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Affinity chromatography and capillary electrophoresis for analysis of the yeast ribosomal proteins

  • Goyder, Miriam S. (The Single Cell Proteomics Group, Institute of Chemical Biology, Department of Chemistry, Imperial College London) ;
  • Willison, Keith R. (Institute of Cancer Research, Chester Beatty Laboratories, Section of Cell and Molecular Biology) ;
  • Klug, David R. (The Single Cell Proteomics Group, Institute of Chemical Biology, Department of Chemistry, Imperial College London) ;
  • DeMello, Andrew J. (Institute of Chemical and Biochemical Engineering, ETH Zurich) ;
  • Ces, Oscar (The Single Cell Proteomics Group, Institute of Chemical Biology, Department of Chemistry, Imperial College London)
  • Received : 2011.12.13
  • Accepted : 2011.12.21
  • Published : 2012.04.30

Abstract

We present a top down separation platform for yeast ribosomal proteins using affinity chromatography and capillary electrophoresis which is designed to allow deposition of proteins onto a substrate. FLAG tagged ribosomes were affinity purified, and rRNA acid precipitation was performed on the ribosomes followed by capillary electrophoresis to separate the ribosomal proteins. Over 26 peaks were detected with excellent reproducibility (<0.5% RSD migration time). This is the first reported separation of eukaryotic ribosomal proteins using capillary electrophoresis. The two stages in this workflow, affinity chromatography and capillary electrophoresis, share the advantages that they are fast, flexible and have small sample requirements in comparison to more commonly used techniques. This method is a remarkably quick route from cell to separation that has the potential to be coupled to high throughput readout platforms for studies of the ribosomal proteome.

Keywords

Affinity chromatography;Capillary electrophoresis;EVV 2DIR;Proteomics;Ribosomal proteins

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