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

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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.

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A. Brown

Lomonosov Moscow State University, Faculty of Chemistry

编辑信件的主要联系方式.
Email: igorrodin@yandex.ru
俄罗斯联邦, Moscow

U. Bliznyuk

Lomonosov Moscow State University, Faculty of Physics; Skobeltsyn Institute of Nuclear Physics

Email: igorrodin@yandex.ru
俄罗斯联邦, Moscow; Moscow

P. Borshchegovskaya

Lomonosov Moscow State University, Faculty of Physics; Skobeltsyn Institute of Nuclear Physics

Email: igorrodin@yandex.ru
俄罗斯联邦, Moscow; Moscow

V. Ipatova

Skobeltsyn Institute of Nuclear Physics

Email: igorrodin@yandex.ru
俄罗斯联邦, Moscow

A. Chernyaev

Lomonosov Moscow State University, Faculty of Physics; Skobeltsyn Institute of Nuclear Physics

Email: igorrodin@yandex.ru
俄罗斯联邦, Moscow; Moscow

I. Ananyeva

Lomonosov Moscow State University, Faculty of Chemistry

Email: igorrodin@yandex.ru
俄罗斯联邦, Moscow

I. Rodin

Lomonosov Moscow State University, Faculty of Chemistry; I.M. Sechenov First Moscow State Medical University of the Ministry of Health of the Russian Federation (Sechenov University)

Email: igorrodin@yandex.ru
俄罗斯联邦, Moscow; Moscow

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2. Fig. 1. Mass spectra of fragmentation of protonated molecular ions of peptides selected for detection of BSA after trypsinolysis in the positively charged ion registration mode: (a) – peptide T66–75 (precursor ion charge 2+); (b) – peptide T402–412 (precursor ion charge 2+); (c) – T549–557 (precursor ion charge 2+).

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3. Fig. 2. Diagram of the relative concentration of identified unique peptides for radiation doses of 0–8 kGy at an X-ray dose rate of 2 Gy/s.

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