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Deciphering the Acute Cellular Phosphoproteome Response to Irradiation with X-rays, Protons and Carbon Ions*

  • Martin Winter
    Affiliations
    From the Functional Proteome Analysis, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany;

    Translational Radiation Oncology, National Center for Tumor diseases (NCT), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 460, D-69120 Heidelberg, Germany;

    German Cancer Consortium (DKTK), Heidelberg, Germany;
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  • Ivana Dokic
    Affiliations
    Translational Radiation Oncology, National Center for Tumor diseases (NCT), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 460, D-69120 Heidelberg, Germany;

    German Cancer Consortium (DKTK), Heidelberg, Germany;

    Heidelberg Ion Beam Therapy Center (HIT), Department of Radiation Oncology, University of Heidelberg Medical School, Im Neuenheimer Feld 450, D-69120 Heidelberg, Germany;

    Heidelberg Institute of Radiation Oncology (HIRO), National Center for Radiation Research in Oncology (NCRO), Heidelberg, Germany
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  • Julian Schlegel
    Affiliations
    Translational Radiation Oncology, National Center for Tumor diseases (NCT), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 460, D-69120 Heidelberg, Germany;

    German Cancer Consortium (DKTK), Heidelberg, Germany;

    Heidelberg Ion Beam Therapy Center (HIT), Department of Radiation Oncology, University of Heidelberg Medical School, Im Neuenheimer Feld 450, D-69120 Heidelberg, Germany;

    Heidelberg Institute of Radiation Oncology (HIRO), National Center for Radiation Research in Oncology (NCRO), Heidelberg, Germany
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  • Uwe Warnken
    Affiliations
    From the Functional Proteome Analysis, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany;
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  • Jürgen Debus
    Affiliations
    Translational Radiation Oncology, National Center for Tumor diseases (NCT), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 460, D-69120 Heidelberg, Germany;

    German Cancer Consortium (DKTK), Heidelberg, Germany;

    Heidelberg Ion Beam Therapy Center (HIT), Department of Radiation Oncology, University of Heidelberg Medical School, Im Neuenheimer Feld 450, D-69120 Heidelberg, Germany;

    Heidelberg Institute of Radiation Oncology (HIRO), National Center for Radiation Research in Oncology (NCRO), Heidelberg, Germany
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  • Author Footnotes
    §§ Shared senior authors.
    Amir Abdollahi
    Footnotes
    §§ Shared senior authors.
    Affiliations
    Translational Radiation Oncology, National Center for Tumor diseases (NCT), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 460, D-69120 Heidelberg, Germany;

    German Cancer Consortium (DKTK), Heidelberg, Germany;

    Heidelberg Ion Beam Therapy Center (HIT), Department of Radiation Oncology, University of Heidelberg Medical School, Im Neuenheimer Feld 450, D-69120 Heidelberg, Germany;

