Journal
/
Biology
/
Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry
JoVE Journal
Biology
A subscription to JoVE is required to view this content.  Sign in or start your free trial.
JoVE Journal Biology
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

•

14:58 min

•

November 12, 2012

•

, , , ,
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

Chapters

  • 00:05Title
  • 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
  • 11:02Results
  • 14:27Conclusion

Summary

Automatic Translation

Automatic Translation

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

Article

Embed

ADD TO PLAYLIST

Usage Statistics

Related Videos

check_url/4057?article_type=v

Identification of protein complexes with quantitative proteomics in S. cerevisiae
check_url/4057?article_type=v

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid
check_url/4057?article_type=v

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamAΔ41
check_url/4057?article_type=v

Analyzing Large Protein Complexes by Structural Mass Spectrometry
check_url/4057?article_type=v

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry
check_url/4057?article_type=v

Identification of Protein Interacting Partners Using Tandem Affinity Purification
check_url/4057?article_type=v

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation
check_url/4057?article_type=v

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)
check_url/4057?article_type=v

Identification of Post-translational Modifications of Plant Protein Complexes
check_url/4057?article_type=v

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Read Article