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Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry
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Biologia
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JoVE Journal Biologia
Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

DOI:
10.3791/4057-v

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14:58 min

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November 12, 2012

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, , , ,
1Banting and Best Department of Medical Research, Donnelly Centre,University of Toronto, 2Deparment of Biochemistry, Research and Innovation Centre,University of Regina, 3Department of Medical Genetics and Microbiology,University of Toronto

Capítulos

  • 00:05Título
  • 01:47Construction of Gene-specific SPA-tagging in E. Coli DY330 Strain
  • 03:54Culturing and Sonication
  • 05:26Affinity Purification
  • 07:53Proteolysis and Sample Preparation for Mass Spectrometry
  • 09:38Protein Identification by LTQ Orbitap Velos Mass Spectrometer
  • 11:02Resultados
  • 14:27Conclusion

Summary

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Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Tags

Protein ComplexesEscherichia ColiSequential Peptide Affinity PurificationTandem Mass SpectrometryProtein-protein InteractionsSubunit CompositionGene FunctionBiological SystemsAffinity PurificationMass SpectrometryChromosomally-tagged ProteinsCarboxy-terminal Sequential Peptide Affinity (SPA) Dual Tagging SystemGram-negative Escherichia ColiGenetically-tractable Host Laboratory StrainsGenome-wide InvestigationsProkaryotesTandem Affinity Purification MethodCalmodulin Binding Peptide (CBP)Tobacco Etch Virus

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