Summary

Определение белковых комплексов в<em> Кишечной палочки</em> С помощью последовательного аффинной очистки пептидов в сочетании с тандемной масс-спектрометрии

Published: November 12, 2012
doi:

Summary

Affinity очистки меченых белков в сочетании с масс-спектрометрии (ППМ) является мощным методом для систематического отображения сети взаимодействия белка и для исследования механистической основе биологических процессов. Здесь мы описываем оптимизированный последовательных пептидов близости (SPA) ППМ процедуры, разработанные для бактерии<em> Кишечной палочки</em>, Который может быть использован для выделения и характеризуют стабильное мульти-белковых комплексов почти до однородности, даже начиная с низким числом копий на клетку.

Abstract

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).

Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Protocol

1. Строительство ген-специфического SPA-пометки в E. Штамм кишечной DY330 Плазмиды pJL148 охватывает SPA-теги последовательности ДНК и канамицин антибиотиков кассеты маркер устойчивости (Кан R) используется в качестве матрицы в полимеразной цепной реакции (ПЦР) 7. 45 ну?…

Representative Results

Once tagged bait proteins, which are expressed at endogenous levels are affinity-purified from logarithmic phase cultures the samples were run on a silver-stain gel to visualize the individual polypeptide components of the isolated stable complexes. We also subjected a second portion of the affinity-purified protein samples to gel-free tandem mass spectrometry (LCMS) to identify the corresponding polypeptide sequences. The effectiveness of this APMS procedure is shown with a representative SDS-PAGE analysis of the compon…

Discussion

Ключевым аспектом SPA-подхода ППМ описано здесь является то, что пометки осуществляется в пределах естественной хромосомной контексте, обеспечивая тем самым нормальное регулирование генов сохраняется (то есть. Родной промоутер приманки сохранились, следовательно, уровни экспрес?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Эта работа была поддержана за счет средств от Канадского фонда инноваций, Геном Канада, Онтарио геномики Института, Онтарио Министерство инноваций и Канадский институт исследований в области здравоохранения гранта JG и AE Красный выражения E. Штамм DY330 был своего рода подарок от Donald L. суд (Национальный институт рака, Фредерик, Мэриленд).

Materials

Materials Vendor and Catalog Numbers  
      I. Antibiotics
Kanamycin Bioshop #KAN201  
Ampicillin Bioshop #AMP201  
      2. Terrific-Broth medium
Bio-Tryptone Bioshop #TRP 402  
Yeast extract Bioshop #YEX 555  
Glycerol Bioshop #GLY 002  
K2HPO4 Bioshop #PPM 302  
KH2PO4 Bioshop #PPM 303  
      3. Bacterial Strain and Plasmid
DY330   Yu et al. (2000)10  
pJL148   Zeghouf et al. (2004)7  
      4. PCR and Electrophoresis Reagents
Taq DNA polymerase Fermentas # EP0281  
10 X PCR buffer Fermentas # EP0281  
10 mM dNTPs Fermentas # EP0281  
25 mM MgCl2 Fermentas # EP0281  
Agarose Bioshop # AGA002  
Loading dye NEB #B7021S  
Ethidium bromide Bioshop # ETB444  
10X TBE buffer Thermo Scientific #28355  
Tris Base Bioshop #TRS001  
Boric acid Bioshop #BOR001  
0.5 M EDTA (pH 8.0) Sigma # E6768  
DNA ladder NEB #N3232L  
      5. Plasmid isolation and Clean-up Kits
Plasmid Midi kit Qiagen #12143  
QIAquick PCR purification kit Qiagen #28104  
      6. PCR and Transformation Equipments
Thermal cycler BioRad iCycler  
Agarose gel electrophoresis BioRad    
Electroporator Bio-Rad GenePulser II  
0.2 cm electroporation cuvette Bio-Rad    
42 °C water bath shaker   Innova 3100  
Beckman Coulter TJ-25 centrifuge Beckman Coulter TS-5.1-500  
32 °C Shaker New Brunswick Scientific, USA    
32 °C large Shaker New Brunswick Scientific, USA    
32 °C plate incubator Fisher Scientific    
      7. Electrophoresis and Western blotting
Acrylamide monomer, N,N’- methylenebis-acrylamide Bio-Rad #161-0125  
Ammonium persulfate Bioshop # AMP001  
n-butanol Sigma # B7906  
TEMED Bioshop #TEM001  
Whatman No. 1 filter paper Fischer Scientific #09-806A  
Mini protean 3 cell Bio-Rad #165-3301  
iBlot gel transfer device Invitrogen #IB1001  
Nitrocellulose membranes Bio-Rad #162-0115  
Monoclonal Anti-Flag M2 antibody Sigma #F3165  
Horseradish peroxidase Amersham #NA931V  
Pre-stained protein molecular weight standards Bio-Rad #161-0363  
Chemiluminescence reagent PIERCE #1856136  
Autoradiography film Clonex Corp #CLEC810  
Quick Draw blotting paper Sigma #P7796  
C2 platform rocking shaker New Brunswick Scientific, USA    
      8. Sonication Equipment and Reagents
Sonicator Branson Ultrasonic #23395  
NaCl Bioshop #SOD001  
Protease inhibitors Roche #800-363-5887  
0.5 mM TCEP-HCl Thermo Scientific #20490  
      9. Affinity Purification Reagents and Equipment
0.8 x 4 cm Bio-Rad polypropylene column Bio-Rad #732-6008  
Benzonase nuclease Novagen #70746  
Anti-FLAG M2 agarose beads Sigma #A2220  
Calmodulin-sepharose beads GE Healthcare #17-0529-01  
TEV protease Invitrogen #12575-015  
Triton X-100 Sigma #T9284  
CaCl2 Sigma #C2661  
EGTA Sigma #E3889  
LabQuake Shaker Thermolyne #59558  
      10. Silver Staining Reagents
Methanol Bioshop #MET302  
Acetic acid Bioshop t#ACE222  
Sodium-thiosulfate Sigma #S-7143  
Silver nitrate Fischer Scientific #S181-100  
Formaldehyde Bioshop #FOR201  
Sodium carbonate Bioshop #SOC512  
      11. Reagents and Equipment for Protein Identification
Trypsin Gold, Mass Spectrometry Grade Promega # V5280  
50 mM NH4HCO3 Bioshop #AMC555  
1 mM CaCl2 Bioshop #CCL302  
Acetonitrile Sigma #A998-4  
Formic acid Sigma #F0507  
HPLC grade water Sigma #95304  
Iodoacetamide Sigma #16125  
Millipore Zip-Tip Millipore # ZTC18M960  
~10 cm of 3 μm Luna-C18 resin Phenomenex    
Proxeon nano HPLC pump Thermo Fisher Scientific    
LTQ Orbitrap Velos mass spectrometer   Thermo Fisher Scientific  
      12. Labware
4 liter conical flasks VWR #89000-372  
50 ml polypropylene falcon tubes Any Vendor    
1.5 ml micro-centrifuge tubes Any Vendor    
250 ml conical flaks VWR #29140-045  
15 ml sterile culture tubes Thermo Scientific #366052  
Cryogenic vials VWR #479-3221  
-80 °C freezer Fisher Scientific #13-990-14  
Speed vacuum system Thermo Scientific    
     

