- inoculate 2 x 3ml media with the yeast strain to be analyzed
- shake or rotate overnight at 30 °C
- use this overnight cultures to adjust 2 x 30ml yeast media to an OD600 of 0.1 - 0.2
- grow the cells to mid-log phase, record OD600 value
There are two possibilities how to proceed a) lyse the complete 30 ml culture. This has the advantage of yielding in higher concentrated metabolite extracts or b) analyze 1.5 ml aliquots, which is the method of choice if a fast sample handling is required. The lysate procedure requires breaking the cells in a buffer containing 2% perchloric acid; this guarantees, that metabolic enzymes are immediately inactivated upon lysis
Lysate Procedure A (30 ml culture)
i) centrifuge the 30 ml culture in a 50 ml tube, 3 min, 3000 g, RT
ii) re-suspend the pellet in 1 ml HBSS buffer
iii) transfer the suspension to a 1.5 ml screw tube
iv) centrifuge 30 sec, 5000g-7000g in a table-top centrifuge
v) remove the supernatant quickly
vi) freeze the sample immediately in liquid nitrogen
vii) (at this point, the pellets can be transferred to -80 °C for storage)
viii) place the tubes on dry-ice, open the tubes
ix) add to the cell pellets an equal volume of glass beads (Hint: Use a 1.5ml-tube-lid as bucket)
x) transfer the tubes to normal ice
xi) add 800 μl of 98% HBSS / 2% perchloric acid
xii) close the tubes, place them into the Fast-Prep-24 machine (alternatively, a Vortex with Turbo-Mix equipment can be used, see Procedure B)
xiii) start the machine, 6.5 m/s, 40 sec
xiv) cool the tubes on ice for 5 min
xv) repeat the fast-prep procedure
xvi) incubate the samples for 30min on ice
xvii) centrifuge the samples for 2 min, at greater than or equal to 14000g, 4 °C
xviii) transfer the supernatant to a fresh tube
xix) freeze and store the samples at -80 °C until the LC-MS/MS analysis, continue at step 6
Procedure B (1.5 ml culture aliquots)
i) aliquot the mid-log culture to a desired number of 1.5ml screw tubes
ii) incubate the cultures in a thermo block for at least 5 min at 30 °C
iii) centrifuge 5000g-7000g, 30sec
iv) remove the supernatant quickly
v) freeze the pellet immediately in liquid nitrogen
vi) (at this point, the pellets can be transferred to -80 °C for storage)
vii) place the tubes on dry-ice and open them
viii) add to the cell pellet an equal volume of glass beads (Hint: Use a 1.5 ml-tube-lid as bucket)
ix) transfer the tubes to normal ice
x) add 400 μl of 98% HBSS /2 % perchloric acid
xi) in the coldroom, transfer the tubes to the Vortex with Turbo-Mix equipment
xii) vortex 4 min, max speed
xiii) incubate on ice for 5 min
xiv) vortex again, 4 min, max speed
xv) incubate the samples for 30 min on ice
xvi) centrifuge the samples for 2 min at greater than of equal to 14000g, 4 °C
xvii) transfer the supernatant to a fresh tube
xviii) freeze and store the tubes at -80 °C until LC-MS/MS analysis, continue at step 6
LC-MS/MS analysis
- thaw the samples
- Prepare calibrator curves for dihydroxacetone phosphate (dhap), ribose 5-phosphate (r5p), erythrose 4-phosphate (e4p), xylulose 5-phosphate or ribulose 5-phosphate (x5p), glucose 6-phosphate or fructose 6-phosphate (g6p), sedoheptulose 7-phosphate (s7p) and 6-phosphogluconate in the range of the expected concentrations
- as internal standard, add 50 μl 10 μM 13C6-glucose-6-phosphate to 50 μl of the lysates (and to the calibrators)
- neutralize the samples with 1 M phosphate buffer (pH 11.5) to pH 7-8
- centrifuge for 5 min, at greater than or equal to 14000g, 4 °C.
- transfer the supernatant to appropriate HPLC vials
- cap the vials
- prepare the solvents for the gradient-chromatography. We recommend to use 12.5% acetonitrile in water supplemented with 500 mg/l octylammonium acetate (pH 7.5) as binary solvent A and 50% acetonitrile in water supplemented with 500 mg/l octylammonium acetate (pH 7.5) as binary solvent B
- equilibrate your C18 HPLC column with solvent A for several minutes with a flow rate of 1 ml/min.
- Set a linear gradient from 100% solvent A to 40% solvent A and 60% solvent B in 8 min (at a flow rate of 1 ml/min)
- If necessary, split the flow post-column dependent on your mass spectrometer
- Set the MS to multiple reaction monitoring mode (MRM) with the electrospray source operating in the negative-ion mode.
- Configure your MS/MS to record the following MRM transitions: dhap: m/z -169/-97; e4p -199/-97; r5p and x5p: m/z -229/-97; g6p: m/z -259/-97, 13C6-glucose 6-P (IS): m/z -265/-97; 6pg: m/z -275/-97 and s7p: m/z -289/-97. Optimized MS settings for an ABI PE-Sciex API-3000 tandem mass spectrometer equipped with an electrospray source (Turbo Ion Spray) as well as the detection limits with this setting have been reported earlier 4
For a relative quantification of the PPP intermediates we suggest to normalize the metabolites to the OD600 value of the starter culture rather than to a cell number count.
Note: xylulose 5-phosphate/ ribulose 5-phosphate and glucose 6-phosphate/fructose 6-phosphate can not be separately quantified with this method due to chromatographic co-elution.