    Heidelberg Institute of Radiation Oncology (HIRO), National Center for Radiation Research in Oncology (NCRO), Heidelberg, Germany
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  • Author Footnotes
    §§ Shared senior authors.
    Martina Schnölzer
    Correspondence
    To whom correspondence should be addressed:German Cancer Research Center (DKFZ), Functional Proteome Analysis, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany. Tel.:+49 6221 42-2723; Fax:+49 6221 42 4562;.
    Footnotes
    §§ Shared senior authors.
    Affiliations
    From the Functional Proteome Analysis, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany;
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  • Author Footnotes
    * This work was supported by German Research Council (DFG-KFO214), Deutsche Krebshilfe (Max-Eder 108876) and intramural grants from National Center for Tumor diseases (NCT/DKFZ-DKTK, Heidelberg, Germany). M.W. was financially supported by the Helmholtz International Graduate School for Cancer Research. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
    This article contains supplemental material.
    §§ Shared senior authors.
Open AccessPublished:March 16, 2017DOI:https://doi.org/10.1074/mcp.M116.066597
      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.
      More than 50% of all cancer patients receive radiotherapy during their course of treatment (
      • Delaney G.
      • Jacob S.
      • Featherstone C.
      • Barton M.
      The role of radiotherapy in cancer treatment.
      ). Conformal X-ray irradiation techniques such as intensity-modulated radiation therapy (IMRT) combine the application of a curative dose to the tumor while sparing adjacent radiosensitive organs keeping side-effects to healthy surrounding tissue within tolerable limits (
      • Baumann M.
      • Krause M.
      • Overgaard J.
      • Debus J.
      • Bentzen S.M.
      • Daartz J.
      • Richter C.
      • Zips D.
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      Radiation oncology in the era of precision medicine.
      ). Contrary to X-rays, which show an exponential dose decrease with increasing tissue depth, charged particles deposit most of their energy to a small region within the tissue with a sharp dose fall-off after the so-called Bragg peak (
      • Durante M.
      • Loeffler J.S.
      Charged particles in radiation oncology.
      ). This precise dose localization enables further dose escalation within the tumor while sparing healthy tissue (
      • Schulz-Ertner D.
      • Tsujii H.
      Particle radiation therapy using proton and heavier ion beams.
      ). Worldwide, more than 135.000 patients have been treated with particles until the end of 2014 and the number is growing steadily (
      • Jermann M.
      Particle Therapy Statistics in 2014.
      ).
      An important term in the field of radiobiology is the relative-biological effectiveness (RBE)
      The abbreviations used are: RBE, relative biological effectiveness;AA, acetic acid;ABC, ammonium bicarbonate;ATM, ataxia telangiectasia mutated;ATR, ataxia telangiectasia and Rad3-related protein;CDK1, cyclin-dependent kinase 1;CHK2, checkpoint kinase 2;DDR, DNA damage response;DSB, double-strand break;FA, formic acid;FLNB, Filamin-B;GAPDH, glyceraldehyde-3-phosphate dehydrogenase;GOBP, gene ontology biological process;GOCC, gene ontology cellular compartment;GOMF, gene ontology molecular function;H2AX, histone H2AX;HFIP, hexafluoro-2-propanol;HIT, Heidelberg Ion Beam Therapy Center;KPNA2, importin subunit alpha-1;LET, linear energy transfer;MAP1B, microtubule-associated protein 1B;MKI67, antigen KI-67;MOPS, 3-(N-morpholino)propanesulfonic acid;NHEJ, nonhomologous end joining;NUMA1, nuclear mitotic apparatus protein 1;PRKDC, DNA-dependent protein kinase catalytic subunit;RAD50, DNA repair protein RAD50;RIPA, radioimmunoprecipitation assay;ROS, reactive oxygen species;RRBP1, ribosome-binding protein 1;SF30, survival fraction 30%;SILAC, stable isotope labeling by amino acids in cell culture;SPE, solid phase extraction;SRC, proto-oncogene tyrosine-protein kinase Src;STMN1, stathmin;TP53BP1, tumor suppressor p53-binding protein 1;VIM, vimentin.
