Radiotherapy is a cornerstone of
cancer therapy. The recently established particle
therapy with raster-scanning
protons and
carbon ions landmarks a new era in the field of high-precision
cancer medicine. However, molecular mechanisms governing radiation induced intracellular signaling remain elusive. Here, we present the first comprehensive proteomic and phosphoproteomic study applying stable
isotope labeling by
amino acids in cell culture (SILAC) in combination with high-resolution mass spectrometry to decipher cellular response to irradiation with X-rays,
protons and
carbon ions. At
protein expression level limited alterations were observed 2 h post irradiation of human
lung adenocarcinoma cells. In contrast, 181 phosphorylation sites were found to be differentially regulated out of which 151 sites were not hitherto attributed to radiation response as revealed by crosscheck with the PhosphoSitePlus database.Radiation-induced phosphorylation of the p(S/T)Q motif was the prevailing regulation pattern affecting
proteins involved in DNA damage response signaling. Because radiation doses were selected to produce same level of cell kill and
DNA double-strand breakage for each radiation quality, DNA damage responsive phosphorylation sites were regulated to same extent. However, differential phosphorylation between radiation qualities was observed for 55 phosphorylation sites indicating the existence of distinct signaling circuitries induced by X-ray versus particle (
proton/
carbon) irradiation beyond the canonical DNA damage response. This unexpected finding was confirmed in targeted spike-in experiments using synthetic
isotope labeled
phosphopeptides. Herewith, we successfully validated uniform DNA damage response signaling coexisting with altered signaling involved in apoptosis and metabolic processes induced by X-ray and particle based treatments.In summary, the comprehensive insight into the radiation-induced phosphoproteome landscape is instructive for the design of functional studies aiming to decipher cellular signaling processes in response to
radiotherapy, space radiation or ionizing radiation per se Further, our data will have a significant impact on the ongoing debate about patient treatment modalities.