Uncovering the unfoldome: enriching cell extracts for unstructured proteins by acid treatment.
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 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 microg 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.
Cortese
Marc S
MS
Department of Biochemistry and Molecular Biology and Center for Computational Biology and Bioinformatics, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Baird
Jason P
JP
Uversky
Vladimir N
VN
Dunker
A Keith
AK
D001426:Bacterial Proteins / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D010449:Peptide Mapping; D010472:Perchlorates / Q000737:chemistry; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D016622:Silver Staining; D014238:Trichloroacetic Acid / Q000737:chemistry
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D010472:Perchlorates; D020543:Proteome; D014238:Trichloroacetic Acid