Project description:After a week of acclimatization, 8-week-old mice were randomly divided into 4 groups consisting of more than 8 mice per group, and subjected to varying total body irradiation (TBI) doses of 0, 0.5, 1 or 3 Gy of X-rays (150 kVp, 20 mA, 0.5-mm aluminum and 0.3-mm copper filters) at a dose rate of 1.0 Gy/min using an MBR-1520R X-ray generator (Hitachi Medical, Tokyo, Japan). Peripheral blood was harvested 24 h after TBI from orbital venous plexus of mice anesthetized using isoflurane (Powerful Isoful; Zoetis, London, UK) by capillary tube, and placed at room temperature for at least 30 min to allow blood-clotting. Sera was collected by centrifugation at 1,200 ×g for 10 min and stored at - 80°C until the analysis. In addition, Sera collected from non-irradiated mice was subjected to varying TBI doses of 0, 0.5, 1 or 3 Gy of X-rays and incubated at 37°C for 24 h and stored at - 80°C until the analysis. 2 μl of sera was diluted with 198 μl of 50 mM ammonium bicarbonate. The serum proteins were precipitated by adding 1 ml of acetone. The precipitated proteins were collected by centrifugation at 15,000 ×g for 10 min. The precipitate was resuspended in 10 μl of 500 mM ammonium bicarbonate and denatured with an equivalent volume of trifluoroethanol and 5 μl of 200 mM DTT. The samples were incubated at 90°C for 30 min and cooled to room temperature. Free cysteine residues were alkylated with 10 μl of 200 mM iodoacetamide for 60 min at room temperature in the dark and the remaining iodoacetamide was quenched by adding 5 μl of 200 mM DTT. The samples were then mixed with 320 μl of 100 mM ammonium bicarbonate. The samples were incubated with 5 μg trypsin (TPCK treated, AB Sciex) at 37°C for 18 h. The samples were desalted with C18 ZipTip (Millipore, Bedford, MA, USA) and eluted with H2O/acetonitrile (5/5; v/v). The ZipTip eluates were dried in a vacuum centrifuge. Desalted samples were rehydrated in 0.1% formic acid and were analyzed by LC-MS using a nanoLC Eksigent 400 system (Eksigent, AB Sciex), coupled online to a TripleTOF6600 mass spectrometer (AB Sciex). Peptide separation was performed using liquid chromatography on a trap and elution conﬁguration using a nano trap column (350 μm × 0.5 mm, 3 μm, 120 Å, AB Sciex) and a nano ChromXP C18 reverse-phase column (75 μm × 15 cm, 3 μm, 120 Å, AB Sciex) at 300 nl/min with a 90 min linear gradient of 8% to 30% acetonitrile in 0.1% formic acid, and then, with a 10 min linear gradient of 30% to 40% acetonitrile in 0.1% formic acid. The mass spectrometer operated in information-dependent acquisition mode, scanning full spectra (400-1500 m/z) for 250 ms, followed by up to 30 MS/MS scans (100-1800 m/z for 50 ms each), for a cycle time of 1.8 s. Candidate ions with a charge state between +2 and + 5 and counts above a minimum threshold of 125 counts per second were isolated for fragmentation, and one MS/MS spectrum was collected before adding those ions to the exclusion list for 12 s. Rolling collision energy was used with a collision energy spread of 15. The mass spectrometer was operated by Analyst TF 1.7.1 software (AB Sciex).

