Proximity biotinylation to define the local environment of the protein kinase A catalytic subunit in adrenal cells.
Proximity biotinylation to define the local environment of the protein kinase A catalytic subunit in adrenal cells.
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DOI：
10.1016/j.xpro.2022.101992
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发表时间：
2023-03-17
期刊：
影响因子：
--
通讯作者：
Scott JD
中科院分区：
文献类型：
--
作者：
Omar MH;Lauer SM;Lau HT;Golkowski M;Ong SE;Scott JD
突变蛋白激酶A催化亚基（PKAc）驱动肾上腺库欣综合征，但其信号相互作用仍不清楚。该方案详细说明了使用活细胞邻近标记来鉴定与人肾上腺细胞中PKAc变体密切相关的亚细胞区室和蛋白质的步骤。我们包括克隆细胞系生成，活生物素标记的近端蛋白质，生物素化蛋白质的分离，和使用生物素连接酶miniTurbo与野生型和突变体PKAc蛋白质组学分析的样品处理的说明。有关本方案使用和执行的完整详细信息，请参见Omar等人。（2022年）。通过检测瞬时和弱相互作用来改善免疫共沉淀描述慢病毒细胞系生成和诱饵蛋白表达优化详细信息标记和分离活细胞中PKA催化亚基近端的蛋白质出版商注：进行任何实验方案都需要遵守当地实验室安全和伦理机构指南。突变蛋白激酶A催化亚基（PKAc）驱动肾上腺库欣综合征，但其信号相互作用仍不清楚。该方案详细说明了使用活细胞邻近标记来鉴定与人肾上腺细胞中PKAc变体密切相关的亚细胞区室和蛋白质的步骤。我们包括克隆细胞系生成，活生物素标记的近端蛋白质，生物素化蛋白质的分离，和使用生物素连接酶miniTurbo与野生型和突变体PKAc蛋白质组学分析的样品处理的说明。
Mutant protein kinase A catalytic subunit (PKAc) drives adrenal Cushing’s syndrome, though its signaling interactions remain unclear. This protocol details steps to use live-cell proximity labeling to identify subcellular compartments and proteins closely associated with variants of PKAc in human adrenal cells. We include instructions for clonal cell line generation, live biotin labeling of proximal proteins, isolation of biotinylated proteins, and sample processing for proteomic analysis using the biotin ligase miniTurbo with wild-type and mutant PKAc. For complete details on the use and execution of this protocol, please refer to Omar et al. (2022). Improves on co-immunoprecipitation by detecting transient and weak interactions Describes lentiviral cell line generation and bait protein expression optimization Details labeling and isolating proteins proximal to PKA catalytic subunit in live cells Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Mutant protein kinase A catalytic subunit (PKAc) drives adrenal Cushing’s syndrome, though its signaling interactions remain unclear. This protocol details steps to use live-cell proximity labeling to identify subcellular compartments and proteins closely associated with variants of PKAc in human adrenal cells. We include instructions for clonal cell line generation, live biotin labeling of proximal proteins, isolation of biotinylated proteins, and sample processing for proteomic analysis using the biotin ligase miniTurbo with wild-type and mutant PKAc.
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影响因子：
5.9
作者：
通讯作者：
--
影响因子：
3.3
作者：
HAROOTUNIAN, AT;ADAMS, SR;TSIEN, RY
通讯作者：
TSIEN, RY
使用 TurboID 在活细胞和生物体中进行高效邻近标记。
影响因子：
46.9
作者：
Branon TC;Bosch JA;Sanchez AD;Udeshi ND;Svinkina T;Carr SA;Feldman JL;Perrimon N;Ting AY
通讯作者：
Ting AY
影响因子：
7.4
作者：
Rappsilber, J;Ishihama, Y;Mann, M
通讯作者：
Mann, M
影响因子：
8.8
作者：
Omar, Mitchell H.;Byrne, Dominic P.;Jones, Kiana N.;Lakey, Tyler M.;Collins, Kerrie B.;Lee, Kyung-Soon;Daly, Leonard A.;Forbush, Katherine A.;Lau, Ho-Tak;Golkowski, Martin;McKnight, G. Stanley;Breault, David T.;Lefrancois-Martinez, Anne-Marie;Martinez, Antoine;Eyers, Claire E.;Baird, Geoffrey S.;Ong, Shao-En;Smith, F. Donelson;Eyers, Patrick A.;Scott, John D.
通讯作者：
Scott, John D.