High-Performance Liquid Chromatography–Mass Spectrometry Study of the Effect of Accelerated Electrons on the Structure of Bovine Serum Albumin

IF 0.9 4区材料科学 Q4 MATERIALS SCIENCE, MULTIDISCIPLINARY Inorganic Materials Pub Date : 2024-07-19 DOI:10.1134/s002016852470002x

A. V. Brown, U. A. Bliznyuk, P. Yu. Borshchegovskaya, V. S. Ipatova, O. Yu. Khmelevsky, A. P. Chernyaev, I. A. Ananyeva, I. A. Rodin

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Abstract

A method is proposed for the high-performance liquid chromatography–mass spectrometry quantification of the effect of an ionizing radiation dose on the structural characteristics of bovine serum albumin (BSA) in an aqueous solution by identifying unique peptides of the protein domain structures. BSA with an initial concentration of 500 mg/L in the physiological solution is irradiated with an accelerated electron beam with the maximum energy of 1 MeV and an average beam current of 1 pA at a dose rate of 18.5 Gy/s. The dose absorbed in the sample volume is estimated with a Fricke (ferrous sulfate) dosimeter. After irradiation of the BSA solution at doses of 0.3, 0.6, 1, 8, and 20 kGy, the structural integrity of the protein native form is analyzed and the protein content is quantified. To do this, the compounds with a mass of more than 30 kDa are removed by centrifugation, and then, BSA is subjected to enzymatic hydrolysis with addition of the trypsin solution, and the resulting peptides with a mass of more than 10 kDa are repeatedly removed. The samples obtained are examined by high-performance liquid chromatography–mass spectrometry. After that, the content of intact protein molecules is estimated by determining the concentrations of unique peptides corresponding to each of the three domains formed from the BSA amino acid sequence. Using the developed technique, a change in the natural conformation of the investigated protein (its denaturation) in water samples induced by ionizing radiation at a dose ranging from 0.3 to 20 kGy is established, on average, for 71% of protein molecules exposed to doses of up to 1 kGy, 79% of molecules exposed to doses of 4 kGy, and 99–100% of molecules exposed to doses of 8 and 20 kGy.

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加速电子对牛血清白蛋白结构影响的高效液相色谱-质谱法研究

摘要提出了一种高效液相色谱-质谱法，通过识别蛋白质结构域的独特肽段，定量测定电离辐射剂量对水溶液中牛血清白蛋白（BSA）结构特征的影响。用最大能量为 1 MeV、平均束流为 1 pA 的加速电子束以 18.5 Gy/s 的剂量率辐照生理溶液中初始浓度为 500 mg/L 的 BSA。样品体积中吸收的剂量用 Fricke（硫酸亚铁）剂量计估算。以 0.3、0.6、1、8 和 20 kGy 的剂量辐照 BSA 溶液后，分析蛋白质原生形态的结构完整性并量化蛋白质含量。为此，先离心除去质量大于 30 kDa 的化合物，然后加入胰蛋白酶溶液对 BSA 进行酶水解，反复除去质量大于 10 kDa 的肽。得到的样品通过高效液相色谱-质谱法进行检测。然后，通过测定 BSA 氨基酸序列形成的三个结构域中每个结构域对应的独特肽的浓度，估算出完整蛋白质分子的含量。利用所开发的技术，在剂量为 0.3 至 20 kGy 的电离辐射诱导下，平均 71% 的蛋白质分子受到 1 kGy 剂量的辐射，79% 的分子受到 4 kGy 剂量的辐射，99%-100% 的分子受到 8 kGy 和 20 kGy 剂量的辐射，水样中被研究蛋白质的天然构象发生了变化（变性）。

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来源期刊

Inorganic Materials 工程技术-材料科学：综合

CiteScore

1.40

自引率

25.00%

发文量

审稿时长

3-6 weeks

期刊介绍： Inorganic Materials is a journal that publishes reviews and original articles devoted to chemistry, physics, and applications of various inorganic materials including high-purity substances and materials. The journal discusses phase equilibria, including P–T–X diagrams, and the fundamentals of inorganic materials science, which determines preparatory conditions for compounds of various compositions with specified deviations from stoichiometry. Inorganic Materials is a multidisciplinary journal covering all classes of inorganic materials. The journal welcomes manuscripts from all countries in the English or Russian language.