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.
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August 2010
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Conference Article|
July 26 2010
Interaction proteomics: characterization of protein complexes using tandem affinity purification–mass spectrometry
Pamela Völkel;
Pamela Völkel
1Chromatinomics, Interdisciplinary Research Institute, Université de Lille 1 – Sciences et Technologies/CNRS USR 3078, Parc Scientifique de la Haute Borne, 50 Avenue Halley, F-59658 Villeneuve d'Ascq, France
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Perrine Le Faou;
Perrine Le Faou
1Chromatinomics, Interdisciplinary Research Institute, Université de Lille 1 – Sciences et Technologies/CNRS USR 3078, Parc Scientifique de la Haute Borne, 50 Avenue Halley, F-59658 Villeneuve d'Ascq, France
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Pierre-Olivier Angrand
Pierre-Olivier Angrand
1
1Chromatinomics, Interdisciplinary Research Institute, Université de Lille 1 – Sciences et Technologies/CNRS USR 3078, Parc Scientifique de la Haute Borne, 50 Avenue Halley, F-59658 Villeneuve d'Ascq, France
1To whom correspondence should be addressed (email pierre-olivier.angrand@iri.univ-lille1.fr).
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Publisher: Portland Press Ltd
Received:
December 16 2009
Online ISSN: 1470-8752
Print ISSN: 0300-5127
© The Authors Journal compilation © 2010 Biochemical Society
2010
Biochem Soc Trans (2010) 38 (4): 883–887.
Article history
Received:
December 16 2009
Citation
Pamela Völkel, Perrine Le Faou, Pierre-Olivier Angrand; Interaction proteomics: characterization of protein complexes using tandem affinity purification–mass spectrometry. Biochem Soc Trans 1 August 2010; 38 (4): 883–887. doi: https://doi.org/10.1042/BST0380883
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