      1The abbreviations used are: RBE, relative biological effectiveness;AA, acetic acid;ABC, ammonium bicarbonate;ATM, ataxia telangiectasia mutated;ATR, ataxia telangiectasia and Rad3-related protein;CDK1, cyclin-dependent kinase 1;CHK2, checkpoint kinase 2;DDR, DNA damage response;DSB, double-strand break;FA, formic acid;FLNB, Filamin-B;GAPDH, glyceraldehyde-3-phosphate dehydrogenase;GOBP, gene ontology biological process;GOCC, gene ontology cellular compartment;GOMF, gene ontology molecular function;H2AX, histone H2AX;HFIP, hexafluoro-2-propanol;HIT, Heidelberg Ion Beam Therapy Center;KPNA2, importin subunit alpha-1;LET, linear energy transfer;MAP1B, microtubule-associated protein 1B;MKI67, antigen KI-67;MOPS, 3-(N-morpholino)propanesulfonic acid;NHEJ, nonhomologous end joining;NUMA1, nuclear mitotic apparatus protein 1;PRKDC, DNA-dependent protein kinase catalytic subunit;RAD50, DNA repair protein RAD50;RIPA, radioimmunoprecipitation assay;ROS, reactive oxygen species;RRBP1, ribosome-binding protein 1;SF30, survival fraction 30%;SILAC, stable isotope labeling by amino acids in cell culture;SPE, solid phase extraction;SRC, proto-oncogene tyrosine-protein kinase Src;STMN1, stathmin;TP53BP1, tumor suppressor p53-binding protein 1;VIM, vimentin.
      being defined as the ratio of X-ray dose to an alternative radiation quality dose which produces the same biological effect such as cell survival or other surrogates such as the number of double-strand breaks (DSBs). Although the RBE for protons is assumed to be comparable to X-rays, carbon ions are more effective in inducing unrepairable DNA damage (
      • Dokic I.
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      • Niklas M.
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      • Tessonnier T.
      • Ferrari A.
      • Parodi K.
      • Jakel O.
      • Debus J.
      • Haberer T.
      • Abdollahi A.
      Next generation multi-scale biophysical characterization of high precision cancer particle radiotherapy using clinical proton, helium-, carbon- and oxygen ion beams.
      ) and may therefore efficiently eradicate formerly radioresistant tumors (
      • Chiblak S.
      • Tang Z.
      • Campos B.
      • Gal Z.
      • Unterberg A.
      • Debus J.
      • Herold-Mende C.
      • Abdollahi A.
      Radiosensitivity of Patient-Derived Glioma Stem Cell 3-Dimensional Cultures to Photon, Proton, and Carbon Irradiation.
      ,
      • Peschke P.
      • Karger C.P.
      • Scholz M.
      • Debus J.
      • Huber P.E.
      Relative biological effectiveness of carbon ions for local tumor control of a radioresistant prostate carcinoma in the rat.
      ,
      • Debus J.
      • Abdollahi A.
      For the next trick: new discoveries in radiobiology applied to glioblastoma.
      ). This is in part because of the fact, that carbon ions predominantly induce clustered and direct DNA damage, which is considered to be less dependent on cell cycle stage, oxygen level, genetic background and is less well repaired by DNA repair mechanisms (
      • Durante M.
      • Loeffler J.S.
      Charged particles in radiation oncology.
      ).
      So far, limited studies exist comparing X-rays to particle based radiations and are mainly focused on investigating the alterations of single or panel genes (
      • Kamlah F.
      • Hänze J.
      • Arenz A.
      • Seay U.
      • Hasan D.
      • Juricko J.
      • Bischoff B.
      • Gottschald O.R.
      • Fournier C.
      • Taucher-Scholz G.
      • Scholz M.
      • Seeger W.
      • Engenhart-Cabillic R.
      • Rose F.
      Comparison of the effects of carbon ion and photon irradiation on the angiogenic response in human lung adenocarcinoma cells.
      ,
      • Girdhani S.
      • Lamont C.
      • Hahnfeldt P.
      • Abdollahi A.
      • Hlatky L.
      Proton irradiation suppresses angiogenic genes and impairs cell invasion and tumor growth.
      ). However, cells are equipped with versatile signaling cascades mainly transduced by post-translational modifications (PTMs) in order to minimize adverse effects of DNA damage (
      • Polo S.E.
      • Jackson S.P.
      Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications.
      ). Post-translationally modified proteins are key targets especially to improve the understanding of acute signaling events. In DNA damage response (DDR), protein phosphorylation plays a crucial role to trigger a chain of events, starting with sensing DNA lesions, signaling cascade activation, cell cycle arrest and recruitment of DNA repair factors (
      • Bennetzen M.V.
      • Larsen D.H.
