A Yeast Metabolite Extraction Protocol Optimised for Time-Series Analyses

Sasidharan, Kalesh; Soga, Tomoyoshi; Tomita, Masaru; Murray, Douglas B.

doi:10.1371/journal.pone.0044283

Cited by 31 publications

(29 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…AMP, ADP and ATP were extracted by bead-beating and measured by CE-MS, cell number and volume were measured by electronic cell counter (CDA500, Sysmex, Japan) and metabolite concentrations calculated to mM 48.…”

Section: Methodsmentioning

confidence: 99%

Time resolved DNA occupancy dynamics during the respiratory oscillation uncover a global reset point in the yeast growth program

Amariei

Machné

Štolc

et al. 2014

MIC

Self Cite

View full text Add to dashboard Cite

The structural dynamics of chromatin have been implicated in the regulation of fundamental eukaryotic processes, such as DNA transcription, replication and repair. Although previous studies have revealed that the chromatin landscape, nucleosome remodeling and histone modification events are intimately tied into cellular energetics and redox state, few studies undertake defined time-resolved measurements of these state variables. Here, we use metabolically synchronous, continuously-grown yeast cultures to measure DNA occupancy and track global patterns with respect to the metabolic state of the culture. Combined with transcriptome analyses and ChIP-qPCR experiments, these paint an intriguing picture where genome-wide nucleosome focusing occurs during the recovery of energy charge, followed by clearance of the promoter regions and global transcriptional slow-down, thus indicating a nucleosome-mediated “reset point” for the cycle. The reset begins at the end of the catabolic and stress-response transcriptional programs and ends prior to the start of the anabolic and cell-growth transcriptional program, and the histones on genes from both the catabolic and anabolic superclusters are deacetylated.

show abstract

Section: Methodsmentioning

confidence: 99%

Time resolved DNA occupancy dynamics during the respiratory oscillation uncover a global reset point in the yeast growth program

Amariei

Machné

Štolc

et al. 2014

MIC

Self Cite

View full text Add to dashboard Cite

show abstract

“…Details of the remaining CE‐MS‐based metabolomics applications of the past two years can be found in Table .…”

Section: Applicationsmentioning

confidence: 99%

CE‐MS for metabolomics: Developments and applications in the period 2012–2014

2014

View full text Add to dashboard Cite

In the field of metabolomics, CE-MS is now regarded as a useful complementary analytical technique for the profiling of (highly) polar ionogenic metabolites in biological samples. Over the past few years, significant advancements have been made in CE-MS approaches for metabolic profiling studies. This paper, which is a follow-up of three previous review papers covering the years 2000-2012 [Electrophoresis 2009, 30, 276-291; Electrophoresis 2011, 32, 52-65; Electrophoresis 2013, 34, 86-98], provides an update of these developments covering the scientific literature from July 2012 to June 2014. Attention will be paid to novel interfacing techniques for coupling CE to MS and their implications for metabolomics studies. The potential of CEC-MS and MEKC-MS are also considered, and CE-MS systems for high-throughput metabolic profiling are discussed. The applicability of CE-MS for metabolomics studies is demonstrated by representative examples in the fields of biomedical, clinical, microbial, plant, environmental, and food metabolomics. An overview of recent CE-MS-based metabolomics studies is given in a table, which provides information on sample type and pretreatment, capillary coatings, and MS detection mode. Finally, general conclusions and perspectives are given.

show abstract

“…Among the most widely used extraction procedures for a number of organisms including yeast is boiling in ethanol . Alternatives are methods using cold methanol and/or chloroform in combination with cycles of freeze–thawing or vigorous shaking for metabolite extraction . According to Canelas et al., near‐complete extraction of a number of metabolites with good reproducibility was possible using boiling ethanol, hot water, and cold methanol extraction .…”

Section: Introductionmentioning

confidence: 99%

Sample preparation workflow for the liquid chromatography tandem mass spectrometry based analysis of nicotinamide adenine dinucleotide phosphate cofactors in yeast^†

et al. 2014

View full text Add to dashboard Cite

The accurate quantification of the highly unstable intracellular cofactor nicotinamide adenine dinucleotide phosphate in its oxidized and reduced forms demands a thorough evaluation of the analytical workflow and dedicated methods reflecting their solution chemistry as well as the biological importance of their ratio. In this work, we present a workflow for the analysis of intracellular levels of oxidized and reduced nicotinamide adenine dinucleotide phosphate in the yeast Pichia pastoris, including hot aqueous extraction, chromatographic separation in reversed-phase conditions employing a 100% wettable stationary phase, and subsequent tandem mass spectrometric analysis. A thorough evaluation and optimization of the sample preparation procedure resulted in excellent biological repeatabilities (on average <10%, N = 3) without employing an internal standardization approach. As a consequence, the methodology proved to be appropriate for the relative assessment of intracellular levels of oxidized and reduced nicotinamide adenine dinucleotide phosphate in different P. pastoris strains. The ratio of reduced versus oxidized nicotinamide adenine dinucleotide phosphate was significantly higher in an engineered strain overexpressing glucose-6-phosphate dehydrogenase than in the corresponding wildtype strain. Interestingly, a difference was also observed in the nicotinamide adenine dinucleotide phosphate pool size, which was significantly higher in the wildtype than in the modified strain.

show abstract

A Yeast Metabolite Extraction Protocol Optimised for Time-Series Analyses

Cited by 31 publications

References 33 publications

Time resolved DNA occupancy dynamics during the respiratory oscillation uncover a global reset point in the yeast growth program

Time resolved DNA occupancy dynamics during the respiratory oscillation uncover a global reset point in the yeast growth program

CE‐MS for metabolomics: Developments and applications in the period 2012–2014

Sample preparation workflow for the liquid chromatography tandem mass spectrometry based analysis of nicotinamide adenine dinucleotide phosphate cofactors in yeast^†

Contact Info

Product

Resources

About

A Yeast Metabolite Extraction Protocol Optimised for Time-Series Analyses

Cited by 31 publications

References 33 publications

Time resolved DNA occupancy dynamics during the respiratory oscillation uncover a global reset point in the yeast growth program

Time resolved DNA occupancy dynamics during the respiratory oscillation uncover a global reset point in the yeast growth program

CE‐MS for metabolomics: Developments and applications in the period 2012–2014

Sample preparation workflow for the liquid chromatography tandem mass spectrometry based analysis of nicotinamide adenine dinucleotide phosphate cofactors in yeast†

Contact Info

Product

Resources

About

Sample preparation workflow for the liquid chromatography tandem mass spectrometry based analysis of nicotinamide adenine dinucleotide phosphate cofactors in yeast^†