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

Мұқаба

Дәйексөз келтіру

Толық мәтін

Ашық рұқсат Ашық рұқсат
Рұқсат жабық Рұқсат берілді
Рұқсат жабық Тек жазылушылар үшін

Аннотация

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.

Толық мәтін

Рұқсат жабық

Авторлар туралы

A. Brown

Lomonosov Moscow State University, Faculty of Chemistry

Хат алмасуға жауапты Автор.
Email: igorrodin@yandex.ru
Ресей, Moscow

U. Bliznyuk

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

Email: igorrodin@yandex.ru
Ресей, Moscow; Moscow

P. Borshchegovskaya

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

Email: igorrodin@yandex.ru
Ресей, Moscow; Moscow

V. Ipatova

Skobeltsyn Institute of Nuclear Physics

Email: igorrodin@yandex.ru
Ресей, Moscow

A. Chernyaev

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

Email: igorrodin@yandex.ru
Ресей, Moscow; Moscow

I. Ananyeva

Lomonosov Moscow State University, Faculty of Chemistry

Email: igorrodin@yandex.ru
Ресей, 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
Ресей, Moscow; Moscow

Әдебиет тізімі

  1. Черняев А.П. Радиационные технологии. Наука. Народное хозяйство. Медицина. М.: Издательство Московского университета, 2019. С. 231.
  2. Черняев А.П., Колыванова М.А., Борщеговская П.Ю. Радиационные технологии в медицине. Часть 1. Медицинские ускорители // Вестн. Моск. ун-та. Серия 3: Физика, астрономия. 2015. № 6. С. 28. (Chernyaev A.P., Kolyvanova M.A., Borshchegovskaya P.Yu. Radiation technology in medicine. Рart 1. medicine accelerators // Moscow Univ. Phys. Bull. 2015. V. 70. № 6. Р. 457.)
  3. ISO 11137-3-2006 Sterilization of health care products Radiation Part 3: Guidance on dosimetric aspects. Стерилизация медицинской продукции. Облучение. Часть 3. Руководство по вопросам дозиметрии.
  4. ISO 14470-2011 Food irradiation – Requirements for the development, validation and routine control of the process of irradiation using ionizing radiation for the treatment of food. Радиационная обработка пищевых продуктов. Требования к разработке, валидации и повседневному контролю процесса облучения пищевых продуктов ионизирующим излучением.
  5. Zhang Rong Ke, Lyu Jia Hua, Li Tao. Effects of radiation on protein // J. Nutr. Oncol. 2020. V. 5. № 3. P. 116. https://doi.org/10.34175/jno202003002
  6. Пузан Н.Д., Чешик И.А. Молекулярные механизмы действия ионизирующего излучения. Влияние облучения на белок (обзор литературы) // Медико-биологические проблемы жизнедеятельности. 2023. № 1. С. 14. (Puzan N.D., Cheshik I.A. Molecular mechanisms of effects of ionizing radiation action. Irradiation effect on protein (literary review) // Medical and Biological Problems of Life Activity. 2023. № 1. P. 14 (in Russ.)) https://doi.org/10.58708/2074-2088.2023-1(29)-14-26
  7. Тимакова Р.Т. Влияние ионизирующего излучения на биологическую ценность белков говядины // Пищевая промышленность. 2020. № 5. C. 12.
  8. Rozhko T.V., Nemtseva E.V., Gardt M.V., Raikov A.V., Lisitsa A.E., Badun G.A., Kudryasheva N.S. Enzymatic responses to low-intensity radiation of tritium // Int. J. Mol. Sci. 2020. V. 21. № 22. P. 1. Article 8464. https://doi.org/10.3390/ijms21228464
  9. Adibian M., Mami Y. Effect of electron-beam irradiation on enzyme activities in Agaricus brunnescens // J. Pure Appl. Microbiol. 2018. V. 12. № 3. P. 1435. https://doi.org/10.22207/JPaM.12.3.46
  10. Павлов А.Н., Чиж Т.В., Снегирев А.С., Санжарова Н.И., Черняев А.П., Борщеговская П.Ю. и др. Технологический процесс радиационной обработки пищевой продукции и дозиметрическое обеспечение // Радиационная гигиена. 2020. Т. 13. № 4. С. 40. https://doi.org/10.21514/1998-426X-2020-13-4-40-50
  11. Giroux M., Lacroix M. Nutritional adequacy of irradiated meat – A review // Food Res. Int. 1998. V. 31. № 4. С. 257.
