Ketola, Raimo A.

Kiuru, Jari

Kokkonen, Juha T.

Penttilä, Merja

Rintala, Eija

Ruohonen, Laura

Simolin, Helena

In system biology, it is important to develop many kinds of analytical tools for elucidating and understanding cell physiology. Different kind of analytical data from transcriptome, proteome and metabolome levels of the cells are needed. For example, in a metabolome level, analysing of the concentrations of adenosine 5´-monophosphate (AMP), adenosine 5´-diphosphate (ADP), adenosine 5´-triphosphate (ATP), provides valuable information for understanding cellular energy metabolism. In our work, an ion-pairing liquid chromatography-electrospray ionisation mass spectrometry (LC-MS) method was developed for the analysis of AMP, ADP and ATP from Saccharomyces cerevisiae chemostats. The analytes were retained and separated by using a C18-reversed-phase microbore column, diisopropyl amine (DIPA) as an ion-pairing reagent and isocratic LC-program (60 mM DIPA/MeOH). Isopropanol was post column added for improving the ionisation efficiency and sensitivity in the electrospray ionisation. Positive electrospray ionisation and selected ion monitoring (SIM) modes were used for detection and quantitation. Because the adduct signals of analytes with DIPA were the base peaks in the mass spectrum during ESI in the used conditions, [AMP + 2DIPA + H]+, [ADP + 3DIPA + H]+ and [ATP + 3DIPA + H]+ ions were used as quantitation ions. The quantitation limits for the adenosine nucleotides were 0.1 µmol/g dry weight (DW) of cell extract for AMP, and 0.5 µmol/g DW for both ADP and ATP. Interday RSD with a control sample was 12-20 % and recoveries ranged from 90 to 130 %. The samples for the analysis were taken from Saccharomyces cerevisiae chemostats at different oxygen levels (aerobic, microaerobic, nanoaerobic and anaerobic). Depending on the oxygen level, the nucleotide concentrations in the samples varied between 0.5-3 µmol/g DW for AMP, 1-4 µmol/g DW for ADP and 1-7 µmol/g DW for ATP. Normally, the energy state of the cell is investigated by using energy charge (EC) defined as EC = (ATP + ADP/2)/(AMP + ADP + ATP). Based on the nucleotide analyses, it was observed that at low oxygen levels, energy charge was low but in anaerobic conditions, it increased to the normal level

Analysis of adenosine nucleotides from Saccharomyces cerevisiae chemostats by liquid chromatography-electrospray ionisation mass spectrometry

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