Overview
This protocol is used to extract and quantify intracellular metabolites from yeast. In particular, it's designed to extract highly thermostable metabolites with fast transport kinetics.
Procedure
Materials
- Boiling buffered ethanol (75% ethanol, 10mM Hepes pH 7.0)
- PBS (can be non-sterile)
- Vacuum filtration unit (e.g. VWR 22001-784)
- Filter disks
- I've used 25mm diameter, 0.45μm pore, cellulose nitrate (VWR 28454-053)
Method
- Grow cells to desired density. Ideally, you want roughly 20 OD*mL of yeast (e.g. 4mL at OD 5, or 20mL at OD 1).
- More yeast will clog the filter disk and slow down your wash steps.
- Place a new filter disk on the vacuum filter unit and turn on the vacuum.
- Apply your cell sample to the disk. Wash with 3 x 30 mL of PBS.
- The cells should appear as a thin paste on top of the disk.
- Remove filter disk and place in 15mL Falcon tube.
- Add 500μL boiling buffered ethanol and vortex
- The cells should wash off the disk into the buffer. If they don't, add more buffer (more buffer means more time concentrating the solution later)
- Incubate for 5 minutes at 80°C
- Spin down sample, max speed for 10 minutes
- Remove supernatant to eppendorf tube
- Concentrate to <50μL in vacufuge
- Be careful not to completely dry out your sample
- Spin down sample, max speed for 10 minutes
- Concentration will cause more compounds to crash out of solution. These precipitates need to be cleared before the sample can be run on the LC.
- Remove supernatant, adjust volume to 50uL with NP water
- Assay sample on LC-MS
Analysis
- CSY3 is ~0.53 mg DCW/(OD*mL)
- Hans et al. quote a value of 2.38 mL/g DCW as the intracellular volume of S. cerevisiae[1]
- We arrive at similar numbers (within a factor of 1.5) by reasoning from first principles (cell number per OD and average cell size).
- For 20 OD*mL, this gives an approximate intracellular volume of 25μL. So measured concentrations in the final sample are roughly half that inside the cell.
- Reproducibility between extracts of a given sample is ~15-20%, similar to the reproducibility between samples.
References
- Hans MA, Heinzle E, and Wittmann C. Quantification of intracellular amino acids in batch cultures of Saccharomyces cerevisiae. Appl Microbiol Biotechnol. 2001 Sep;56(5-6):776-9. DOI:10.1007/s002530100708 | PubMed ID:11601629 | HubMed [hans]
- Villas-Bôas SG, Højer-Pedersen J, Akesson M, Smedsgaard J, and Nielsen J. Global metabolite analysis of yeast: evaluation of sample preparation methods. Yeast. 2005 Oct 30;22(14):1155-69. DOI:10.1002/yea.1308 | PubMed ID:16240456 | HubMed [villas-boas]
- Maharjan RP and Ferenci T. Global metabolite analysis: the influence of extraction methodology on metabolome profiles of Escherichia coli. Anal Biochem. 2003 Feb 1;313(1):145-54. DOI:10.1016/s0003-2697(02)00536-5 | PubMed ID:12576070 | HubMed [maharjan]
All Medline abstracts: PubMed | HubMed
Contact
Josh Michener
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