Search for breast cancer proteomic markers in total blood exosomes

O. S. Tutanov; Y. S. Bakakina; K. V. Proskura; A. E. Grigoryeva; V. E. Syakhovich; S. A. Beliaev; E. I. Ryabchikova; Y. P. Tsentalovich; P. P. Laktionov; S. N. Tamkovich

doi:10.21294/1814-4861-2020-19-2-49-61

Search for breast cancer proteomic markers in total blood exosomes

Переведенное название: Поиск протеомных маркеров рака молочной железы в составе суммарных экзосом крови

O. S. Tutanov, Y. S. Bakakina, K. V. Proskura, A. E. Grigoryeva, V. E. Syakhovich, S. A. Beliaev, E. I. Ryabchikova, Y. P. Tsentalovich, P. P. Laktionov, S. N. Tamkovich

Результат исследования: Научные публикации в периодических изданиях › статья › рецензирование

Аннотация

Актуальность. Актуальной задачей повышения эффективности ранней диагностики онкологических заболеваний является поиск высокоспецифичных опухолевых маркеров в биологических жидкостях организма. Значительная часть экзосом ассоциирована с поверхностью форменных элементов крови, однако белковый спектр таких экзосом ранее не исследовался. Использование суммарных экзосом крови (экзосом плазмы и экзосом, ассоциированных с поверхностью клеток крови) может не только значительно повысить специфичность и чувствительность существующих методов, но и выявить новые онкомаркеры для жидкостной биопсии. Цель работы - поиск кандидатных белковых онкомаркеров рака молочной железы (РМЖ) путем сравнения 2D-протеомных карт суммарных экзосом крови здоровых женщин и больных РМЖ. Материал и методы. Экзосомы выделены из крови здоровых женщин и больных РМЖ методом ультрафильтрации с последующим ультрацентрифугированием и охарактеризованы при помощи трансмиссионной электронной микроскопии и иммуноцитохимии. Концентрацию белка в экзосомах определяли при помощи коммерческого набора NanoOrange Protein Quantitation kit (Invitrogen). Протеом экзосом исследован с помощью 2D-электрофореза с последующей идентификацией белков методом масс-спектрометрии. Результаты. Получены высокоочищенные препараты микровезикул суммарной крови размером не более 100 нм, на поверхности которых иммуноцитохимически были выявлены специфические для экзосом маркеры (CD63, CD9 и CD24). Сравнительный анализ протеомных карт экзосомальных белков здоровых женщин и больных РМЖ, полученных с помощью 2D-электрофореза, позволил установить значимые различия в уровне экспрессии и наборе белков в норме и патологии. Методом пептидного фингерпринта идентифицировано 11 перспективных протеомных маркеров РМЖ, из них LRG и у-цепь FGB выявлены в составе экзосом впервые (согласно базе Exocarta). Методом MALDI-TOF масс-спектрометрии идентифицировано 99 белков в препаратах экзосом крови здоровых женщин и больных РМЖ, из них 35 % выявлены в составе экзосом впервые (согласно базе Exocarta). В составе суммарных экзосом крови онкологических больных выявлено 17 (53 %) белков, ассоциированных с РМЖ. Заключение. Полученные результаты свидетельствуют о том, что суммарные экзосомы крови являются перспективным источником диагностического материала для поиска протеомных маркеров РМЖ. Идентифицированные протеомные онкомаркеры в их составе требуют дальнейшей валидации

Переведенное название	Поиск протеомных маркеров рака молочной железы в составе суммарных экзосом крови
Язык оригинала	английский
Страницы (с-по)	49-61
Число страниц	13
Журнал	Siberian Journal of Oncology
Том	19
Номер выпуска	2
DOI	https://doi.org/10.21294/1814-4861-2020-19-2-49-61
Состояние	Опубликовано - 1 фев 2020

Ключевые слова

Breast cancer
Exosomes
Mass-spectrometry
Proteomic markers
Transmission electron microscopy

