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2D-Gel Analysis

2D-Gel Analysis. Jennifer Wagner. Image retrieved from http://en.wikipedia.org/wiki/File:Coomassie-2D-Gels.jpg. 2D-gel analysis. Goals: 1)To characterize and quantify all the proteins in a particular sample

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2D-Gel Analysis

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  1. 2D-Gel Analysis Jennifer Wagner Image retrieved from http://en.wikipedia.org/wiki/File:Coomassie-2D-Gels.jpg

  2. 2D-gel analysis Goals: 1)To characterize and quantify all the proteins in a particular sample 2)To identify mechanisms linking the genotype and environment together into the phenotype “a snapshot in time” Fey, et al., 2001

  3. 2D-gel analysis Uses? Large scale identification of all proteins in a sample Comparison of two samples to find differences in protein expression From Jefferies, et al., http://www.aber.ac.uk/parasitology/Proteome/Tut_2D.html#Section%201

  4. 2D-gel analysis “Typical” steps: • Isolate sample • Separate proteins by 2DGE • Visualize proteins and excise spots of interest • Digest proteins with trypsin • Use MALDI-MS to measure molecular mass • Use LC-MS/MS or MALDI-MS/MS to obtain sequence information Hu, et al., 2005

  5. 2D-gel analysis “Typical” steps: • Isolate sample • Separate proteins by 2DGE • Visualize proteins and excise spots of interest • Digest proteins with trypsin • Use MALDI-MS to measure molecular mass • Use LC-MS/MS or MALDI-MS/MS to obtain sequence information Hu, et al., 2005

  6. 2DGE What is it?

  7. 2DGE What is it? a method for separating and identifying the proteins in a sample by displacement in 2 dimensions oriented at right angles to one another From Jefferies, et al., http://www.aber.ac.uk/parasitology/Proteome/Tut_2D.html#Section%201

  8. 2DGE Load sample Isoelectric SDS-PAGE focusing Images retrieved from http://genome.wellcome.ac.uk/doc_wtd021045.html

  9. Visualization of proteins Coomassie blue staining Detect 36-47ng Silver staining Detect 0.5-1.2ng Fluorescent staining Detect 1-2 ng From Jefferies, et al., http://www.aber.ac.uk/parasitology/Proteome/Tut_2D.html#Section%201 Images from http://www.kendricklabs.com/2d+CoomassieBlue.htm http://www.unil.ch/dbcm/page48211_fr.html

  10. Advantages of 2D-gel analysis 1) Very sensitive 2) High resolution >10,000 different proteins 3) Unbiased search Fey, et al., 2001

  11. Limitations of 2D-gel analysis 1) Lack of resolution of all proteins present 2) Irreproducibility of results 3) Biased Fey, et al., 2001

  12. Possible Solutions 1) narrow range gels, sample prefractionation 2) immobilized pH gradients, standardized conditions 3) Better visualization Fey, et al., 2001 Images from http://www.kendricklabs.com/2d+autorad.htm and http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Protein-Expression-and-Analysis/Protein-Gel-Electrophoresis/2D-Gel-Electrophoresis.html?cid=invggl123000000000704s&

  13. Alternatives to 2DGE Large scale peptide or protein arrays Image from http://microarray.swmed.edu/p_protein.html Fey, et al., 2001

  14. Alternatives to 2DGE Capillary isoelectric focusing Fey, et al., 2001 Image from http://www.convergentbiosci.com/revolution.html

  15. Large-scale identification of proteins in human salivary proteome by liquid chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry Hu, et al., 2005

  16. Hu, et al., 2005 “Typical” proteomics methods, using 2DGE vs. “shotgun” proteomics

  17. Sample preparation “Whole saliva from a healthy, non-smoking male in the morning at least two hours after eating and rinsing mouth with water” Hu, et al., 2005 Image retrieved from http://www.healthjockey.com/2008/04/17/heart-attack-detected-through-saliva-and-nano-bio-chip/

  18. “Typical” method: Isolate sample Separate proteins by 2DGE Visualize proteins and excise spots of interest Digest proteins with trypsin Use MALDI-MS to measure molecular mass Use LC-MS/MS or MALDI-MS/MS to obtain sequence information “Shotgun” method: Isolate sample Prefractionate sample using microcon filter Digest proteins with trypsin 4) LC-MS/MS to obtain sequence information Proteomic analysis Hu, et al., 2005

  19. Shotgun proteomics Figure 1 from Hu, et al., 2005

  20. Shotgun proteomics LC-ESI mass spectrum MS/MS Mass/charge ratio used to identify proteins Figures 3 and 4 from Hu, et al., 2005

  21. Proteins identified with shotgun proteomics Hu, et al., 2005

  22. Typical proteomics 2D gel Proteins were visualized with SYPRO Ruby Figure 5 from Hu, et al., 2005

  23. Typical proteomics MALDI-MS analysis mass/charge ratio used to identify proteins Figure 6 from Hu, et al., 2005

  24. Proteins identified withtypical proteomics Hu, et al., 2005

  25. “Typical” method: Visualized 300 protein spots 105 were characterized 64 proteins identified <10 kDa – ~100 kDa “Shotgun” method: 600 candidate sequence tags generated 266 proteins identified 2.9 kDa – 590 kDa Wider range of isoelectric points 2D-gel vs. shotgun Hu, et al., 2005

  26. Hu, et al., 2005 Figure 7 from Hu, et al., 2005

  27. Conclusionsfrom Hu, et al., 2005 Shotgun proteomics was successful! Combination of “typical” and “shotgun” approaches most effective

  28. Future directionsfrom Hu, et al., 2005 2D LC-MS/MS Use of affinity columns Apply technology to: Look at differential protein composition from stratified gland secretions Develop proteome fingerprints for diagnosis of oral diseases

  29. Useful 2D-gel websites GELBANK: http://www.gelbank.anl.gov GelScape: http://www.gelscape.ualberta.ca:8080/htm/index.html NCI Flicker: http://www.lecb.ncifcrf.gov/flicker/ World-2D PAGE repository: http://world-2dpage.expasy.org/repository/

  30. World-2DPAGE Repository http://world-2dpage.expasy.org/repository/

  31. Search by gene name No results were found http://world-2dpage.expasy.org/repository/

  32. Search by pI/Mw range

  33. Student Questions The end of the Hu paper mentioned proteomic analysis and fingerprinting being used as a diagnostic tool for certain diseases. Along those lines, would it be possible to use these types of analyses for personalized medicine?

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