Smolke:Protocols/Yeast metabolite extraction

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Contents

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

  1. 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.
  2. Place a new filter disk on the vacuum filter unit and turn on the vacuum.
  3. 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.
  4. Remove filter disk and place in 15mL Falcon tube.
  5. 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)
  6. Incubate for 5 minutes at 80°C
  7. Spin down sample, max speed for 10 minutes
  8. Remove supernatant to eppendorf tube
  9. Concentrate to <50μL in vacufuge
    • Be careful not to completely dry out your sample
  10. 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.
  11. Remove supernatant, adjust volume to 50uL with NP water
  12. 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

  1. Hans MA, Heinzle E, and Wittmann C. . pmid:11601629. PubMed HubMed [hans]
  2. Villas-Bôas SG, Højer-Pedersen J, Akesson M, Smedsgaard J, and Nielsen J. . pmid:16240456. PubMed HubMed [villas-boas]
  3. Maharjan RP and Ferenci T. . pmid:12576070. PubMed HubMed [maharjan]
All Medline abstracts: PubMed HubMed

Contact

Josh Michener

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