  12. Дриль А.А., Рождественская Л.Н. Повышение биологической ценности белка и увеличение сроков хранения полуфабриката из вешенки обыкновенной методом электронной стерилизации // Изв. вузов. Прикл. химия и биотехнология. 2019. Т. 9. № 3. С. 500.
  13. Zhao L., Zhang Y., Pan Z., Venkitasamy C., Zhang L., Xiong W. et al. Effect of electron beam irradiation on quality and protein nutrition values of spicy yak jerky // LWT. 2017. № 87. Р. 1.
  14. Zarei H., Bahreinipour M., Eskandari K., Eskandari K., Zarandi S.-M., Ardestani S. Spectroscopic study of gamma irradiation effect on the molecular structure of bovine serum albumin // Vacuum. 2016. № 136. Р. 91.
  15. Liu G.X., Liu J., Tu Z.C., Sha X.M., Wang H., Wang Z.X. Investigation of conformation change of glycated ovalbumin obtained by Co-60 gamma-ray irradiation under drying treatment // Innov. Food Sci. Emerg. Technol. 2018. № 47. P. 286.
  16. Liu Y.-F., Oey I., Bremer P., Carne A., Silcock P. Modifying the functional properties of egg proteins using novel processing techniques: A review // Comprehensive Reviews in Food Science and Food Safety. 2019. V. 18. № 4. P. 986.10.1111/1541-4337.12464
  17. Olsen J.V., Ong S.E., Mann M. Trypsin cleaves exclusively C-terminal to arginine and lysine residues // Mol. Cell Proteomics. 2004. V. 3. № 6. P. 608. https://doi.org/10.1074/mcp.T400003-MCP200
  18. Keil B. Proteolysis data bank: Specificity of alpha-chymotrypsin from computation of protein cleavages // Protein Seq. Data Anal. 1987. V. 1. № 1. P. 13.
  19. Walmsley S.J., Rudnick P.A., Liang Y., Dong Q., Stein S.E., Nesvizhskii A.I. Comprehensive analysis of protein digestion using six trypsins reveals the origin of trypsin as a significant source of variability in proteomics // J Proteome Res. 2013. V. 12. № 12. P. 5666. https://doi.org/10.1021/pr400611h
  20. Saveliev S., Bratz M., Zubarev R., Szapacs M., Budamgunta H., Urh M. Trypsin/Lys-C protease mix for enhanced protein mass spectrometry analysis // Nat. Methods. 2013. V. 10. № 11. https://doi.org/10.1038/nmeth.f.371
  21. Браун А.В., Близнюк У.А., Борщеговская П.Ю., Ипатова В.С., Хмелевский О.Ю., Черняев А.П. и др. Исследование влияния ускоренных электронов на структурные характеристики бычьего сывороточного альбумина с использованием жидкостной хромато-масс-спектрометрии высокого разрешения // Заводск. лаборатория. Диагностика материалов. 2023. Т. 89. № 3. С. 14.
  22. Sam K.C. Chang, Yan Zhang. Protein analysis / Food Analysis. New York: Springer. 2017. P. 315. https://doi.org/10.1007/978-3-319-45776-5_18
  23. Марри Р., Греннер Д., Мейес П., Родуэлл В. Биохимия человека. М.: Мир, 1993. Т. 1. С. 384. ISBN 5-03-001774-7
  24. Gonzalez V.D., Gugliotta L.M., Giacomelli C.E., Meira G.R. Latex of immunodiagnosis for detecting the Chagas disease: II. Chemical coupling of antigen Ag36 onto carboxylated latexes // J. Mater. Sci. Mater. Med. 2008. № 19. С. 789.
  25. Doumas B.T., Bayse D.D., Carter R.J., Peters T., Schaffer R. A candidate reference method for determination of total protein in serum. I. Development and validation // Clin. Chem. 1981. V. 27. № 10. P. 1642.
  26. Francis G. Albumin and mammalian cell culture: Implications for biotechnology applications // Cytotechnology. 2010. № 62. Р. 1.
  27. Спектрофотометр УФ-3000. https://istina.msu.ru/equipment/card/615320740/ (дата обращения сентябрь 2024).
  28. Swissprot. Swiss Institute of Bioinformatics: Geneva, Switzerland. http://www.uniprot.org/contact (дата обращения 17.01.2018).
  29. Huang B.X., Kim H.Y., Dass C. Probing three-dimensional structure of bovine serum albumin by chemical cross-linking and mass spectrometry // J. Am. Soc. Mass Spectrom. 2004. № 15. P. 1237.

Қосымша файлдар

Қосымша файлдар
Әрекет
1. JATS XML
Жүктеу
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+).

Жүктеу (418KB)
Метадеректер
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.

Жүктеу (205KB)
Метадеректер

© Russian Academy of Sciences, 2025