Re-evaluating adenosine-nucleoside polyphosphate levels in human cells by mass spectrometry

Dinucleoside polyphosphates (Np_nNs) are known as alarmones but their functions in cellular metabolism remain largely unexplored till to date. Here, we report new data concerning their cellular quantification using mass spectrometry-based methods. Key for this approach were ¹³C-isotope-labeled internal standards of eight different compounds (¹³C-Ap_nN, n = 3,4; N = Adenosine, Cytidine, Guanosine, Uridine) that were chemically synthesized from ¹³C₅-adenosine. For this, a novel synthesis strategy was developed. These compounds were used to account for losses during the extraction for the determination of intracellular Ap_3/4N-levels. Cell samples from two human cell lines, HEK293T and H1299, were measured using a triple quad mass spectrometer (TQ-MS). Additionally, menadione was added to the cell dishes to generate oxidative stress. We were able to reproduce previous findings that all Ap_3/4N levels increase in stressed cells. In addition, we showed that cells lacking the Np₃N hydrolase Fhit (fragile histidine triad) (H1299, FHIT-negative) exhibit hundred-fold increased levels of Ap₃Ns but also ten-fold increased levels of Ap₄Ns. This finding contradicts previous data, where no impact of the expression of Fhit on Ap₄N-levels was detected. For FHIT-negative cells, no significant increase in Ap_3/4N levels was observed when oxidative stress was applied, suggesting that a change in hydrolase activity could be the primary stress response rather than increased biosynthesis.