DOI: 10.5061/dryad.9p8cz8wm7
title: Analysis of the proteomic profile in serum of irradiated nonhuman primates treated with Ex-Rad, a radiation medical countermeasure

description: There are currently four radiation medical countermeasures that have been
 approved by the United States Food and Drug Administration to mitigate
 hematopoietic acute radiation syndrome, all of which are repurposed
 radiomitigators. The evaluation of additional candidate drugs that may
 also be helpful for use during a radiological/nuclear emergency is
 ongoing. A chlorobenzyl sulfone derivative (organosulfur compound) known
 as Ex-Rad, or ON01210, is one such candidate medical countermeasure, being
 a novel, small-molecule kinase inhibitor that has demonstrated efficacy in
 the murine model. In this study, nonhuman primates exposed to ionizing
 radiation were subsequently administered Ex-Rad as two treatment schedules
 (Ex-Rad I administered 24 and 36 h post-irradiation, and Ex-Rad II
 administered 48 and 60 h post-irradiation) and the proteomic profiles of
 serum using a global molecular profiling approach were assessed. We
 observed that administration of Ex-Rad post-irradiation is capable of
 mitigating radiation-induced perturbations in protein abundance,
 particularly in restoring protein homeostasis and immune response and
 mitigating hematopoietic damage, at least in part after acute exposure.
 Taken together, restoration of functionally significant pathway
 perturbations may serve to protect damage to vital organs and provide
 long-term survival benefits to the afflicted population.
description: Data Acquisition Nano-liquid
 chromatography-tandem mass spectrometry (nano-LC-MS/MS) was used to
 analyze the peptides from human serum digests, performed on a ThermoFisher
 Ultimate 3000 HPLC System (ThermoFisher Scientific, Bremen, Germany)
 connected to a quadrupole-Orbitrap mass spectrometer (Q-Exactive,
 ThermoFisher Scientific, Bremen, Germany) equipped with an online
 nano-electrospray flexi ion source. Firstly, the
 peptides were redissolved in 40 μl solvent (100% water with 0.1% formic
 acid) and then 1 μg peptide sample was loaded on the analytical column
 (Acclaim PepMap C18, 75 mm and 25 cm). Subsequently, the peptides were
 separated and identified under a 65 min method, which was composed of 2
 min in 25% B, 40 min of 95% B, 48 min of 95% B, 50 min of 1% B, 50.1 min
 of 95% B, 55 min of 95%, and 57 min of 1% B. The column was then
 reequilibrated at initial conditions for 8 min with a constant flow rate
 at 300 nl/min. The column temperature kept at ambient temperature and the
 electrospray voltage of 2.2 kV versus the inlet of the mass spectrometer
 was applied. For the library development on the fractions, the Q-Exactive
 mass spectrometer was operated in data-dependent mode and each scan cycle
 consisted of one full-MS scan at the m/z ranging from m/z 375 to 2,000
 with a mass resolution of 70 K. Then the selected parent ions were
 fragmented in ten sequential high-energy collisional dissociations (HCD)
 MS/MS scans with a resolution of 17.5 K. In addition, for MS/MS, the
 precursor ions were activated with 30% normalized collision energy and 2
 m/z of the isolation window. In all cases, single charge state was
 rejected and a micro-scan was recorded using dynamic exclusion of 30
 s. Samples were acquired in the Data Independent
 Acquisition (DIA) mode after a full precursor ion scan (range between 375
 – 2,000 m/z). The resolution of the instrument was set to 35,000 and the
 isolation window was 20 Da. For both DDA and DIA, the normalized collision
 energy (NCE) was 30 ev. Database retrieval and
 statistical tests Tandem mass spectra were searched by
 Pulsar search engine (Biognosys) and the database searched was the Human
 UniProt obtained on 11/23/2021. The data retrieval parameters for the
 Q-Exactive instrument were as follows: precursor mass tolerance: 5 ppm;
 fragment mass tolerance: 50 mmu; enzyme: trypsin; missed cleavages: 2;
 fixed modification: Carbamidomethyl on cysteine (C); variable
 modifications: Oxidation on methionine (M). The percolator algorithm was
 used to control the false discovery rate (FDR) of peptides below
 1%. The test of radiation effect and radiomitigative
 effects of Ex-Rad were calculated either by Mann–Whitney U test or
 two-tailed unpaired Student t-test, whereas p-values of less than 0.05
 exclusively were considered significant. Furthermore, p-values were
 corrected for multiple testing using Benjamini–Hochberg procedure which
 limits the FDRs. All statistical analyses were performed using R (version
 4.2.0).