Purifying and Analysing Cytosolic Protein Complexes

Theresa M Phillips, ProServe Technologies, Toronto, Ontario, Canada

Published online: December 2009

DOI: 10.1002/9780470015902.a0021883

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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 References
    Amero C, Schanda P, Dura M et al. (2009) Fast two-dimensional NMR spectroscopy of high molecular weight protein assemblies. Journal of the American Chemical Society 131(10): 3448–3449. doi:10.1021/ja809880p.
    book Auerbach D and Stagljar I (2005) "Yeast two-hybrid protein–protein interaction networks". In: Waksman G (ed.) Proteomics and Protein–Protein Interactions: Biology, Chemistry, Bioinformatics, and Drug Design, pp. 19–31. London, England: Springer Science and Business Media, Inc.
    Blobel J, Bernado P, Svergun D, Tauler R and Pons M (2009) Low-resolution structures of transient protein-protein complexes using small-angle X-ray scattering. Journal of the American Chemical Society 131(12): 4378–4386. doi:10.1021/ja808490b.
    Bousquet-Dubouch M, Baudelet E, Guerin F et al. (2009) Affinity purification strategy to capture human endogenous proteasome complexes diversity and to identify proteasome interacting proteins. Molecular and Cellular Proteomics 8(5): 1150–1164. Published online 3 February, manuscript #M800193-MCP200.
    other Chittleborough D, Tadjiki S, Ranville J, Shanks F and Beckett R (2004) Soil colloid analysis by flow field-flow fractionation. SuperSoil 2004: Third Australian New Zealand Soils Conference, 5–9 December, University of Sydney, Australia.
    Diviani D, Lattion A, Abuin L, Staub O and Cotecchia S (2003) The adaptor complex 2 directly interacts with the 1b-adrenergic receptor and plays a role in receptor endocytosis. Journal of Biological Chemistry 278(21): 19331–19340.
    Fenn JB, Mann M, Meng CK, Wong SF and Whitehouse CM (1989) Electrospray ionization for mass spectrometry of large biomolecules. Science 246: 64–71.
    Guerrero C, Tagwerker C, Kaiser P and Huang L (2006) An integrated mass spectrometry-based proteomic approach. Molecular & Cellular Proteomics 5.2: 366–378. doi:10.1074/mcp.M500303-MCP200.
    Lopato S, Bazanova N, Morran S et al. (2006) Isolation of plant transcription factors using a modified yeast one-hybrid system. BioMed Central Plant Methods 2: 3. doi: 10.1186/1746-4811-2-3.
    Petrotchenko E, Xiao K, Cable J et al. (2009) BiPS, a photocleavable, isotopically coded, fluorescent cross-linker for structural proteomics. Molecular and Cellular Proteomics 8(2): 273–286.
    Radhashree M and Sadofsky M (2009) A WW-like module in the RAG1 N-terminal domain contributes to previously unidentified protein-protein interactions. Nucleic Acids Research 37(10): 3301–3309. Advance access published 20 March 25, doi:10.1093/nar/gkp192.
    book Rehm H (2006) Protein Biochemistry and Proteomics. New York: Elsevier Academic Press.
    Reschiglian P and Moon MH (2008) Flow field-flow fractionation: a pre-analytical method for proteomics. Journal of Proteomics 71: 265–276.
    Rigaut G, Shevchenko A, Rutz B et al. (1999) A generic protein purification method for protein complex characterization and proteome exploration. Nature Biotechnology 17: 1030–1032.
    Rohrbough J, Galgiani J and Wysocki V (2007) The application of proteomic techniques to fungal protein identification and quantification. Annals of the New York Academy of Sciences 1111: 133–146. doi:10.1196/annals.1406.034.
    book Romier C (2005) "Protein complexes assembly by multi-expression in bacterial and eukaryotic hosts". In: Waksman G (ed.) Proteomics and Protein–Protein Interactions: Biology, Chemistry, Bioinformatics, and Drug Design, pp. 233–249. London, England: Springer Science and Business Media, Inc.
