Smolke:Protocols/Yeast metabolite extraction

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.

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).
 * 2) *More yeast will clog the filter disk and slow down your wash steps.
 * 3) Place a new filter disk on the vacuum filter unit and turn on the vacuum.
 * 4) Apply your cell sample to the disk. Wash with 3 x 30 mL of PBS.
 * 5) *The cells should appear as a thin paste on top of the disk.
 * 6) Remove filter disk and place in 15mL Falcon tube.
 * 7) Add 500μL boiling buffered ethanol and vortex
 * 8) *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)
 * 9) Incubate for 5 minutes at 80°C
 * 10) Spin down sample, max speed for 10 minutes
 * 11) Remove supernatant to eppendorf tube
 * 12) Concentrate to <50μL in vacufuge
 * 13) *Be careful not to completely dry out your sample
 * 14) Spin down sample, max speed for 10 minutes
 * 15) *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.
 * 16) Remove supernatant, adjust volume to 50uL with NP water
 * 17) 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 hans
 * 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.

Contact
Josh Michener