期刊
JOURNAL OF PROTEOME RESEARCH
卷 11, 期 2, 页码 564-575出版社
AMER CHEMICAL SOC
DOI: 10.1021/pr200459c
关键词
quantitative proteomics; protein interactions; RNA polymerases; mass spectrometry; MudPIT; cellular imaging; normalized spectral abundance factor
资金
- Stowers Institute for Medical Research
- Promega Corporation
Efficient determination of protein interactions and cellular localization remains a challenge in higher order eukaryotes and creates a need for robust technologies for functional proteomics studies. To address this, the Halo Tag technology was developed for highly efficient and rapid isolation of intracellular complexes and correlative in vivo cellular imaging. Here we demonstrate the strength of this technology by simultaneous capture of human eukaryotic RNA polymerases (RNAP) 1, II, and III using a shared subunit, POLR2H, fused to the Halo Tag. Affinity purifications showed successful isolation, as determined using quantitative proteomics, of all RNAP core subunits, even at expression levels near endogenous. Transient known RNAP II interacting partners were identified as well as three previously uncharacterized interactors. These interactions were validated and further functionally characterized using cellular imaging. The multiple capabilities of the Halo Tag technology demonstrate the ability to efficiently isolate highly challenging multiprotein complexes, discover new interactions, and characterize cellular localization.
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