Metabolite extraction from S. cerevisiae

Reagents required:

Cell growth and media:

YPD: 1% yeast extract, 2% peptone and 2% glucose

SGE: Yeast nitrogen base without amino acids with 1% glycerol and 2% ethanol

Metabolite quenching and extraction: quenching solution - 60% Methanol, extraction solution - 75% Ethanol, dry ice/methanol/water bath

Other abbreviations: RT – room temperature, OD₆₀₀ – optical density at 600 nm

Note: While the protocol is described for yeast, this protocol will work for most microbial cells (and not only S. cerevisiae) grown in a variety of liquid medium.

1. Start an overnight culture of desired strain in a culture tube in 4ml YPD media. Grow the strains at 30°C and 250rpm.
2. Start a 30 ml secondary culture in a 100ml flask at OD₆₀₀ ~ 0.15, and let it grow till OD₆₀₀ is in mid-log phase (OD₆₀₀ ~ 1.0, it takes around 4hrs at 30°C and 250 rpm).
3. If media shifting has to be done, pellet around 15ml of log-phase growing culture by centrifuging at 1500g for 2 min at RT, and wash with sterile water (resuspend in 5ml water, and spin at 1500g for 2 min, RT). Resuspend the pellet in the desired media (e.g. SGE) and culture at 30°C and 250rpm for desired time.
4. Start preparing the methanol bath ~1.5hrs prior to the metabolite extraction time-points. For this, in a small thermocol/insulated box, add 60% methanol and lower the temperature by intermittently adding dry ice. The temperature should be around -40°C. During the whole process of extraction, ensure that the temperature is maintained between -38 to 42°C, and not does not exceed 45°C (otherwise the cells will freeze).
5. At the time point when metabolite extraction is desired, measure OD₆₀₀ and quench 10 OD₆₀₀ of cells in 40ml of 60% Methanol in a 50 ml falcon tube maintained in the methanol bath at -40°C for 5 min. Gently invert the tube twice for universal mixing. Note: this ratio of cells to quenching solution can be scaled appropriately
6. Centrifuge the suspension at 1000g for 3 min at -8°C (or the lowest temp the centrifuge goes (up to -20°C)).
7. Carefully discard the supernatant (do not pipette), and wash the pellet in ~800ml of cold 60% methanol (-40°C), and transfer it to a chilled 2ml vial (-40°C) kept in a floater in the methanol bath. Be careful to resuspend all the cell-pellet.
8. Centrifuge the vials at the 1000g for ~2min at -8°C (or the lowest temp the centrifuge goes (up to -20C)).
9. Discard the supernatant carefully, and gently tap on a tissue paper to remove excess methanol.
10. Resuspend the pellet in 75% ethanol at RT by vortexing the sample. Do not use pipette for resuspension. Heat the suspension at 80°C for 3min and transfer it to ice for 5 min. At this stage if needed, the samples can be kept on ice for ~1hr, if other immediate extractions have to be performed for next time-points.
    Optional Step: If required at this step, add U-¹³CAsp or any other labelled compound as internal standard, established for your protocol (the concentration to be added depends on the sensitivity of the Mass-Spec used for detection).
11. Centrifuge the suspension at 20000g for 1 min at RT, and transfer 950ml of the supernatant to another fresh tube.
12. Centrifuge the 950ml of supernatant again at 20000g for 10 min at RT. Transfer the supernatant into 4 vials: 300ml into 1^st tube (for amino acid), 300ml into 2^nd tube (for glycolytic intermediates), 200ml into 3^rd tube (extra, can be used as a technical replicate) and 100ml into 4^th tube (for TCA). If extraction is being performed specifically for detection of TCA intermediates, prepare the metabolite extract from 3OD₆₀₀ cells (volume of quenching solution to sample is in the ratio 4:1 ), and divide it into triplicate by resuspending 300ml each into 3 vials.
13. Dry the tubes using a speed-vac at 30°C for ~2hrs, and store at -80°C.
14. For TCA intermediates, derivatization is required before detection. The detailed protocol of this derivatization is available in (Walvekar et al., 2018). The analysis parameters for LC-MS/MS, as well as Q1/Q3 parameters for different metabolites can also be found in (Walvekar et al., 2018). On ABSciex Qtrap6500, for detection of amino acids, samples can be dissolved in 750ml of LC-MS grade water; derivatized TCA samples in 1ml of 50% Methanol in water; and for glycolytic intermediates, samples are dissolved in 200ml of 30% Acetonitrile in water with 5mM ammonium acetate.

References:

Walvekar, A., Rashida, Z., Maddali, H., and Laxman, S. (2018). A versatile LC-MS / MS approach for comprehensive , quantitative analysis of central metabolic pathways. Wellcome Open Res. 3, 1–11.

https://wellcomeopenresearch.org/articles/3-122/v1

Category