      • Bunkenborg J.
      • Bartek J.
      • Lukas J.
      • Andersen J.S.
      Site-specific phosphorylation dynamics of the nuclear proteome during the DNA damage response.
      ). Although phosphoproteome data exist elucidating the cellular response to X-rays per se (
      • Bennetzen M.V.
      • Larsen D.H.
      • Bunkenborg J.
      • Bartek J.
      • Lukas J.
      • Andersen J.S.
      Site-specific phosphorylation dynamics of the nuclear proteome during the DNA damage response.
      ) as well as to various X-ray dose levels (
      • Yang F.
      • Stenoien D.L.
      • Strittmatter E.F.
      • Wang J.
      • Ding L.
      • Lipton M.S.
      • Monroe M.E.
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      • Gristenko M.A.
      • Tang K.
      • Fang R.
      • Adkins J.N.
      • Camp D.G. 2.
      • Chen D.J.
      • Smith R.D.
      Phosphoproteome profiling of human skin fibroblast cells in response to low- and high-dose irradiation.
      ,
      • Yang F.
      • Waters K.M.
      • Miller J.H.
      • Gritsenko M.A.
      • Zhao R.
      • Du X.
      • Livesay E.A.
      • Purvine S.O.
      • Monroe M.E.
      • Wang Y.
      • Camp D.G.
      • Smith R.D.
      • Stenoien D.L.
      Phosphoproteomics profiling of human skin fibroblast cells reveals pathways and proteins affected by low doses of ionizing radiation.
      ), radiation quality dependent phosphoproteome studies are urgently needed.
      In the present study, we aimed to systematically decipher acute signaling events induced by different radiation qualities using high-resolution mass spectrometry based proteomics. To this end, we irradiated SILAC-labeled human lung adenocarcinoma cells (A549) with X-rays, protons and carbon ions in clinical-like setting. A549 cells have an intact DNA damage repair machinery (e.g. p53 wild-type) leading to a relatively low background of DNA damage foci compared with a panel of well-known cancer cell lines (
      • Dokic I.
      • Mairani A.
      • Niklas M.
      • Zimmermann F.
      • Chaudhri N.
      • Krunic D.
      • Tessonnier T.
      • Ferrari A.
      • Parodi K.
      • Jakel O.
      • Debus J.
      • Haberer T.
      • Abdollahi A.
      Next generation multi-scale biophysical characterization of high precision cancer particle radiotherapy using clinical proton, helium-, carbon- and oxygen ion beams.
      ). Therefore, A549 cells are particularly suitable to study radiation induced DNA damage response. Proteomic and phosphoproteomic analyses performed 2 h post irradiation showed extensive alterations of the phosphorylation status whereas the protein expression itself remained largely unaffected. Phosphorylation events behaved similar upon proton and carbon irradiation, however a distinct number of sites responded differentially to X-rays versus particle based treatments. To validate selected candidates, we used synthetic isotope labeled phosphopeptides in a targeted spike-in experiment as recently proposed by Kennedy et al. as an alternative to extensive Western blotting, which requires phosphosite specific antibodies (
      • Kennedy J.J.
      • Yan P.
      • Zhao L.
      • Ivey R.G.
      • Voytovich U.J.
      • Moore H.D.
      • Lin C.
      • Pogosova-Agadjanyan E.L.
      • Stirewalt D.L.
      • Reding K.W.
      • Whiteaker J.R.
      • Paulovich A.G.
      Immobilized metal affinity chromatography coupled to multiple reaction monitoring enables reproducible quantification of phospho-signaling.
      ). By this approach, we validated the prevailing pattern of phosphorylation sites associated with DDR to be regulated in equal measure by all radiation qualities. Moreover, phosphorylation sites responding differentially to X-ray and particle based treatments were confirmed. Herewith, we demonstrate the existence of radiation quality dependent signaling events in the acute cell response.
      In summary, we established a robust and efficient workflow for the investigation and validation of phosphorylation changes in response to X-rays and particle based irradiations. We show for the first time, that differential acute signaling events are triggered by different radiation qualities. With this we provide valuable information to better understand molecular effects of radiation qualities and offer potential drug targets for modulation and optimization of cancer radiotherapy.

