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

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.
On the basis of the result of the information-dependent acquisition, an assay for a high-resolution multiple reaction monitoring (MRM-HR) experiment targeted to the serum albumin oxidative modification was developed using Skyline software (MacCoss Lab, University of Washington, WA). The transitions of MRM-HR targeting each peptide were shown in Table 3. A TOF-MS scan in the mass range of 400-2,250 Da with 0.1 sec accumulation time was acquired followed by the product ion of target peptide precursors mass with 100 ms accumulation time. Collision energies were calculated with the rolling collision energy as in the information-dependent acquisition experiment. For MRM-HR data analysis, MultiQuant 3.0 Software (AB Sciex) was used to calculate integrated area of each peptide. All peak selections were checked manually after the automated matches.