БИОХИМИЯ, 2026, том 91, вып. 3, с. 511–536
УДК 577.12
Анализ чистоты и качества малых внеклеточных везикул плазмы крови методами протеомики и рамановской спектроскопии
1 Федеральное государственное бюджетное учреждение «Национальный медицинский исследовательский центр акушерства, гинекологии и перинатологии имени академика В.И. Кулакова» Минздрава России, 117997 Москва, Россия
2 Сколковский институт науки и технологий, 143026 Москва, Россия
3 Московский физико-технический институт (государственный университет), 141700 Московская область, Россия
4 Физический институт имени П.Н. Лебедева РАН, 119991 Москва, Россия
5 Институт биохимической физики имени Н.М. Эмануэля РАН, 119334 Москва, Россия
6 Московский государственный университет имени М.В. Ломоносова, Научно-исследовательский институт физико-химической биологии имени А.Н. Белозерского, 119234 Москва, Россия
7 Московский государственный университет имени М.В. Ломоносова, биологический факультет, 119234 Москва, Россия
8 Институт высшей нервной деятельности и нейрофизиологии Российской академии наук, 117485 Москва, Россия
9 ФГАОУ ВО Первый МГМУ имени И.М. Сеченова Минздрава России, кафедра акушерства, гинекологии, перинатологии и репродуктологии, 119991 Москва, Россия
Поступила в редакцию 18.11.2025
После доработки 13.02.2026
Принята к публикации 14.02.2026
DOI: 10.7868/S3034529426030047
КЛЮЧЕВЫЕ СЛОВА: малые внеклеточные везикулы, выделение, плазма крови, протеомика, масс-спектрометрия, рамановская спектроскопия.
Аннотация
Ультрацентрифугирование (UC) долгое время считалось «золотым стандартом» для выделения внеклеточных везикул (ВВ). Однако из‑за таких недостатков, как высокая стоимость ультрацентрифуги и роторов, трудоёмкий и длительный протокол, низкий выход с учётом исходного объёма биожидкости и низкая пропускная способность, продолжается разработка новых подходов к выделению ВВ. В настоящей работе мы сравнили три метода выделения наиболее изученного подтипа внеклеточных везикул, а именно малых внеклеточных везикул (мВВ), из плазмы крови человека: ультрацентрифугирование (UC), экспресс-асимметричную глубинную фильтрацию (ExADFi) и иммуноаффинный захват анти‑CD9 (AS‑CD9), с фокусом на их рамановские и протеомные профили. Для всех трёх методов чистоту и качество выделения мВВ оценивали по уровню контаминации фракции мВВ основными белками плазмы крови, такими как альбумин и аполипопротеины (APOA1, APOH, APOA4, APOC2, APOC1 и APOC4). Метод UC показал наибольшее соотношение концентрации белка к концентрации наночастиц. AS‑CD9 и ExADFi обеспечили чистоту и уровни невезикулярных загрязнений, сопоставимые с UC, при этом AS‑CD9 требовал минимальных затрат времени на выделение. Метод ExADFi продемонстрировал промежуточные результаты между UC и AS‑CD9 по таким характеристикам, как чистота образца мВВ, выход и время выделения. Рамановская спектроскопия дополнила подробности о составе выделенных мВВ и подтвердила различия, наблюдавшиеся в протеомных профилях. Полученные результаты показали, что AS‑CD9 и ExADFi могут служить альтернативой классического UC и могут применяться в зависимости от конечных требований и цели применения очищенных мВВ, включая функциональные исследования и задачи поиска биомаркеров.
Сноски
* Адресат для корреспонденции.
Вклад авторов
Е.Н. Николаев, В.Е. Франкевич, М.А. Кепша, Е.А. Межевитинова, Н.М. Назарова, В.Н. Прилепская, Г.Т. Сухих – концепция и руководство работой; Е.Н. Римская, А.Е. Бугрова, В.С. Чернышев, А.С. Кононихин, А.О. Токарева, Е.А. Евтушенко, А.А. Яковлев, А.М. Юрин – проведение экспериментов; В.С. Чернышев, Н.Л. Стародубцева, Е.Н. Римская, Д.Н. Силачев – обсуждение результатов исследования; В.С. Чернышев, Н.Л. Стародубцева, Е.Н. Римская, А.Е. Бугрова, А.С. Кононихин, Д.Н. Силачев, А.О. Токарева, Е.А. Евтушенко, А.А. Яковлев, А.М. Юрин – написание текста; М.А. Кепша, Е.А. Межевитинова, Е.Н. Николаев, В.Е. Франкевич, Н.М. Назарова, В.Н. Прилепская, Г.Т. Сухих – редактирование текста статьи.
Финансирование
Работа выполнена при финансовой поддержке государственного задания Министерства здравоохранения Российской Федерации (регистрационный номер 12503063216‑9 «Разработка тест-системы для ранней и малоинвазивной диагностики предраковых и онкологических заболеваний аногенитальной области, ассоциированных с папилломавирусной инфекцией у женщин репродуктивного возраста методом жидкостной биопсии»).
Благодарности
Авторы выражают благодарность лаборатории масс-спектрометрии Сколковского института науки и технологий за помощь в проведении протеомного ВЭЖХ-МС-анализа.
Конфликт интересов
Авторы заявляют об отсутствии конфликта интересов.
Соблюдение этических норм
Исследование было одобрено комиссией по этике биомедицинских исследований ФГБУ «НМИЦ АГП им. В.И. Кулакова» Минздрава России (протокол № 1 от 30 января 2025 г.).
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