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In the event of a nuclear detonation, thousands of people will be exposed to non-lethal radiation doses. There are multiple long-term health concerns for exposed individuals who receive non-lethal radiation exposures. Low doses of radiation, especially of high linear energy transfer (LET) radiation, can lead to the development of neurocognitive defects. The identification of serum biomarkers that can be used to monitor the emergence of the long-term biological sequelae of radiation exposure, such as neurocognitive defects, would greatly help the post-exposure health monitoring of the affected population. The authors have determined the impact that cranial irradiation with 2 Gy of high LET (150 keV um) has on the ability of rats to perform spatial memory tasks, and identified serum protein changes that are biomarkers of radiation exposure and of radiation-induced neurocognitive impairment. Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectroscopy (MALDI TOF-TOF) analysis of weak cation exchange (WCX) enriched serum protein preparations identified 23 proteins of interest: 10 were biomarkers of physical radiation dose, with six showing increased expression and four being undetectable in the irradiated rat serum. Four proteins were uniquely expressed in those rats that had good spatial memory and nine proteins were markers of bad spatial memory. This study provides proof of the concept that serum protein profiling can be used to identify biomarkers of radiation exposure and the emergence of radiation-sequelae in this rat model, and this approach could be easily applied to other systems to identify radiation biomarkers.

MALDI-TOF mass spectrometry is a widely used technique for serum protein expression profiling and biomarker discovery. Many profiling strategies typically employ chemical affinity beads or surfaces to decrease sample complexity of dynamic fluids such as serum or plasma. However, many of the proteins captured on a particular surface or bead are not resolved in the lower mass ranges where time-of-flight mass spectrometers are most effective. Thus, a majority of reported protein expression profiling studies primarily interrogate the native low molecular mass constituents of the target sample. We report an expression profiling workflow that utilizes immobilized trypsin paramagnetic beads following an initial affinity bead fractionation step, thereby reducing large mass proteins to peptides that are better suited to analysis and sequencing determinations. Our bead-based trypsin approach resulted in more efficient digestion of complex serum protein extracts at short incubation times. This method was reproducible and readily adaptable to robotic sample handling and may be combined in tandem with other bead fractionation surfaces. When weak cationic and weak anionic bead surfaces were used, experimental conditions were optimized for tandem combinations of these beads with the immobilized trypsin step to produce an efficient serum fractionation strategy. A proof-of-concept pilot experiment using pooled human serum samples demonstrating reproducibility is presented, along with the sequence determination of selected tryptic peptides of serum proteins.