Determination of the Cellular Ion Concentration in Saccharomyces cerevisiae Using ICP-AES

Materials and Reagents

1. Petri dishes (Sigma-Aldrich, catalog number: P5606-400EA )
2. Toothpicks
3. 5 ml pipette (Gilson, catalog number: F123607 )
4. 25 mm diameter Whatman^® glass microfiber filters, grade GF/F (GE Healthcare, catalog number: 1825-025 )
5. Saccharomyces cerevisiae strains to be analyzed (performed here on BY4741 or BY4742 S. cerevisiae strains)
6. Yeast extract KAT (Ohly, catalog number: OHLY^® KAT)
7. Glucose (Merck, catalog number: 1083469029 )
8. EGTA (Acros Organic, catalog number: 409910250 )
9. HCl 37% (VWR, catalog number: 20255.29 )
10. Suprapur^® nitric acid 65% (Merck, catalog number: 100441 )
11. Certipur^® single-element standards for inductively coupled plasma spectroscopy (Merck, catalog number: depends on the ion to be analyzed)
12. Milli Q water
13. NaOH (Sigma-Aldrich, catalog number: S5881 )
14. YD plates (see Recipes)
15. YD medium (see Recipes)
16. 1 mM EGTA (1 L) (see Recipes)
    

Equipment

1. 500 ml Erlenmeyer (VWR, catalog number: 734-1833 )
2. 250 ml glass beakers (VWR, catalog number: 213-1124P )
3. 100 ml graduated cylinders (VWR, catalog number: 612-3836 )
4. Haldenwanger^TM porcelain crucibles (Thermo Fisher Scientific, catalog number: 12306507 )
5. Analytical balance (Mettler Toledo, catalog number: AB104-S )
6. Filter holding manifold (Hoefer, catalog number: FH225V )
7. Laboratory oven (Utest, catalog number: UTD )
8. Desiccator (VWR, catalog number: 467-0071P )
9. Carbolite^® muffle furnace (Sigma-Aldrich, catalog number: Z654973 )
10. iCAP 6500 ICP-OES CID spectrometer (Thermo Fisher Scientific, discontinued)
    

Procedure

1. Sample preparation
   1. Streak the S. cerevisiae strains to be analyzed on YD plates (Recipe 1). Incubate for two days at 28 °C.
   2. Using a sterile toothpick, select individual colonies.
   3. Re-suspend cells in 100 ml YD medium (in 500 ml Erlenmeyer) (Recipe 2) and grow them at 28 °C under agitation (120 rpm) to an OD₆₀₀ of 3 (OD₆₀₀ = 1 corresponds to a density of 1.25 x 10⁷ cells/ml).
      Note: Wash the 500 ml Erlenmeyer with 10% HCl before yeast growth to avoid any impact of the detergents used for washing the glassware on the ionic content measurement. Rinse them with Milli Q water and allow them to dry.
   4. Filter the 100 ml culture using glass microfiber filters on the filter holding manifold at 4 °C.
   5. Wash cells twice on the filter with 2 ml EGTA 1 mM (Recipe 3) at 4 °C.
   6. Wash cells twice with 2 ml Milli Q water at 4 °C.
   7. Immerge each filter in a heat-resistant porcelain crucible containing 10 ml Milli Q water for 10 min. Remove the filter from the crucible.
      Note: Before introducing any sample in the porcelain crucibles, weigh them when empty for further determination of the mass of dry matter. 
   8. Place the porcelain crucibles in an oven at 95 °C overnight to dry the sample. Figure 1 summarizes the protocol Steps A4 to A8.
   9. Place the porcelain crucibles in a desiccator for 8 h for further drying.
   10. Weigh the porcelain crucibles for further determination of the mass of dry matter.
   Note: Steps A4 to A8 can be carried out with 100 ml YD medium (without cells). This sample will be used as a blank for further measurement.
   
   
   Figure 1. Experimental Steps A4 to A8. The 100 ml yeast culture is first filtered. Then, cells on the filter are washed with EGTA 1 mM (twice) and Milli Q water (twice). The filter with washed cells is subsequently immerged in a porcelain crucible containing 10 ml Milli Q water. Finally, the filter is removed and the porcelain crucible is placed in an oven for further drying.
   