    book Romier C (2008) "Protein complexes assembly by multi-expression in bacterial and eukaryotic hosts". In: Sussman J and Silman I (eds) Structural Proteomics and its Impact on the Life Sciences, pp. 233–250. Singapore: World Scientific Publishing Co. Pte. Ltd.
    Salih E (2004) Phosphoproteomics by mass spectrometry and classical protein chemistry approaches. Mass Spectrometry Reviews 24: 828–846. doi:10.1002/mas.20042.
    book Schuck P (ed.) (2007) Protein Reviews Volume 5: Protein Interactions: Biophysical Approaches for the Study of Complex Reversible Systems. New York: Springer Science and Business Media, Inc.
    book Steven AC and Belnap DM (2005) "Cryo-electron microscopy in the era of structural proteomics". In: Waksman G (ed.) Proteomics and Protein–Protein Interactions: Biology, Chemistry, Bioinformatics, and Drug Design, pp. 269–306. London, England: Springer Science and Business Media, Inc.
    Sutherland B, Toews J and Kast J (2008) Utility of formaldehyde cross-linking and mass spectrometry in the study of protein-protein interactions. Journal of Mass Spectrometry 43(6): 699–715.
    book Szyperski T (2005) "On NMR-based structural proteomics". In: Waksman G (ed.) Proteomics and Protein–Protein Interactions: Biology, Chemistry, Bioinformatics, and Drug Design, pp. 307–329. London, England: Springer Science and Business Media, Inc.
    Volkov A, Worrall J, Holtzmann E and Ubbink M (2006) Solution structure and dynamics of the complex between cytochrome c and cytochrome c peroxidise determined by paramagnetic NMR. Proceedings of the National Academy of Science of the USA 103(50): 18945–18950. doi:10.1073/pnas.0603551103.
    book Waksman G (2005) "Preface". In: Waksman G (ed.) Protein Reviews Volume 3: Proteomics and Protein-Protein Interactions: Biology, Chemistry, Bioinformatics, and Drug Design, pp. V–VI. London, England: Springer Science and Business Media, Inc.
    book Waksman G and Sansom C (2005) "Introduction: Proteomics and protein-protein interactions: biology chemistry, bioinformatics, and drug design". In: Waksman G (ed.) Protein Reviews Volume 3: Proteomics and Protein-Protein Interactions: Biology, Chemistry, Bioinformatics, and Drug Design, pp. 1–18. London, England: Springer Science and Business Media, Inc.
    Wysocki V, Resing K, Zhang Q and Cheng G (2005) Mass spectrometry of peptides and proteins. Methods 35: 211–222. doi:10.1016/j.ymeth.2004.08.013.
    Zheng J, Anderson C, Miller K, Cheatham M and Dallos P (2009) Identifying components of the hair-cell interactome involved in cochlear amplification. BioMed Central Genomics 10: 127. doi:10.1186/1471-2164-10-127.
 Further Reading
    Chiang T and Scholtens D (2009) A general pipeline for quality and statistical assessment of protein interaction data using R and bioconductor. Nature Protocols 4(4): 535–539 doi: 10.1038/nprot.2009.26.
    book Tang X and Bruce J (2009) "Chemical cross-linking for protein-protein interaction studies". In: Lipton M and Pasa-Tolic L (eds) Mass Spectrometry of Proteins and Peptides, vol. 492, pp. 283–293. (Springer Science and Business Media, Inc.). doi:10.1007/978-1-59745-493-3_17. London, England: Humana Press.
    book Wang Yi, Yazdi P and Qin J (2005) "The use of mass spectrometry in studying protein-protein interaction". In: Waksman G (ed.) Proteomics and Protein–Protein Interactions: Biology, Chemistry, Bioinformatics, and Drug Design, v–vi, pp. 33–48. London, England: Springer Science and Business Media, Inc.
    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.

Related Sub-Topics:

Basic Cell Biology, Protein, RNA and DNA | Techniques in Cell Biology | Protein Structure | Biochemical and Biophysical Techniques | Proteins: Structure, Function, Metabolism
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Article Title: Purifying and Analysing Cytosolic Protein Complexes
 
How to Cite close
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]