      DISCUSSION

      Precision radiotherapy such as particle radiation exhibits physical and biological advantages in comparison to conventional X-ray therapy (
      • Durante M.
      • Loeffler J.S.
      Charged particles in radiation oncology.
      ). The application of particle radiation is especially of interest for difficult to reach tumors such as those in close proximity to organs at risk, where highly precise conformity of irradiation beams is needed and tumors being resistant to conventional X-ray therapy (i.e. hypoxic tumors) (
      • Tsujii H.
      • Mizoe J.-E.
      • Kamada T.
      • Baba M.
      • Kato S.
      • Kato H.
      • Tsuji H.
      • Yamada S.
      • Yasuda S.
      • Ohno T.
      • Yanagi T.
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      • Sugawara T.
      • Ezawa H.
      • Kandatsu S.
      • Yoshikawa K.
      • Kishimoto R.
      • Miyamoto T.
      Overview of clinical experiences on carbon ion radiotherapy at NIRS.
      ,
      • Austin-Seymour M.
      • Griffin T.
      • Laramore G.
      • Maor M.
      • Parker R.
      High-LET radiation therapy of non-small cell lung cancer.
      ,
      • Okada T.
      • Kamada T.
      • Tsuji H.
      • Mizoe J.-E.
      • Baba M.
      • Kato S.
      • Yamada S.
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      • Kiyohara H.
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      • Shinoto M.
      • Tsujii H.
      Carbon ion radiotherapy: clinical experiences at National Institute of Radiological Science (NIRS).
      ). Although particle radiation is already applied for patient treatment, there is still only limited knowledge about the molecular and cellular effects induced by the different radiation qualities. In our study, we compared the acute proteome and phosphoproteome response of human lung adenocarcinoma (A549) cells 2h after irradiation with X-rays, protons and carbon ions, respectively. Our goal was to characterize the triggered signaling events and decipher aberrant signaling following treatment with the applied radiation qualities.
      The depicted study represents a robust and efficient strategy to elucidate proteomic and phosphoproteomic changes in response to ionizing radiation. Alterations of protein abundance were observed at this acute time point for eight proteins only. In contrast, the prevailing pattern of cellular response was mediated by phosphorylation as a fast mechanism of signal transduction. In line with our observation, 6 Gy X-ray irradiation of GM00130 cells was also found to elicit limited alterations in proteome abundance within a relatively short time frame (5min - 8h) as recently published by Bennetzen et al. (
      • Bennetzen M.V.
      • Larsen D.H.
      • Bunkenborg J.
      • Bartek J.
      • Lukas J.
      • Andersen J.S.
      Site-specific phosphorylation dynamics of the nuclear proteome during the DNA damage response.
      ) confirming our decision to focus on changes at phosphorylation level. Single or fractionated high radiation doses as employed in the area of stereotactic ablative radiotherapy (SABR, radiosurgery) may result in higher number of differentially expressed proteins as suggested by Cho et al. (
      • Cho Y.-S.
      • Song K.-B.
      Effect of γ-irradiation on the molecular properties of bovine serum albumin and β-lactoglobulin.
      ). In contrast, in this study we focused on investigating dose ranges relevant to clinical routine applications.
      Applying SILAC based phosphoproteomics, we identified a total of 5107 phosphorylation sites, including 2818 quantified sites that were subject of statistical analysis because of stringent exclusion thresholds. A phosphopeptide enrichment protocol was adopted which required only small sample amount (150 μg per SILAC state). Other enrichment strategies such as extensive sample fractionation would enhance the number of identifications and deepen the coverage of regulated signaling pathways (
      • Loroch S.
      • Zahedi R.P.
      • Sickmann A.
      Highly Sensitive Phosphoproteomics by Tailoring Solid-Phase Extraction to Electrostatic Repulsion-Hydrophilic Interaction Chromatography.
      ,
      • Batth T.S.
      • Francavilla C.
      • Olsen J.V.
      Off-Line High-pH Reversed-Phase Fractionation for In-Depth Phosphoproteomics.
      ,
      • Gruhler A.
      • Olsen J.V.
      • Mohammed S.
      • Mortensen P.
      • Faergeman N.J.
      • Mann M.
      • Jensen O.N.
      Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway.
      ,
      • McNulty D.E.
      • Annan R.S.
      Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection.
      ), however this would require a larger amount of starting material. Moreover, our enrichment protocol was optimized for peptides containing one phosphorylation site only. Enhancement of multiple phosphorylated peptides would necessitate adjustments of the protocol such as reduction of IMAC material during enrichments (
      • Ye J.
      • Zhang X.
      • Young C.
      • Zhao X.
      • Hao Q.
      • Cheng L.
      • Jensen O.N.
      Optimized IMAC-IMAC protocol for phosphopeptide recovery from complex biological samples.
      ). An additional extension by a single-step immunoaffinity purification would enlarge the proportion of phosphotyrosine residues (
      • Kettenbach A.N.
      • Gerber S.A.