ГРНТИ

34 БИОЛОГИЯ

Доступ к документу

10.21294/1814-4861-2020-19-2-49-61

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@article{925280cd1de9495e959fbd8062f83782,

title = "Search for breast cancer proteomic markers in total blood exosomes",

abstract = "To improve early detection of cancer, search for tumor markers in biological fluids is of great importance. a significant portion of exosomes is associated with the surface of blood cells, however, the protein spectrum of such exosomes has not been previously studied. the use of total blood exosomes (plasma exosomes and exosomes associated with the surface of blood cells) can not only significantly increase the specificity and sensitivity of existing methods, but also suggest new tumor markers for liquid biopsy. Objective. search for candidate protein tumor markers of breast cancer by comparing 2d-proteomic maps of total blood exosomes of healthy females (HFs) and breast cancer patients (BCPs). Methods. Exosomes were isolated from plasma and total blood of HFs and BCPs by ultrafiltration followed by ultracentrifugation and were characterized using transmission electron microscopy (tEM) and immunocytochemistry. Protein concentration in exosomes was determined using the NanoOrange Protein Quantitation kit (Invitrogen) commercial kit. Proteomes of exosomes were studied using 2d electrophoresis followed by protein identification by mass spectrometry. Results. Highly purified samples of vesicles from plasma and total blood of no more than 100 nm in size were obtained, on the surface of which markers specific for exosomes were detected by monoclonal antibodies Cd9. a comparative analysis of the proteomic maps of exosomal proteins of the HFs and BCPs obtained by 2d-electrophoresis allowed us to establish significant differences in the expression level and protein set in normal and pathological conditions. the 11 proteomic markers of breast cancer were identified by the peptide fingerprint method, of which lRG и Y-chain FGB were detected in the composition of exosomes for the first time (according to the Exocarta database). using MaldI-tOF mass spectrometry, 99 proteins were identified in the exosome preparations of the blood of HFs and BCPs, of which 35% were detected in the composition of the exosomes for the first time (according to the Exocarta database). 17 (53%) proteins associated with breast cancer were detected in total blood exosomes of cancer patients. Conclusion. the results obtained indicate that total blood exosomes are a promising source of diagnostic material for the search for proteomic markers of breast cancer. the identified proteomic tumor markers require further validation.",

keywords = "Breast cancer, Exosomes, Mass-spectrometry, Proteomic markers, Transmission electron microscopy",

author = "Tutanov, {O. S.} and Bakakina, {Y. S.} and Proskura, {K. V.} and Grigoryeva, {A. E.} and Syakhovich, {V. E.} and Beliaev, {S. A.} and Ryabchikova, {E. I.} and Tsentalovich, {Y. P.} and Laktionov, {P. P.} and Tamkovich, {S. N.}",

note = "Тутанов О.С., Бакакина Ю.С., Проскура К.В., Григорьева А.E., Сяхович В.Э., Беляев С.А., Рябчикова Е.И., Центалович Ю.П., Лактионов П.П., Тамкович С.Н. Поиск протеомных маркеров рака молочной железы в составе суммарных экзосом крови // Сибирский онкологический журнал. - 2020. - Т. 19. - № 2. - С. 49-61",

year = "2020",

month = feb,

day = "1",

doi = "10.21294/1814-4861-2020-19-2-49-61",

language = "English",

volume = "19",

pages = "49--61",

journal = "Siberian Journal of Oncology",

issn = "1814-4861",

publisher = "Tomsk National Research Medical Center of the Russian Academy of Sciences",

number = "2",

}

Search for breast cancer proteomic markers in total blood exosomes. / Tutanov, O. S.; Bakakina, Y. S.; Proskura, K. V.; Grigoryeva, A. E.; Syakhovich, V. E.; Beliaev, S. A.; Ryabchikova, E. I.; Tsentalovich, Y. P.; Laktionov, P. P.; Tamkovich, S. N.

В: Siberian Journal of Oncology, Том 19, № 2, 01.02.2020, стр. 49-61.

Результат исследования: Научные публикации в периодических изданиях › статья › рецензирование

TY - JOUR

T1 - Search for breast cancer proteomic markers in total blood exosomes

AU - Tutanov, O. S.

AU - Bakakina, Y. S.

AU - Proskura, K. V.

AU - Grigoryeva, A. E.

AU - Syakhovich, V. E.

AU - Beliaev, S. A.

AU - Ryabchikova, E. I.

AU - Tsentalovich, Y. P.

AU - Laktionov, P. P.

AU - Tamkovich, S. N.

