Purifying and Analysing Cytosolic Protein Complexes

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

Figure 1.

Yeast two‐hybrid system. (a) The system depicted using the Gal4 protein. (b) Building gene fusions for this system. Reproduced by permission of Prof. David B. Collinge, University of Copenhagen, Denmark.

Figure 2.

Gel overlay assay. (a) The assay depicted using barley–powdery mildew interactions. (b) The 14‐3‐3 proteins have been shown to accumulate in barley–powdery mildew interactions. They are known to bind other proteins in other plants (and animals). Do they bind something in barley? Panel A is stained for protein. Panels C and E show Western blots from total leaf and leaf epidermis (where the fungus lives) showing the presence of a protein (H+ATPase) in inoculated not uninoculated epidermis. Panels B and D are identical membranes to C and E which have been probed with DIG‐14‐3‐3 protein as described in the earlier depiction. Thus it seems very likely that 14‐3‐3 is binding the H+ATPase in the inoculated epidermis, confirming yeast two‐hybrid results. Bgh: Blumeria graminis f sp hordei (Barley–powdery mildew infection). Reproduced by permission of Prof. David B. Collinge, University of Copenhagen, Denmark.

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Further Reading

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Yang W, Steen H and Freeman M (2008) Proteomic approaches to the analysis of multiprotein signaling complexes. Proteomics 8: 832–851. doi:10.1002/pmic.200700650.

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Phillips, Theresa M(Dec 2009) Purifying and Analysing Cytosolic Protein Complexes. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0021883]