DOI: 10.20348/storedb/1155/1220
title: Radiation exposure of peripheral mononuclear blood cells alters the composition and function of released extracellular vesicles
title: Proteomic analysis on the extracellular vesicles of irradiated peripheral mononuclear blood cells

description: Normal tissue toxicity is a dose-limiting factor in radiation therapy. One component of normal tissue that is continuously exposed during therapeutic irradiation is the circulating population of blood mononuclear cells (PBMC). PBMCs are highly sensitive to IR, however little is known how ionizing radiation (IR) affects the PBMC response on a systemic level. Therefore it was the aim of this study to investigate whether IR was capable to induce changes in the composition and function of extracellular vesicles secreted from PBMCs after radiation exposure to different doses. Whole blood samples from healthy donors were irradiated with 0 Gy, 0,1 Gy, 2 Gy or 6 Gy X-rays and PBMC-secreted EVs were isolated 72 h later. Proteome analysis of EVs by label-free proteomics identified 606 proteins, of which 283 significantly changed their abundance after irradiation. microRNA expression analysis by small RNA sequencing revealed 379 microRNAs, of which 34 were changed after irradiation. For both, proteome and microRNA data, principal component analysis showed a dose-dependent separation of control and exposed groups. An IPA network analysis of the radiation-regulated EV proteins and microRNAs consistently predicted an association of deregulated components with apoptosis, cell death and survival. Functional studies identified endothelial cells as efficient EV recipient system, whereby irradiation of recipient cells further increased the uptake. Furthermore we detected an apoptosis suppressive effect of EVs from irradiated PBMCs in endothelial recipient cells. In summary, our study demonstrates that IR triggers the communication between PBMCs and endothelial cells. We identified EVs from irradiated PBMC donors as transmitters of protective signals to irradiated endothelial cells. Thus our data may lead to the discovery of biomarker candidates for radiation dosimetry and even more importantly, they suggest EVs a novel systemic communication pathway between irradiated and non-irradiated normal, non-cancer tissues.
description: Filter aided sample preparation (FASP) digest Each 10 µg were digested using a modified FASP procedure (Wiśniewski et al., 2009). After reduction and alkylation using DTT and IAA, the proteins were centrifuged on a 30 kDa cutoff filter device (Sartorius), washed thrice with UA buffer (8 M urea in 0.1 M Tris/HCl pH 8.5) and twice with 50 mM ammoniumbicarbonate. The proteins were digested for 2 hours at room temperature using 1 µg Lys-C (Wako Chemicals, Neuss, Germany) and for 16 hours at 37°C using 2 µg trypsin (Promega, Mannheim, Germany). After centrifugation (10 min at 14 000 g) the eluted peptides were acidified with 0.5% TFA and stored at -20°C. LC-MSMS measurements LC-MS/MS analysis was performed on a Q-Exactive HF mass spectrometer (Thermo Scientific) online coupled to an Ultimate 3000 nano-RSLC (Dionex). Tryptic peptides were automatically loaded on a C18 trap column (300 µm inner diameter (ID) × 5 mm, Acclaim PepMap100 C18, 5 µm, 100 Å, LC Packings) at 30µl/min flow rate prior to C18 reversed phase chromatography on the analytical column (nanoEase MZ HSS T3 Column, 100Å, 1.8 µm, 75 µm x 250 mm, Waters) at 250nl/min flow rate in a 95 minutes non-linear acetonitrile gradient from 3 to 40% in 0.1% formic acid. Profile precursor spectra from 300 to 1500 m/z were recorded at 60000 resolution with an automatic gain control (AGC) target of 3e6 and a maximum injection time of 50 ms. TOP10 fragment spectra of charges 2 to 7 were recorded at 15000 resolution with an AGC target of 1e5, a maximum injection time of 50 ms, an isolation window of 1.6 m/z, a normalized collision energy of 28 and a dynamic exclusion of 30 seconds. Quantitative data analysis using Progenesis QI for proteomics Generated raw files were analyzed using Progenesis QI for proteomics (version 3.0, Nonlinear Dynamics, part of Waters) for label-free quantification as described previously (Hauck et al., 2010; Merl et al., 2012). Features of charges 2-7 were used and all MSMS spectra were exported as mgf file. Peptide search was performed using Mascot search engine (version 2.5.1) against the Ensembl human protein database (83462 sequences, 31286148 residues). Search settings were: 10 ppm precursor tolerance, 0.02 Da fragment tolerance, one missed cleavage allowed. Carbamidomethyl on cysteine was set as fixed modification, deamidation of glutamine and asparagine allowed as variable modification, as well as oxidation of methionine. Applying the percolator algorithm (Brosch et al., 2009) resulted in a peptide false discovery rate (FDR) of 0.54%. Search results were reimported in the Progenesis QI software. Proteins were quantified by summing up the abundances of all unique peptides per protein. Resulting normalized protein abundances were used for calculation of fold-changes and statistical values were exported from the Progenesis QI software. For final quantifications, proteins were identified with at least 2 unique peptides and with ratios greater than 1.30-fold or less than 0.77-fold (p 0.05) were defined as being significantly differentially expressed.