N1 - Тутанов О.С., Бакакина Ю.С., Проскура К.В., Григорьева А.E., Сяхович В.Э., Беляев С.А., Рябчикова Е.И., Центалович Ю.П., Лактионов П.П., Тамкович С.Н. Поиск протеомных маркеров рака молочной железы в составе суммарных экзосом крови // Сибирский онкологический журнал. - 2020. - Т. 19. - № 2. - С. 49-61

PY - 2020/2/1

Y1 - 2020/2/1

N2 - To improve early detection of cancer, search for tumor markers in biological fluids is of great importance. a significant portion of exosomes is associated with the surface of blood cells, however, the protein spectrum of such exosomes has not been previously studied. the use of total blood exosomes (plasma exosomes and exosomes associated with the surface of blood cells) can not only significantly increase the specificity and sensitivity of existing methods, but also suggest new tumor markers for liquid biopsy. Objective. search for candidate protein tumor markers of breast cancer by comparing 2d-proteomic maps of total blood exosomes of healthy females (HFs) and breast cancer patients (BCPs). Methods. Exosomes were isolated from plasma and total blood of HFs and BCPs by ultrafiltration followed by ultracentrifugation and were characterized using transmission electron microscopy (tEM) and immunocytochemistry. Protein concentration in exosomes was determined using the NanoOrange Protein Quantitation kit (Invitrogen) commercial kit. Proteomes of exosomes were studied using 2d electrophoresis followed by protein identification by mass spectrometry. Results. Highly purified samples of vesicles from plasma and total blood of no more than 100 nm in size were obtained, on the surface of which markers specific for exosomes were detected by monoclonal antibodies Cd9. a comparative analysis of the proteomic maps of exosomal proteins of the HFs and BCPs obtained by 2d-electrophoresis allowed us to establish significant differences in the expression level and protein set in normal and pathological conditions. the 11 proteomic markers of breast cancer were identified by the peptide fingerprint method, of which lRG и Y-chain FGB were detected in the composition of exosomes for the first time (according to the Exocarta database). using MaldI-tOF mass spectrometry, 99 proteins were identified in the exosome preparations of the blood of HFs and BCPs, of which 35% were detected in the composition of the exosomes for the first time (according to the Exocarta database). 17 (53%) proteins associated with breast cancer were detected in total blood exosomes of cancer patients. Conclusion. the results obtained indicate that total blood exosomes are a promising source of diagnostic material for the search for proteomic markers of breast cancer. the identified proteomic tumor markers require further validation.

AB - To improve early detection of cancer, search for tumor markers in biological fluids is of great importance. a significant portion of exosomes is associated with the surface of blood cells, however, the protein spectrum of such exosomes has not been previously studied. the use of total blood exosomes (plasma exosomes and exosomes associated with the surface of blood cells) can not only significantly increase the specificity and sensitivity of existing methods, but also suggest new tumor markers for liquid biopsy. Objective. search for candidate protein tumor markers of breast cancer by comparing 2d-proteomic maps of total blood exosomes of healthy females (HFs) and breast cancer patients (BCPs). Methods. Exosomes were isolated from plasma and total blood of HFs and BCPs by ultrafiltration followed by ultracentrifugation and were characterized using transmission electron microscopy (tEM) and immunocytochemistry. Protein concentration in exosomes was determined using the NanoOrange Protein Quantitation kit (Invitrogen) commercial kit. Proteomes of exosomes were studied using 2d electrophoresis followed by protein identification by mass spectrometry. Results. Highly purified samples of vesicles from plasma and total blood of no more than 100 nm in size were obtained, on the surface of which markers specific for exosomes were detected by monoclonal antibodies Cd9. a comparative analysis of the proteomic maps of exosomal proteins of the HFs and BCPs obtained by 2d-electrophoresis allowed us to establish significant differences in the expression level and protein set in normal and pathological conditions. the 11 proteomic markers of breast cancer were identified by the peptide fingerprint method, of which lRG и Y-chain FGB were detected in the composition of exosomes for the first time (according to the Exocarta database). using MaldI-tOF mass spectrometry, 99 proteins were identified in the exosome preparations of the blood of HFs and BCPs, of which 35% were detected in the composition of the exosomes for the first time (according to the Exocarta database). 17 (53%) proteins associated with breast cancer were detected in total blood exosomes of cancer patients. Conclusion. the results obtained indicate that total blood exosomes are a promising source of diagnostic material for the search for proteomic markers of breast cancer. the identified proteomic tumor markers require further validation.

KW - Breast cancer

KW - Exosomes

KW - Mass-spectrometry

KW - Proteomic markers

KW - Transmission electron microscopy

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