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Purifying and Analysing Cytosolic Protein Complexes

  1. Theresa M Phillips

Published Online: 15 DEC 2009

DOI: 10.1002/9780470015902.a0021883

eLS

eLS

How to Cite

Phillips, T. M. 2009. Purifying and Analysing Cytosolic Protein Complexes. eLS. .

Author Information

  1. ProServe Technologies, Toronto, Ontario, Canada

Publication History

  1. Published Online: 15 DEC 2009

Abstract

The study of cytosolic protein complexes, called systems or functional proteomics, is complicated by the challenges associated with purifying unadulterated, functional complexes, and with developing analytical methods for studying protein structure that can accommodate high molecular masses, or weak and transient protein–protein interactions. Development of new analytical techniques has progressed considerably in recent years, with the adaptation of mass spectrometry (MS) and nuclear magnetic resonance (NMR) for large complexes, in particular, adaptations using the popular electrospray ionization (ESI) coupled with a ToF (time-of-flight) analyser. Purification methods that have been adapted for the study of protein–protein interactions include electromobility shift assays, formaldehyde crosslinking, tandem affinity purification (TAP) and coexpression purification techniques, whereas flow field-flow fractionation (F4) has been cited as having many significant advantages over all of these. New approaches to study posttranslational modifications, and adaptation of detection methods for quantitative analyses, are also being used to advance the study of protein complexes.

Key Concepts:

  • Functional proteomics aims to identify how proteins interact with each other and other macromolecules.

  • Purification of protein complexes is more difficult than that of individual proteins.

  • Techniques for protein complex purification include methods such as formaldehyde crosslinking and tandem affinity purification.

  • The introduction of electrospray ionization made it possible to create aerosols of proteins that could be analysed by mass spectrometry.

  • Methods for isolation of a complex for study include in vitro reconstitution, coexpression and endogenous complex purification.

  • Protein complexes with transient interactions can be studied using nuclear magnetic resonance.

  • Formaldehyde crosslinking stabilizes protein interactions during purification.

  • The yeast two-hybrid technique is usually used to isolate transcriptional proteins from the nucleus.

  • A modification of the Western blot, called a gel overlay assay, is used to detect proteins with binding affinity for one another.

Keywords:

  • cytosolic proteins;
  • protein complexes;
  • functional proteomics;
  • formaldehyde cross-linking;
  • electrospray ionization