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.

Start an overnight culture of desired strain in a culture tube in 4ml YPD media. Grow the strains at 30°C and 250rpm.
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).
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.
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).
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
Centrifuge the suspension at 1000g for 3 min at -8°C (or the lowest temp the centrifuge goes (up to -20°C)).
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.
Centrifuge the vials at the 1000g for ~2min at -8°C (or the lowest temp the centrifuge goes (up to -20C)).
Discard the supernatant carefully, and gently tap on a tissue paper to remove excess methanol.
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).
Centrifuge the suspension at 20000g for 1 min at RT, and transfer 950ml of the supernatant to another fresh tube.
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.
Dry the tubes using a speed-vac at 30°C for ~2hrs, and store at -80°C.
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