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Experimental Approaches to Protein–Protein Interactions

Proteomic Complex Detection using Sedimentation (ProCoDeS): screening for proteins in stable complexes and their candidate interaction partners

Marcelo P. Segura, Kathryn S. Lilley, Paul Dupree
Biochemical Society Transactions Jul 26, 2010, 38 (4) 923-927; DOI: 10.1042/BST0380923
Marcelo P. Segura
Department of Biochemistry, University Of Cambridge, Tennis Court Road, Cambridge CB2 1QW, U.K.
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Kathryn S. Lilley
Department of Biochemistry, University Of Cambridge, Tennis Court Road, Cambridge CB2 1QW, U.K.
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Paul Dupree
Department of Biochemistry, University Of Cambridge, Tennis Court Road, Cambridge CB2 1QW, U.K.
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  • For correspondence: p.dupree@bioc.cam.ac.uk
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Abstract

Over the last few years, our view of cellular organization has changed from one in which enzymes and proteins usually act independently to the situation at present where we commonly accept that many, if not all, enzymes act in close association with others. Co-precipitation using an antibody against a test protein is the standard assay for the identification of members of protein complexes [Musso, Zhang and Emili (2007) Chem. Rev. 107, 3585–3600]. The introduction of TAP (tandem affinity purification) tagging enhanced original approaches in order to analyse protein complexes on a larger scale with reduced false discoveries of interacting partners due to more efficient purification of complexes. However, this technique has some limitations as a high-throughput tool for systems biology: the requirement for genetic manipulation to express the tagged protein excludes studies of non-transformable organisms and intact tissue. In those cases where TAP is applicable, a considerable amount of work is required to generate the baits and to optimize experimental conditions. A technique developed in our laboratories, ProCoDeS (Proteomic Complex Detection using Sedimentation), focuses on the detection of endogenous complexes. Protein samples are separated by centrifugation and then different fractions from the resulting gradient are analysed using quantitative MS. The identification of possible protein partners is based on statistical analysis of the co-fractionation of proteins, without any need for purification of individual complexes. The prospects of ProCoDeS and similar techniques based on quantitative MS for measurement of protein complex composition are reviewed in the present article.

  • blue native gel electrophoresis
  • high-throughput screen
  • Pearson correlation
  • protein–protein interaction
  • Proteomic Complex Detection using Sedimentation (ProCoDeS)
  • tandem affinity purification (TAP)

Footnotes

  • Experimental Approaches to Protein–Protein Interactions: A Biochemical Society Focused Meeting held at University of Sheffield, Sheffield, U.K., 11–12 January 2010. Organized and Edited by Michael Sutcliffe (Manchester, U.K.) and Mike Williamson (Sheffield, U.K.).

Abbreviations: BN, blue native gel electrophoresis; ICAT, isotope-coded affinity tag; iTRAQ, isobaric tag for relative and absolute quantification; MS/MS, tandem MS; TAP, tandem affinity purification

  • © The Authors Journal compilation © 2010 Biochemical Society
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August 2010

Volume: 38 Issue: 4

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Proteomic Complex Detection using Sedimentation (ProCoDeS): screening for proteins in stable complexes and their candidate interaction partners
Marcelo P. Segura, Kathryn S. Lilley, Paul Dupree
Biochemical Society Transactions Aug 2010, 38 (4) 923-927; DOI: 10.1042/BST0380923
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Proteomic Complex Detection using Sedimentation (ProCoDeS): screening for proteins in stable complexes and their candidate interaction partners
Marcelo P. Segura, Kathryn S. Lilley, Paul Dupree
Biochemical Society Transactions Aug 2010, 38 (4) 923-927; DOI: 10.1042/BST0380923

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Keywords

blue native gel electrophoresis
high-throughput screen
Pearson correlation
protein–protein interaction
Proteomic Complex Detection using Sedimentation (ProCoDeS)
tandem affinity purification (TAP)

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