Interaction proteomics: characterization of protein complexes using tandem affinity purification–mass spectrometry
| Autor: | Pamela Völkel, Perrine Le Faou, Pierre-Olivier Angrand |
|---|---|
| Rok vydání: | 2010 |
| Předmět: |
Proteomics
Tandem affinity purification Chromatography biology Tobacco etch virus biology.organism_classification Biochemistry Chromatography Affinity Mice FLAG-tag Tandem Mass Spectrometry Multiprotein Complexes Protein Interaction Mapping Protein purification Proteome biology.protein Animals Humans Protein A Protein Binding Myc-tag |
| Zdroj: | Biochemical Society Transactions. 38:883-887 |
| ISSN: | 1470-8752 0300-5127 |
| DOI: | 10.1042/bst0380883 |
| Popis: | Most cellular processes are carried out by a multitude of proteins that assemble into multimeric complexes. Thus a precise understanding of the biological pathways that control cellular events relies on the identification and on the biochemical characterization of the proteins involved in such multimeric assemblies. Advances in MS have made possible the identification of multisubunit protein complexes isolated from cell lysates with high sensitivity and accuracy, whereas the TAP (tandem affinity purification) methodology efficiently isolates native protein complexes from cells for proteomics analysis. TAP is a generic method based on the sequential utilization of two affinity tags to purify protein assemblies. During the first purification step, the Protein A moiety of the TAP tag is bound to IgG beads, and protein components associated with the TAP-tagged protein are retrieved by TEV (tobacco etch virus) protease cleavage. This enzyme is a sequence-specific protease cleaving a seven-amino-acid recognition site located between the first and second tags. In the second affinity step, the protein complex is immobilized to calmodulin-coated beads via the CBP (calmodulin-binding peptide) of the TAP tag. The CBP–calmodulin interaction is calcium-dependent and calcium-chelating agents are used in the second elution step to release the final protein complex preparation used for protein identification by MS. The TAP–MS approach has proven to efficiently permit the characterization of protein complexes from bacteria, yeast and mammalian cells, as well as from multicellular organisms such as Caenorhabditis elegans, Drosophila and mice. |
| Databáze: | OpenAIRE |
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