Project description:After a week of acclimatization, 8-week-old mice were randomly divided into 4 groups consisting of more than 8 mice per group, and subjected to varying total body irradiation (TBI) doses of 0, 0.5, 1 or 3 Gy of X-rays (150 kVp, 20 mA, 0.5-mm aluminum and 0.3-mm copper filters) at a dose rate of 1.0 Gy/min using an MBR-1520R X-ray generator (Hitachi Medical, Tokyo, Japan). Peripheral blood was harvested 24 h after TBI from orbital venous plexus of mice anesthetized using isoflurane (Powerful Isoful; Zoetis, London, UK) by capillary tube, and placed at room temperature for at least 30 min to allow blood-clotting. Sera was collected by centrifugation at 1,200 ×g for 10 min and stored at - 80°C until the analysis. In addition, Sera collected from non-irradiated mice was subjected to varying TBI doses of 0, 0.5, 1 or 3 Gy of X-rays and incubated at 37°C for 24 h and stored at - 80°C until the analysis. 2 μl of sera was diluted with 198 μl of 50 mM ammonium bicarbonate. The serum proteins were precipitated by adding 1 ml of acetone. The precipitated proteins were collected by centrifugation at 15,000 ×g for 10 min. The precipitate was resuspended in 10 μl of 500 mM ammonium bicarbonate and denatured with an equivalent volume of trifluoroethanol and 5 μl of 200 mM DTT. The samples were incubated at 90°C for 30 min and cooled to room temperature. Free cysteine residues were alkylated with 10 μl of 200 mM iodoacetamide for 60 min at room temperature in the dark and the remaining iodoacetamide was quenched by adding 5 μl of 200 mM DTT. The samples were then mixed with 320 μl of 100 mM ammonium bicarbonate. The samples were incubated with 5 μg trypsin (TPCK treated, AB Sciex) at 37°C for 18 h. The samples were desalted with C18 ZipTip (Millipore, Bedford, MA, USA) and eluted with H2O/acetonitrile (5/5; v/v). The ZipTip eluates were dried in a vacuum centrifuge. Desalted samples were rehydrated in 0.1% formic acid and were analyzed by LC-MS using a nanoLC Eksigent 400 system (Eksigent, AB Sciex), coupled online to a TripleTOF6600 mass spectrometer (AB Sciex). Peptide separation was performed using liquid chromatography on a trap and elution conﬁguration using a nano trap column (350 μm × 0.5 mm, 3 μm, 120 Å, AB Sciex) and a nano ChromXP C18 reverse-phase column (75 μm × 15 cm, 3 μm, 120 Å, AB Sciex) at 300 nl/min with a 90 min linear gradient of 8% to 30% acetonitrile in 0.1% formic acid, and then, with a 10 min linear gradient of 30% to 40% acetonitrile in 0.1% formic acid. The mass spectrometer operated in information-dependent acquisition mode, scanning full spectra (400-1500 m/z) for 250 ms, followed by up to 30 MS/MS scans (100-1800 m/z for 50 ms each), for a cycle time of 1.8 s. Candidate ions with a charge state between +2 and + 5 and counts above a minimum threshold of 125 counts per second were isolated for fragmentation, and one MS/MS spectrum was collected before adding those ions to the exclusion list for 12 s. Rolling collision energy was used with a collision energy spread of 15. The mass spectrometer was operated by Analyst TF 1.7.1 software (AB Sciex). For data-independent acquisition (SWATH acquisition), the parameters were set as follows: 100 ms TOF MS scan, followed by 200 variable SWATH windows each at 50 ms accumulation time for m/z 400-1250. MS/MS SWATH scans were set at 5 Da window overlapping by 1 Da for m/z 400-1250 and varied on each side of the mass range. The total cycle time was 9.6 s. Rolling collision energy parameter script was used for automatically controlling the collision energy.

Project description:Human deep space and planetary travel is limited by uncertainties regarding the health risks associated with exposure to galactic cosmic radiation (GCR), and in particular the high linear energy transfer (LET), heavy ion component. Here we assessed the impact of two high-LET ions 56Fe and 28Si, and low-LET X rays on genome-wide methylation patterns in human bronchial epithelial cells. We found that all three radiation types induced rapid and stable changes in DNA methylation but at distinct subsets of CpG sites affecting different chromatin compartments. The 56Fe ions induced mostly hypermethylation, and primarily affected sites in open chromatin regions including enhancers, promoters and edges (“shores”) of CpG islands. The 28Si ion-exposure had mixed effects, inducing both hyper and hypomethylation and affecting sites in more repressed heterochromatic environments, whereas X rays induced mostly hypomethylation, primarily at sites in gene bodies and intergenic regions. Significantly, the methylation status of 56Fe ion irradiation sensitive sites, but not those affected by X ray or 28Si ions, could discriminate tumor from normal tissue for human lung adenocarcinomas and squamous cell carcinomas. Thus, high LET radiation exposure leaves a lasting imprint on the epigenome, and affects sites relevant to human lung cancer. The 56Fe ion signature may prove useful in monitoring the cumulative biological impact and associated cancer risks encountered by astronauts in deep space. Genome wide DNA methylation profiling of normal human bronchial epithelial cells irradiated with varying doses of 28Si-ion radiation ( 300 MeV/u at 0, 0.3, 1.0 Gy) , 56Fe-ion radiation (600 MeV/u at 0, 0.1, 0.3, 1.0 Gy) or X rays (320 kV at 0, 1.0 Gy). Triplicate control and irradiated samples were incubated and sampled at 4 timepoints between 2 and 62 days. The Illumina Infinium 450k Human DNA methylation Beadchip was used to obtain DNA methylation profiles across >485,000 CpGs from collected samples. Samples include: 56Fe ions, 4 doses x 4 time points x 3 replicates (4 removed in QC) = 44 samples; 28Si ions = 3 doses x 4 time points x 3 replicates = 36 samples; X ray, 2 doses x 4 time points x 3 replicates (2 removed in QC)= 22 samples.

			Action	DRS
	200715_Kashiwakura_0.5Gy_6_MRM-HR.wiff.scan	Wiff
	200715_Kashiwakura_0.5Gy_7_MRM-HR.wiff.scan	Wiff
	200715_Kashiwakura_0.5Gy_8_MRM-HR.wiff.scan	Wiff
	200715_Kashiwakura_control_1_MRM-HR.wiff.scan	Wiff
	200715_Kashiwakura_control_2_MRM-HR.wiff.scan	Wiff

Dataset Information

Quantification of oxidative modification of mouse serum albumin by MRM-HR

Dataset's files

Publications

Serum Proteomic and Oxidative Modification Profiling in Mice Exposed to Total Body X-Irradiation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets