Uncovering the unfoldome: Enriching cell extracts for unstructured proteins by acid treatment

Marc S. Cortese, Jason P. Baird, Vladimir N. Uversky, A. Dunker

Research output: Contribution to journalArticle

50 Citations (Scopus)

Abstract

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) ortrichloroacetic acid (TCA) treatment where 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 E. coli protein. Despite this huge reduction, 158 and 142 spots were observed from the 5% and the 9% treated samples, respectively, on silver-stained 2-D SDS-PAGE gels loaded with 10 μg of protein. Treatment with 1% PCA was less selective with more visible spots and a greater than 3-fold higher yield. A substantial yield of unprecipitated protein was obtained after 3% TCA treatment, suggesting that the common use of TCA precipitation prior to 2-D 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.

Original languageEnglish
Pages (from-to)1610-1618
Number of pages9
JournalJournal of Proteome Research
Volume4
Issue number5
DOIs
StatePublished - Sep 2005

Fingerprint

Intrinsically Disordered Proteins
Cell Extracts
Acids
Proteins
Gels
Precipitates
Protein Unfolding
Escherichia coli Proteins
Proteome
Silver
Proteomics
Polyacrylamide Gel Electrophoresis
Complement System Proteins
Perchloric Acid

Keywords

  • Acid-soluble protein
  • Disordered proteins
  • Disordered regions
  • Intrinsic disorder

ASJC Scopus subject areas

  • Genetics
  • Biotechnology
  • Biochemistry

Cite this

Uncovering the unfoldome : Enriching cell extracts for unstructured proteins by acid treatment. / Cortese, Marc S.; Baird, Jason P.; Uversky, Vladimir N.; Dunker, A.

In: Journal of Proteome Research, Vol. 4, No. 5, 09.2005, p. 1610-1618.

Research output: Contribution to journalArticle

Cortese, Marc S. ; Baird, Jason P. ; Uversky, Vladimir N. ; Dunker, A. / Uncovering the unfoldome : Enriching cell extracts for unstructured proteins by acid treatment. In: Journal of Proteome Research. 2005 ; Vol. 4, No. 5. pp. 1610-1618.
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