Abstract Diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) are formed in prokaryotic and eukaryotic cells. Since their concentrations increase significantly upon cellular stress they were considered to be alarmones triggering stress adaptive processes. However, their exact role remains elusive. To elucidate the proteome-wide interactome of Ap3A and Ap4A and gain insights into their intracellular roles, we herein report the development of new photoaffinity-labeling probes and their employment in chemical proteomics using eukaryotic and prokaryotic cells. We found the involvement of the ApnA interactors in many fundamental cellular process including carboxylic acid and nucleotide metabolism, gene expression, various regulatory processes and stress response. Our results highlight common roles of these ApnAs across the domains of life, but also roles that are different for Ap3A or Ap4A. This study provides a rich source for further functional studies of these nucleotides and depicts useful tools for characterisation of their regulatory mechanisms in cells.