Buffers and Solutions

1. 1 liter Terrific Broth (TB) media

11 g Bio-Tryptone
22 g Yeast Extract
2% Glycerol
50 ml potassium salt stock solution

2. Potassium Salt Stock Solution

1.5 M K2HPO4
0.35 M KH2PO4

3. Sonication Buffer

20 mM Tris-HCl (pH 7.9)
150 mM NaCl
0.2 mM EDTA
10% Glycerol
Before use add protease inhibitor (PI) and 0.1-0.5 mM TCEP

4. AFC buffer

30 mM Tris-HCl (pH 7.9)
150 mM NaCl
0.1% detergent
Before use add PI and 0.1-0.5 mM TCEP

5. TEV cleavage buffer

30 mM Tris-HCl (pH 7.9)
150 mM NaCl
0.2 mM EDTA
0.1% detergent
Before use add PI and 0.1-0.5 mM TCEP

6. Calmodulin binding buffer

30 mM Tris-HCl (pH 7.9)
150 mM NaCl
2 mM CaCl2
0.1% detergent
Before use add PI and 0.1-0.5 mM TCEP

7. Calmodulin wash buffer

30 mM Tris-HCl pH 7.9
150 mM NaCl
2 mM CaCl2
0.1-0.5 mM TCEP

8. Calmodulin elution buffer

30 mM Tris-HCl (pH 7.9)
100 mM NaCl
10 mM EGTA
0.1-0.5 mM TCEP

9. Developing solution (1L)

37% Formaldehyde
30 g sodium carbonate
1000 ml distilled water

10. Digestion buffer

50 mM NH4HCO3
1 mM CaCl2

11. Wetting and Equilibration solution

70% acetonitrile (ACN) in 0.1% formic acid

12. Washing solution

100% H2O in 0.1% formic acid

References

  1. Butland, G., et al. Interaction network containing conserved and essential protein complexes in Escherichia coli. Nature. 433, 531-537 (2005).
  2. Hu, P., Janga, S. C., Babu, M., Diaz-Mejia, J. J., Butland, G. Global functional atlas of Escherichia coli encompassing previously uncharacterized proteins. PLoS Biol. 7, e1000096 (2009).
  3. Krogan, N. J., et al. Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature. 440, 637-643 (2006).
  4. Mak, A. B., et al. A lentiviral functional proteomics approach identifies chromatin remodeling complexes important for the induction of pluripotency. Mol. Cell Proteomics. 9, 811-823 (2010).
  5. Polanowska, J., et al. Tandem immunoaffinity purification of protein complexes from Caenorhabditis elegans. Biotechniques. 36, 778-782 (2004).
  6. Veraksa, A., Bauer, A., Artavanis-Tsakonas, S. Analyzing protein complexes in Drosophila with tandem affinity purification-mass spectrometry. Dev. Dyn. 232, 827-834 (2005).
  7. Zeghouf, M., et al. Sequential Peptide Affinity (SPA) system for the identification of mammalian and bacterial protein complexes. J. Proteome Res. 3, 463-468 (2004).
  8. Puig, O., et al. The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods. 24, 218-229 (2001).
  9. Rigaut, G., et al. A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 17, 1030-1032 (1999).
  10. Yu, D., et al. An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 97, 5978-5983 (2000).
  11. Kislinger, T., et al. PRISM, a generic large scale proteomic investigation strategy for mammals. Mol. Cell Proteomics. 2, 96-106 (2003).
  12. Vlasblom, J., et al. GenePro: a Cytoscape plug-in for advanced visualization and analysis of interaction networks. Bioinformatics. 22, 2178-219 (2006).
  13. de Berardinis, V., et al. A complete collection of single-gene deletion mutants of Acinetobacter baylyi ADP1. Mol. Syst. Biol. 4, 174 (2008).
  14. Babu, M., et al. Sequential peptide affinity purification system for the systematic isolation and identification of protein complexes from Escherichia coli. Methods Mol. Biol. 564, 373-400 (2009).

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Cite This Article
Babu, M., Kagan, O., Guo, H., Greenblatt, J., Emili, A. Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry. J. Vis. Exp. (69), e4057, doi:10.3791/4057 (2012).

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