Characterization of Protein Complexes Using Chemical Cross-Linking Coupled Electrospray Mass Spectrometry
Identification and characterization of large protein complexes is a mainstay of biochemical toolboxes. Utilization of cross-linking chemicals can facilitate the capture and identification of transient or weak interactions of a transient nature (Huang and Kim, PloS One 8:e61430, 2013; Gao et al., J V...
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| Veröffentlicht in: | Methods in molecular biology (Clifton, N.J.) N.J.), 2018, Vol.1788, p.43-61 |
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| container_title | Methods in molecular biology (Clifton, N.J.) |
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| creator | Cummins, Timothy D. Sapkota, Gopal P. |
| description | Identification and characterization of large protein complexes is a mainstay of biochemical toolboxes. Utilization of cross-linking chemicals can facilitate the capture and identification of transient or weak interactions of a transient nature (Huang and Kim, PloS One 8:e61430, 2013; Gao et al., J Vis Exp doi: 10.3791/51387, 2014). Here we describe a detailed methodology for a cell culture-based proteomic approach. We describe the generation of cells stably expressing green fluorescent protein (GFP)-tagged proteins under the tetracycline-inducible promoter and subsequent proteomic analysis of GFP-interacting proteins. We include a list of proteins that were identified as interactors of GFP. |
| doi_str_mv | 10.1007/7651_2017_85 |
| format | Article |
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| identifier | ISSN: 1064-3745 |
| ispartof | Methods in molecular biology (Clifton, N.J.), 2018, Vol.1788, p.43-61 |
| issn | 1064-3745 1940-6029 |
| language | eng |
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| source | MEDLINE; Springer Books |
| subjects | Amino Acid Sequence Animals Cell Line Cross-Linking Reagents - chemistry Gene Expression GFP affinity purification Green Fluorescent Proteins - analysis Green Fluorescent Proteins - genetics Green Fluorescent Proteins - metabolism Humans Inducible expression Interactome Mass spectrometry Protein complex Protein Interaction Mapping - methods Protein Interaction Maps Proteins - analysis Proteins - genetics Proteins - metabolism Protein–protein interaction Proteomics - methods Spectrometry, Mass, Electrospray Ionization - methods Transfection - methods |
| title | Characterization of Protein Complexes Using Chemical Cross-Linking Coupled Electrospray Mass Spectrometry |
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