   
2. Measurement of the ionic content
   1. Place the samples in the muffle furnace at 500 °C for 4 h.
   2. Resuspend the ashes in 6.5% nitric acid (0.5 ml nitric acid 65% + 9.5 ml Milli Q H₂O).
   3. Dilute the ICP standards from Merck in a range of concentration that covers the expected ionic concentration in the samples to be analyzed. Acidify the standard solutions to 0.5% nitric acid (from 65% nitric acid). The standard concentrations used for measurement of yeast ionic content in our study are mentioned in Table 1.
   4. Measure the emitted light for each standard and sample. Typical instrument parameters are given in Table 2. Table 3 gathers the wavelengths and the viewing mode used for measurement of each cation.
   5. Deduce the ionic content in the samples using the calibration curve established from the standards (see Data analysis).
      
      Table 1. Concentrations of the standards (St) used for the calibration line
      
      
      Table 2. Typical instrument parameters used on the Thermo Scientific ICAP 6500 ICP OES
      
      
      Table 3. Wavelengths and viewing mode used for the twelve elements
      
      

Data analysis

The calibration curve of the emitted light as a function of the concentration of the standards is first modelled using linear regression. The validation parameter for a calibration line is a R² coefficient of minimum 0.999. An example of calibration line is provided in Figure 2.


Figure 2. Calibration line of the emitted light according to the concentration of nickel (ppb) in the five standard solutions

The equation of the linear regression is determined as:



with A the slope of the line and B its intercept. This equation is then used to deduce the concentration (in ppb or ppm) of the samples to be analyzed from the measurement of the intensity of the light emitted by the following equation:



The ionic content obtained in ppm can then be converted into µmol of the ion per gram of yeast dry matter. To do so, first determine the mass of dry matter by subtracting the mass of the empty porcelain crucibles to that of the crucibles containing the cells after drying. Then convert the ionic concentration obtained in ppm to µmol of the ion per gram of dry matter according to the following formula:



With MM the molar mass of the ion in g/mol.

The limit of quantification of each element is mentioned in Table 4.

Table 4. Limit of quantification for the twelve elements

Recipes

1. YD plates
   2 g (2% w/v) yeast extract KAT
   2 g (2% w/v) glucose
   2 g (2% w/v) agar
   Adjust to 100 ml with Milli Q water and autoclave
   Pour in Petri dishes
2. YD medium
   2 g (2% w/v) yeast extract KAT
   2 g (2% w/v) glucose
   Adjust to 100 ml with Milli Q water and autoclave
3. 1 mM EGTA (1 L)
   0.3804 g EGTA
   Adjust pH to 8.0 with NaOH
   

Acknowledgments

The work was supported by grants from the Fonds National de la Recherche Scientifique (FNRS, grant PDR-T.0206.16). L.T. is a research fellow at the ‘Fonds pour le Formation à la Recherche dans l’Industrie et dans l’Agriculture’. We thank Anne-Sophie Colinet for prior protocol development.

Competing interests

The authors declare that they have no conflicts of interest with the contents of this article.

References

1. Thines, L., Deschamps, A., Sengottaiyan, P., Savel, O., Stribny, J. and Morsomme, P. (2018). The yeast protein Gdt1p transports Mn²⁺ ions and thereby regulates manganese homeostasis in the Golgi. J Biol Chem 293(21): 8048-8055.
2. Dahlquist, R. L., and Knoll, J. W. (1978). Inductively Coupled Plasma-Atomic Emission Spectrometry: Analysis of Biological Materials and Soils for Major, Trace, and Ultra-trace Elements. Appl Spectrosc 32(1):1-30.
3. Eide, D. J., Clark, S., Nair, T. M., Gehl, M., Guerinot, M. L., and Harper, J. F. (2005). Characterization of the yeast ionome: a genome-wide analysis of nutrient mineral and trace element homeostasis in Saccharomyces cerevisiae. Genome Biol 6(9):R77.