      Rapid and Reproducible Single-Stage Phosphopeptide Enrichment of Complex Peptide Mixtures: Application to General and Phosphotyrosine-Specific Phosphoproteomics Experiments.
      ,
      • Boersema P.J.
      • Foong L.Y.
      • Ding V.M.Y.
      • Lemeer S.
      • van Breukelen B.
      • Philp R.
      • Boekhorst J.
      • Snel B.
      • den Hertog J.
      • Choo A.B.H.
      • Heck Albert J.R.
      In-depth qualitative and quantitative profiling of tyrosine phosphorylation using a combination of phosphopeptide immunoaffinity purification and stable isotope dimethyl labeling.
      ). However, as irradiated sample material was very limited because of restricted time slots for researchers caused by privileged beam time for patient treatment we could not randomly extend sample amount to apply additional enrichment strategies.
      To examine cellular signaling events differentially regulated by the radiation qualities, application of radiobiological equivalent doses is a prerequisite. Therefore, we determined the RBE utilizing the gold standard clonogenic survival assay. At isoeffective doses of all three radiation qualities, resulting in 30% survival fraction, we were able to identify a large number of regulated phosphorylation sites and most importantly could show for the first time differential signaling induced by X-rays versus the raster-scanning particle irradiations (proton/carbon). Statistical analyses were conducted to: (i) identify phosphorylation sites with common regulation pattern across all radiation qualities and (ii) identify sites with differential phosphorylation pattern between the three radiation qualities. A total of 181 phosphorylation sites are regulated in response to ionizing radiation. One hundred twenty-six sites thereof are regulated regardless of the radiation quality, whereas 55 show differential regulation between the qualities. We found enrichment of five different kinase sequence motifs in our data and could confirm similar enrichment of motif families by analysis of previously published radiation dependent phosphoproteome alterations deposited in the PhosphoSitePlus database.
      A consensus radiation quality independent phosphorylation pattern of p(S/T)Q sites was found mainly enriched for sites in the DDR, in line with current knowledge that DDR is a hallmark of ionizing radiation (
      • Lomax M.E.
      • Folkes L.K.
      • O'Neill P.
      Biological consequences of radiation-induced DNA damage: relevance to radiotherapy.
      ,
      • Santivasi W.L.
      • Xia F.
      Ionizing radiation-induced DNA damage, response, and repair.
      ), which is induced and anticipated by the usage of cell kill based isoeffective doses. Observed downregulation of p(S/T)P and pSPXXK sites representing motifs of the CDK, GSK-3 and MAP kinases have crucial role in the cell response to ionizing radiation per se. CDKs play a key role in maintaining genome integrity (
      • Meyerson M.
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      • Su L.K.
      • Gorka C.
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      A family of human cdc2-related protein kinases.
      ). Our finding that radiation induced DNA damage affect protein substrates of CDKs hence provides a plausible explanation for the phenomenological observation of cell cycle arrest after irradiation. Indeed, radiation induced cell cycle arrest is important for proper DNA repair (
      • Weinert T.
      A DNA damage checkpoint meets the cell cycle engine.
      ,
      • Sanchez Y.
      • Wong C.
      • Thoma R.S.
      • Richman R.
      • Wu Z.
      • Piwnica-Worms H.
      • Elledge S.J.
      Conservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk regulation through Cdc25.
      ). Recent findings indicate that CDKs additionally contribute to further upstream events such as checkpoint control and DNA repair (
      • Johnson N.
      • Shapiro G.I.
      Cyclin-dependent kinases (cdks) and the DNA damage response: rationale for cdk inhibitor-chemotherapy combinations as an anticancer strategy for solid tumors.
      ). GSK-3 executes heterogeneous signaling functions including both cell death and survival signals dependent on the signaling context (
      • Maurer U.
      • Preiss F.
      • Brauns-Schubert P.
      • Schlicher L.
      • Charvet C.
      GSK-3 - at the crossroads of cell death and survival.
      ). The MAP kinases ERK1/ERK2 likewise conduct regulation of cell proliferation, migration and death (
      • Cargnello M.
      • Roux P.P.
      Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases.
      ,
      • Mebratu Y.
      • Tesfaigzi Y.
      How ERK1/2 activation controls cell proliferation and cell death: Is subcellular localization the answer?.
      ). The prevalent downregulation of phosphorylation sites belonging to these kinase motifs implies that cells enter a state of proliferative arrest to enable proper DNA repair and maintain genomic stability. These findings together emphasize, that radiation induced acute signaling pathways are dominated by DNA repair functions, cell cycle regulation and cell survival/death signals as previously reported (
      • Nowsheen S.
      • Yang E.S.
      The intersection between DNA damage response and cell death pathways.
      ). Our data enables the assignment of phosphorylation sites to distinct cellular functions and facilitate further investigations of exact phosphosite functionality.
