Study of the Effect of X-Ray Radiation on the Structural Characteristics of Bovine Serum Albumin Protein Using High-Resolution Liquid Chromatography−Mass Spectrometry
Аннотация
A method for assessing changes in the structural characteristics of the bovine serum albumin (BSA) protein in an aqueous solution as a result of exposure to ionizing radiation is developed and tested. The method consists in identifying unique peptides of the protein domain structures, as well as in establishing modifications of the amino acid sequence using high-resolution liquid chromatography–mass spectrometry. A BSA solution is irradiated by X-rays from a tube of a voltage of 80 kV and average current of 1 mA, the dose rate is 2 Gy/s. The absorbed dose in the sample volume is estimated by the ferrosulfate dosimetry method. An aqueous solution of BSA is irradiated at doses of 0.1, 0.5, 1, 2, 4, and 8 kGy, after which the amount of protein molecules in the solution is quantitatively estimated and the structural integrity of the native form of the protein is analyzed, as well as amino acid modifications in the BSA sequence as a result of exposure to radiation are established. For a more in-depth analysis, the cysteine–cysteine disulfide bonds of BSA are reduced, followed by the alkylation of the resulting thiol residues with bromoacetic acid amide. The enzymatic hydrolysis of BSA is carried out with the addition of a trypsin solution. The resulting samples are analyzed by high-resolution liquid chromatography–mass spectrometry with high-resolution tandem mass spectrometric detection. Next, the change in the number of intact protein molecules is estimated by determining unique peptides corresponding to each of the three domains forming the amino acid sequence of BSA. Taking into account the optimization of the detection conditions for the three domains as markers of the active form of BSA, the limit of detection for each peptide is calculated. The developed approach makes it possible to establish a change in the natural conformation of the bovine serum albumin protein (its denaturation) in water samples as a result of exposure to ionizing radiation at doses of 4−8 kGy at an average power of 2 Gy/s.