Model Organisms Biochemistry

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Authors: Mirjam Wamelink, Erwin Jansen, Eduard Struys, Hans Lehrach, Cornelis Jakobs & Markus Ralser

Introduction

The pentose phosphate pathway (PPP) is a central pathway of the cellular carbohydrate metabolism. The PPP is directly interconnected with glycolysis and composed of a irreversible, oxidative- and a reversible, non-oxidative part. The PPP provides the cell with intermediates for several bioorganic syntheses, and acts as balancer for the redox state in response to oxidant exposures (1,2). Here, we provide a liquid chromatography tandem mass spectrometry (LC-MS/MS) based protocol for accurate quantification of PPP intermediates in S. cerevisiae.

Reagents

  1. Yeast growth media (dependent on the desired strain and experiment)
  2. Hanks Balanced Salt Solution (HBSS) without Phenol red (e.g. Sigma H1387 + 4.2 mM sodium bicarbonate)
  3. Perchloric acid
  4. Acid washed glass beads (425-600 μm, Sigma)
  5. Phosphate buffer, 1 M, pH= 11.5
  6. 13C6-glucose-6-phosphate prepared by glucokinase using 13C6-glucose 3
  7. Acetonitrile (Merck)
  8. Octylammonium acetate prepared from octylamine (Sigma) and acetic acid
  9. 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 (6pg) to generate calibration curves for an accurate quantification.

Equipment

  1. Microcentrifuge
  2. Spectrophotometer
  3. Vortex with TurboMix equipment (e.g. Scientific Industries Vortex Genie 2 with Turbo Mix holder SI-0563), or FastPrep-24 machine (MP Biomedicals)
  4. Triple quadrupole tandem mass spectrometer with classic HPLC-unit (we used a - PE-Sciex API-3000 tandem mass spectrometer equipped with an electrospray source (Turbo Ion Spray) and a Perkin-Elmer series 200 HPLC unit)
  5. Dedicated 3.9×150 mm Symmetry C18 HPLC column, bead size 5 µm (Waters)

Procedure

  1. inoculate 2×3ml media with the yeast strain to be analyzed
  2. shake or rotate overnight at 30 °C
  3. use this overnight cultures to adjust 2×30ml yeast media to an OD600 of 0.1 – 0.2
  4. grow the cells to mid-log phase, record OD600 value
  5. 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

  • 6. thaw the samples
  • 7. 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
  • 8. as internal standard, add 50 μl 10 μM 13C6-glucose-6-phosphate to 50 μl of the lysates (and to the calibrators)
  • 9. neutralize the samples with 1 M phosphate buffer (pH 11.5) to pH 7-8
  • 10. centrifuge for 5 min, at greater than or equal to 14000g, 4 °C.
  • 11. transfer the supernatant to appropriate HPLC vials
  • 12. cap the vials
  • 13. 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
  • 14. equilibrate your C18 HPLC column with solvent A for several minutes with a flow rate of 1 ml/min.
  • 15. 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)
  • 16. If necessary, split the flow post-column dependent on your mass spectrometer
  • 17. Set the MS to multiple reaction monitoring mode (MRM) with the electrospray source operating in the negative-ion mode.
  • 18. 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)
  • 19. 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.

Timing

2 days + yeast overnight culture

References

  1. Wamelink, M.M., Struys, E.A. & Jakobs, C. The biochemistry, metabolism and inherited defects of the pentose phosphate pathway: a review. J Inherit Metab Dis 31, 703-717 (2008).
  2. Ralser, M. et al. Dynamic rerouting of the carbohydrate flux is key to counteracting oxidative stress. J Biol 6, 10 (2007).
  3. Huck, J.H., Struys, E.A., Verhoeven, N.M., Jakobs, C. & van der Knaap, M.S. Profiling of pentose phosphate pathway intermediates in blood spots by tandem mass spectrometry: application to transaldolase deficiency. Clinical chemistry 49, 1375-1380 (2003).
  4. Wamelink, M.M. et al. Quantification of sugar phosphate intermediates of the pentose phosphate pathway by LC-MS/MS: application to two new inherited defects of metabolism. J Chromatogr B Analyt Technol Biomed Life Sci 823, 18-25 (2005).

Acknowledgements

We thank our lab members for critical discussions.

Associated Publications

Metabolic re-configuration precedes transcriptional regulation in the antioxidant response, Markus Ralser, Mirjam M C Wamelink, Simone Latkolik, Erwin E W Jansen, Hans Lehrach, and Cornelis Jakobs, Nature Biotechnology 27 (7) 604 - 605 doi:10.1038/nbt0709-604

Author information

Mirjam Wamelink, Erwin Jansen, Eduard Struys & Cornelis Jakobs, Metabolic Unit, VU University Medical Center Amsterdam, de Boelelaan 1117, 1081 HV Amsterdam, The Netherlands

Hans Lehrach & Markus Ralser, Max Planck Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany

Source: Protocol Exchange (2009) doi:10.1038/nprot.2009.140. Originally published online 14 July 2009.

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