1. 2D-Gel Analysis Jennifer Wagner
Image retrieved from

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
*large scale identification: undertaken when the global protein expression of an organism or a tissue is being investigated and best carried out on model organisms whose genomes have been fully sequenced because proteins can be more readily identified from MS data.
*differential expression: you might like to compare a drug resistant organism to a susceptible one in an attempt to find the changes responsible for the resistance. Here the sequence requirements of the organism are not as important, as you are looking for a relatively small number of differences and can devote more time to the identification of each protein
Point out that two samples could be same person at different times, etc.
From Jefferies, et al.,

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
Red boxes are steps that introduce bias and affect your “snapshot”
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
Red boxes are steps that introduce bias and affect your “snapshot”
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.,

8. 2DGE Load sample Isoelectric SDS-PAGE focusing
Discuss how pH gradient is created: see note sheet
Images retrieved from

9. Visualization of proteins
Coomassie blue staining
Detect 36-47ng
Silver staining
Detect ng
Fluorescent staining
Detect 1-2 ng
Discuss pros and cons of each method: see note sheet
How does this create bias?
From Jefferies, et al.,
Images from

10. Advantages of 2D-gel analysis
1) Very sensitive
2) High resolution
>10,000 different proteins
3) Unbiased search
Unbiased search: is it really?
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
Sample prefractionation: uses affinity columns
Better visualization: fluorescent dyes or radioisotopic detection
Fey, et al., 2001
Images from and

13. Alternatives to 2DGE Large scale peptide or protein arrays
Offer powerful, high-throughput answers to specific questions about particular proteins
Image from
Fey, et al., 2001

14. Alternatives to 2DGE Capillary isoelectric focusing Fey, et al., 2001
Well suited to proteins of low molecular weight; could become powerful if fully automated
Fey, et al., 2001
Image from

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. “Typical” proteomics methods, using 2DGE
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”
How does this create bias? What if….?
Hu, et al., 2005
Image retrieved from

18. Proteomic analysis “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
Hu, et al., 2005

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

20. Shotgun proteomics Mass/charge ratio used to identify proteins 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 with typical proteomics
Ask class to compare and contrast two methods
What is going on in red box? Answer: protein modifications
Hu, et al., 2005

25. 2D-gel vs. shotgun “Typical” method: “Shotgun” 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
Hu, et al., 2005

26. Hu, et al., 2005 Figure 7 from Hu, et al., 2005
Ask why? Possible answers: proteins from bacteria, food, also we don’t know everything about our proteins yet
Certain groups we do see act as control, we would expect there to be high numbers of immune response proteins in saliva
Figure 7 from Hu, et al., 2005

27. Conclusions from Hu, et al., 2005
Shotgun proteomics was successful!
Combination of “typical” and “shotgun” approaches most effective
Shotgun approach: Identified small and large proteins unable to be identified by 2DGE
Typical approach: More sensitive to protein modifications

28. Future directions from 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
2D LC-MS/MS: Another dimension of chromatography
Use of affinity columns: To remove highly abundant proteins

29. Useful 2D-gel websites GELBANK: http://www.gelbank.anl.gov
GelScape:
NCI Flicker:
World-2D PAGE repository:

30. World-2DPAGE Repository

31. Search by gene name No results were found

32. Search by pI/Mw range

36. 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?
To be added