      Despite employing depicted isoeffective doses, 55 phosphorylation sites were identified to respond differentially between the three radiations. We identified distinct p(S/T)P sites with radiation quality dependent regulation being mostly reflected by minor downregulation of phosphorylation sites after X-ray treatment and pronounced downregulation following particle irradiation. This coherent pattern leads to the deduction that a common upstream regulator is responsible for these regulations. Our data points to the CDK family with its versatile regulation of cellular processes including most importantly cell cycle and transcription regulation (
      • Malumbres M.
      • Harlow E.
      • Hunt T.
      • Hunter T.
      • Lahti J.M.
      • Manning G.
      • Morgan D.O.
      • Tsai L.-H.
      • Wolgemuth D.J.
      Cyclin-dependent kinases: a family portrait.
      ). We could show that particle irradiation leads to dephosphorylation of proteins involved in cell mitosis such as MKI67, MAP1B, RRBP1, and KPNA2 to a larger extent than X-ray irradiation. Sites on STMN1, FLNB, and VIM that are involved in cell migration and invasion show a similar pattern. These observations support the previously published data on functional analysis of different cell lines after proton and carbon irradiation in comparison to X-rays, suggesting altered regulation of angiogenesis (
      • Kamlah F.
      • Hänze J.
      • Arenz A.
      • Seay U.
      • Hasan D.
      • Juricko J.
      • Bischoff B.
      • Gottschald O.R.
      • Fournier C.
      • Taucher-Scholz G.
      • Scholz M.
      • Seeger W.
      • Engenhart-Cabillic R.
      • Rose F.
      Comparison of the effects of carbon ion and photon irradiation on the angiogenic response in human lung adenocarcinoma cells.
      ,
      • Girdhani S.
      • Lamont C.
      • Hahnfeldt P.
      • Abdollahi A.
      • Hlatky L.
      Proton irradiation suppresses angiogenic genes and impairs cell invasion and tumor growth.
      ,
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      Effects of carbon-ion beam irradiation on the angiogenic response in lung adenocarcinoma A549 cells.
      ), cell migration (
      • Fujita M.
      • Imadome K.
      • Shoji Y.
      • Isozaki T.
      • Endo S.
      • Yamada S.
      • Imai T.
      Carbon-ion irradiation suppresses migration and invasiveness of human pancreatic carcinoma cells MIAPaCa-2 via Rac1 and RhoA degradation.
      ,
      • Rieken S.
      • Habermehl D.
      • Wuerth L.
      • Brons S.
      • Mohr A.
      • Lindel K.
      • Weber K.
      • Haberer T.
      • Debus J.
      • Combs S.E.
      Carbon ion irradiation inhibits glioma cell migration through downregulation of integrin expression.
      ), and mitosis (
      • Amornwichet N.
      • Oike T.
      • Shibata A.
      • Ogiwara H.
      • Tsuchiya N.
      • Yamauchi M.
      • Saitoh Y.
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      • Ohno T.
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      Carbon-ion beam irradiation kills X-ray-resistant p53-null cancer cells by inducing mitotic catastrophe.
      ) by different radiation qualities. We identified phosphorylation sites corresponding to these processes that were not yet described in the context of ionizing radiation. However, their exact role within the context of radiation biology remains to be elucidated.
      For validation purposes, we selected phosphorylation sites of interest and confirmed their identity in an unbiased spike-in approach using synthetic isotope labeled phosphopeptides. Compared with Western blotting, the use of synthetic peptides has tremendous advantage: (i) no phosphosite specific antibodies are needed, (ii) uncomplicated and inexpensive production of synthetic peptides, (iii) misinterpretation of Western blot signals because of cross-reactivity of the antibodies is avoided (
      • Janes K.A.
      An analysis of critical factors for quantitative immunoblotting.
      ), and (iv) distinct multiplexing possibilities in a targeted spike-in approach (
      • Kennedy J.J.
      • Yan P.
      • Zhao L.
      • Ivey R.G.
      • Voytovich U.J.
      • Moore H.D.
      • Lin C.
      • Pogosova-Agadjanyan E.L.
      • Stirewalt D.L.
      • Reding K.W.
      • Whiteaker J.R.
      • Paulovich A.G.
      Immobilized metal affinity chromatography coupled to multiple reaction monitoring enables reproducible quantification of phospho-signaling.
      ,
      • Stecker E.K.
      • Minkoff B.B.
      • Sussman M.R.
      Phosphoproteomic analyses reveal early signaling events in the osmotic stress response.
      ). Moreover, in future experiments the same pool of synthetic peptides can be used as internal standards in tissue samples or in vivo experiments (
      • Narumi R.
      • Murakami T.
      • Kuga T.
      • Adachi J.
      • Shiromizu T.
      • Muraoka S.
      • Kume H.
      • Kodera Y.
      • Matsumoto M.
      • Nakayama K.
      • Miyamoto Y.
      • Ishitobi M.
      • Inaji H.
      • Kato K.
      • Tomonaga T.
      A strategy for large-scale phosphoproteomics and SRM-based validation of human breast cancer tissue samples.
