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Mass spectrometric analysis of affinity-purified proteasomes from the human myelogenous leukemia K562 cell lineстатья
Информация о цитировании статьи получена из
Web of Science,
Scopus
Статья опубликована в журнале из списка Web of Science и/или Scopus
Дата последнего поиска статьи во внешних источниках: 18 апреля 2017 г.
- Авторы: Artamonova T.O., Khodorkovskii M.A., Tsimokha A.S.
- Журнал: Russian Journal of Bioorganic Chemistry
- Том: 40
- Номер: 6
- Год издания: 2014
- Издательство: Maik Nauka/Interperiodica Publishing
- Местоположение издательства: Russian Federation
- Первая страница: 666
- Последняя страница: 680
- DOI: 10.1134/s1068162014060041
- Аннотация: Proteasomes carry out regulated proteolysis of most proteins in a cell and thereby play a crucial role in the regulation of various cellular processes. Determination of the subunit composition and posttranslational modifications of proteasomes is one of the important stages in understanding of proteasomes functions in the cell and mechanisms of their regulation. To solve this problem a strategy of affinity purification of proteasomes with the subsequent mass spectrometric analysis has been implemented, using human myelogenous leukemia cells. Proteasomes have been purified from the stable K562 cell line expressing beta 7 (PSMB4) subunit of the 20S proteasome tagged with C-terminal HTBH peptide containing two His(6) fragments, the specific site of cleavage by Tobacco Etch Virus (TEV) protease, and signal sequence for biotinylation in vivo, using method of noncovalent binding through formation of biotin complex with streptavidin with the subsequent elution with TEV protease. All known subunits of the 26S proteasome, as well as PA200 and PA28 gamma regulators have been identified using MALDI FT-ICR mass spectrometry. We have demonstrated that the heat shock proteins, components of the ubiquitin-proteasome system, and some cytoskeleton proteins are associated with proteasomes. A number of new sites of phosphorylation, ubiquitination, and N-terminal modification have been found for 16 proteasome subunits. The presented mass spectrometric analysis will be useful for the further proteomic studies of proteasomes under cellular stress.
- Добавил в систему: Цимоха Анна Сергеевна