Study of the effect of x-ray radiation on the structural characteristics of bovine serum albumin protein using high-resolution liquid chromatography-mass spectrometry

A method for assessing changes in the structural characteristics of bovine serum albumin (BSA) protein in aqueous solution as a result of exposure to ionizing radiation has been developed and tested. The method consists of identifying unique peptides of the protein domain structures, as well as establishing amino acid sequence modifications using high-resolution liquid chromatography-mass spectrometry. The BSA solution was irradiated with X-ray radiation with a tube voltage of 80 kV and an average current of 1 mA, the dose rate was 2 Gy/sec. The absorbed dose in the sample volume was estimated by the ferrosulfate dosimetry method. Aqueous solution of BSA was irradiated at doses of 0.1, 0.5, 1, 2, 4, and 8 kGy, after which the content of protein molecules in the solution was quantitatively assessed and the structural integrity of the native form of protein was analyzed, as well as the modifications of amino acids in the BSA sequence as a result of the radiation action were determined. For more in-depth analysis, the reduction of cysteine-cysteine disulfide bonds by BSA followed by alkylation of the resulting thiol residues with bromoacetic acid amide was performed. Enzymatic hydrolysis of BSA was carried out with the addition of trypsin solution. The obtained samples were analyzed by high-resolution liquid chromatography-mass spectrometry with high-resolution tandem mass spectrometric detection. Next, we evaluated the change in the number of intact protein molecules by detecting unique peptides corresponding to each of the three domains that make up the amino acid sequence of BSA. The detection limit of each peptide was calculated taking into account the optimization of conditions for detection of the three domains as markers of the active form of BSA. The developed approach made it possible to determine the change in the natural conformation of bovine serum albumin protein (its denaturation) in aqueous samples as a result of ionizing radiation exposure at doses of 4-8 kGy at an average power of 2 Gy/sec.