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J Biomol Tech. 2010 September; 21(3 Suppl): S4.
PMCID: PMC2918186

Development of a Tagless Strategy for the Identification and Characterization of Native Protein Complexes

Abstract

s1-2

We are developing a novel tagless strategy to purify, identify and characterize water-soluble, stable complexes at high throughput, using the bacterium Desulfovibrio vulgaris as a model. First we fractionate a crude protein extract into a large number of fractions with four optimized orthogonal separation steps, ammonium sulfate precipitation, cation exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography. We then identify polypeptides and monitor their elution patterns by quantitative iTRAQ mass spectrometry (MS). Co-migrating polypeptides are automatically detected as putative protein complexes by mathematical algorithms. We fractionated 10 g of protein extract from a 400 L culture of cells by the four-step purification scheme. So far, we have analyzed 34% of fractionation space (2218 gel filtration chromatography fractions) by MS/MS, from which 833 polypeptides have been identified. As judged by their elution profiles, about 73% of the polypeptides migrate through gel filtration columns as part of multimeric complexes. Fourty heteromeric complexes were inferred on the basis of their homology to E. coli orthologs or a shared operon. Independent analysis by electron microscopy of a number of these DvH protein complexes confirms that their stoichiometries often differ not only from those of E. coli complexes but also in some cases from those of orthologs in more closely related species. This method will be used as part of a larger effort by the Ecosystems and Networks Integrated with Genes and Molecular Assemblies to model stress responses relevant to the detoxification of metal and radionuclide contaminated sites.


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