Large-Scale Identification of Intrinsically Disordered Proteins

Vladimir N. Uversky, Marc S. Cortese, Peter Tompa, Veronika Csizmok, A. Keith Dunker

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Scopus citations


A method to enrich cell extracts in totally unfolded proteins was investigated. A literature search revealed that 14 of 29 proteins isolated by their failure to precipitate during perchloric acid (PCA) or trichloroacetic acid (TCA) treatment were also shown experimentally to be totally disordered. A near 100,000-fold reduction in yield was observed after 5% or 9% PCA treatment of total soluble Escherichia coli protein. Despite this huge reduction, 158 and 142 spots were observed from the 5% and the 9% treated samples, respectively, on silver-stained two-dimensional (2D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels loaded with 10 μ g of protein. Treatment with 1% PCA was less selective with more visible spots and a greater than threefold higher yield. A substantial yield of unprecipitated protein was obtained after 3% TCA treatment, suggesting that the common use of TCA precipitation prior to 2D gel analysis may result in loss of unstructured protein due to their failure to precipitate. Our preliminary analysis suggests that treating total protein extracts with 3-5% PCA and determining the identities of soluble proteins could be the starting point for uncovering unfoldomes (the complement of unstructured proteins in a given proteome). The 100,000-fold reduction in yield and concomitant reduction in number of proteins achieved by 5% PCA treatment produced a fraction suitable for analysis in its entirety using standard proteomic techniques. In this way, large numbers of totally unstructured proteins could be identified with minimal effort. A method of different logic has been recently presented that not only allows the identification of intrinsically disordered proteins (IDPs) but at the same time also provides information about the structural state of an uncharacterized protein. The technique combines a native gel electrophoresis of heat-treated proteins with a second, denaturing gel containing 8 M urea. The heat treatment precipitates most globular proteins and leaves IDPs in solution, providing the first evidence for the lack of a well-defined structure. The subsequent 8 M urea electrophoresis is rationalized by the usual structural indifference of IDPs to chemical denaturation. As urea is uncharged and IDPs are just as "denatured" in 8 M urea as under native conditions, they run the same distance in the second dimension as in the first and end up along the diagonal. However, the heat-stable globular proteins unfold in 8 M urea, which make them run slower, and accumulate above the diagonal. This deviation in their running ensures the separation of IDPs from the globular proteins and enables the MS-based identification of IDPs.

Original languageEnglish (US)
Title of host publicationInstrumental Analysis of Intrinsically Disordered Proteins
Subtitle of host publicationAssessing Structure and Conformation
PublisherJohn Wiley and Sons
Number of pages23
ISBN (Print)9780470343418
StatePublished - Sep 9 2010


  • 2D electrophoresis technique-for de novo recognition and IDPS characterization
  • Cell extract enrichment-in extended IDPS by heat treatment
  • Intrinsically disordered proteins-large-scale identification

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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  • Cite this

    Uversky, V. N., Cortese, M. S., Tompa, P., Csizmok, V., & Dunker, A. K. (2010). Large-Scale Identification of Intrinsically Disordered Proteins. In Instrumental Analysis of Intrinsically Disordered Proteins: Assessing Structure and Conformation (pp. 671-693). John Wiley and Sons.