      ). Easy handling of synthetic peptides makes a distribution between laboratories possible. The availability of synthetic isotope labeled phosphopeptide pools, similar to antibody arrays, would be a valuable tool to study complete signaling pathways with high confidence and accurate quantification in a fast and reproducible manner. When the amount of each synthetic peptide is adjusted to its ionization efficiency as conducted in our study and a list of suitable transitions for the identification is included, it would enable a fast and easy usage of these synthetic phosphopeptide pools.
      Our pool contained 28 synthetic phosphopeptides covering a broad range of characteristic, in order to confirm multiple hypotheses in a single experiment. We confirmed that the DNA damage responsive phosphorylation sites of the p(S/T)Q motif show similar regulation pattern between the radiation qualities. Among these, sites on TP53BP1, PRKDC, and RAD50 with well-known DDR activity were similarly phosphorylated by the different radiation qualities. Moreover, our results indicate TP53BP1 to be a key regulator in response to ionizing radiation, supporting the recently published data which highlighted TP53BP1 in a central position to maintain genome integrity (
      • Panier S.
      • Boulton S.J.
      Double-strand break repair: 53BP1 comes into focus.
      ). In this work, we validated nine phosphopeptides containing ten phosphorylation sites on TP53BP1, which are regulated by crucial kinases simultaneously (ATM, PRKDC, and CDK1), underlining the versatile role of TP53BP1 as one of the main genome guardian molecules (
      • Panier S.
      • Boulton S.J.
      Double-strand break repair: 53BP1 comes into focus.
      ).
      Moreover we confirmed ten phosphorylation sites with differential regulation pattern between the radiation qualities which is because of the radiations mode of action. Although X-ray induced damage is mostly a consequence of increased ROS production and indirect damage, high-LET carbon ion irradiation induces direct complex DNA damages (
      • Dokic I.
      • Mairani A.
      • Niklas M.
      • Zimmermann F.
      • Chaudhri N.
      • Krunic D.
      • Tessonnier T.
      • Ferrari A.
      • Parodi K.
      • Jakel O.
      • Debus J.
      • Haberer T.
      • Abdollahi A.
      Next generation multi-scale biophysical characterization of high precision cancer particle radiotherapy using clinical proton, helium-, carbon- and oxygen ion beams.
      ). Hence differences between these two radiation qualities were anticipated. In contrast, proton irradiation was so far thought to elicit similar biological effects to X-rays reflected by the currently fixed proton RBE of 1.1 for patient treatment. Surprisingly, unsupervised clustering and principle component analysis clearly separated X-ray irradiation from proton and carbon irradiation. Hence, the low-LET proton irradiation shared more common phosphoproteome characteristics with high-LET carbon than with conventional X-ray irradiation. This is an interesting unexpected finding, however in line with more recent data indicating potential less appreciated radiobiological differences attributed to protons beyond the classical cell killing effect (
      • Girdhani S.
      • Lamont C.
      • Hahnfeldt P.
      • Abdollahi A.
      • Hlatky L.
      Proton irradiation suppresses angiogenic genes and impairs cell invasion and tumor growth.
      ). Further, in the field of radiotherapy the paradigm of fixed proton RBE of 1.1 is currently controversially debated and increasing body of data imply that improved radiobiological models are urgently needed to better characterize the exact effect of proton irradiation (
      • Giovannini G.
      • Bohlen T.
      • Cabal G.
      • Bauer J.
      • Tessonnier T.
      • Frey K.
      • Debus J.
      • Mairani A.
      • Parodi K.
      Variable RBE in proton therapy: comparison of different model predictions and their influence on clinical-like scenarios.
      ). Our observations at phosphoproteome level indicate that differences in radiobiology between X-ray and proton irradiation exist even at doses that produce the same level of cell kill.
      In summary, we present a versatile workflow for identification, quantification and validation of phosphorylation sites in response to ionizing radiation that could be employed for a broad spectrum of applications in life sciences. Our data stimulate radiation research at novel frontiers of intracellular signaling and cell-cell communication. These are instrumental for a better understanding of discrepancies found on tissue level responses to different radiation qualities. Further exploration in this direction will supplement and adjust our view on the so far predominantly tumor cell kill centric modeling of radiobiological effects. Especially for the ongoing debate about proton RBE in patient treatment we offer exclusive data. Moreover, the presented phosphorylation sites, that are differentially regulated, offer attractive targets for modulation and improvement of radiotherapy in the clinical setting.

      Acknowledgments

      We thank Claudia Rittmüller for her excellent technical assistance with all cell experiments, Andrea Mairani and Stephan Brons for providing medical physics support at HIT for particle beam irradiation and Ramona Mayer for her excellent technical assistance during sample preparations.

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