A novel two-dimensional electrophoresis technique for the identification of intrinsically unstructured proteins.
Intrinsically unstructured proteins (IUPs) lack a well defined three-dimensional structure under physiological conditions. They constitute a significant fraction of various proteomes, but only a handful of them have so far been identified. Here we report the development of a two-dimensional electrophoresis technique for their de novo recognition and characterization. This technique consists of the combination of native and 8 m urea electrophoresis of heat-treated proteins where IUPs are expected to run into the diagonal, whereas globular proteins either precipitate upon heat treatment or unfold and run off the diagonal in the second dimension. This behavior was born out by a collection of 10 known IUPs and four globular proteins. By running Escherichia coli and Saccharomyces cerevisiae extracts, several novel IUPs were also identified by mass spectrometric analysis of spots at or near the diagonal. By comparing this novel method to several other techniques, such as the PONDR(R) predictor, hydrophobicity-net charge plot, CD analysis, and gel filtration chromatography, it was shown to provide dependable global assessment of disorder even in dubious cases. Overall the reproducibility and ease of performance of this technique may promote the proteomic scale recognition and characterization of protein disorder.
Csizmók
Veronika
V
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, H-1518 Budapest, Hungary.
Szollosi
Edit
E
Friedrich
Peter
P
Tompa
Peter
P
D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D029968:Escherichia coli Proteins / Q000032:analysis / Q000737:chemistry; D005656:Fungal Proteins / Q000032:analysis / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D013058:Mass Spectrometry; D011506:Proteins / Q000032:analysis* / Q000737:chemistry* / Q000302:isolation & purification
D029968:Escherichia coli Proteins; D005656:Fungal Proteins; D011506:Proteins