategy to specific yeast strains and single-ORF yeast deletion mutants generated through the EUROFAN programme are presented.
Castrillo
Juan I
JI
School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT, UK.
Hayes
Andrew
A
Mohammed
Shabaz
S
Gaskell
Simon J
SJ
Oliver
Stephen G
SG
D002021:Buffers; D013058:Mass Spectrometry; D008970:Molecular Weight; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000254:growth & development / Q000378:metabolism*; D012984:Software
D002021:Buffers