2D-Gel Analysis Jennifer Wagner Image retrieved from Coomassie-2D-Gels.jpg.
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Transcript of 2D-Gel Analysis Jennifer Wagner Image retrieved from Coomassie-2D-Gels.jpg.
![Page 1: 2D-Gel Analysis Jennifer Wagner Image retrieved from Coomassie-2D-Gels.jpg.](https://reader035.fdocuments.us/reader035/viewer/2022062314/56649e525503460f94b47e16/html5/thumbnails/1.jpg)
2D-Gel Analysis
Jennifer Wagner
Image retrieved from http://en.wikipedia.org/wiki/File:Coomassie-2D-Gels.jpg
![Page 2: 2D-Gel Analysis Jennifer Wagner Image retrieved from Coomassie-2D-Gels.jpg.](https://reader035.fdocuments.us/reader035/viewer/2022062314/56649e525503460f94b47e16/html5/thumbnails/2.jpg)
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
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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
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2D-gel analysis
“Typical” steps:
1) Isolate sample
2) Separate proteins by 2DGE
3) Visualize proteins and excise spots of interest
4) Digest proteins with trypsin
5) Use MALDI-MS to measure molecular mass
6) Use LC-MS/MS or MALDI-MS/MS to obtain sequence information
Hu, et al., 2005
![Page 5: 2D-Gel Analysis Jennifer Wagner Image retrieved from Coomassie-2D-Gels.jpg.](https://reader035.fdocuments.us/reader035/viewer/2022062314/56649e525503460f94b47e16/html5/thumbnails/5.jpg)
2D-gel analysis
“Typical” steps:
1) Isolate sample
2) Separate proteins by 2DGE
3) Visualize proteins and excise spots of interest
4) Digest proteins with trypsin
5) Use MALDI-MS to measure molecular mass
6) Use LC-MS/MS or MALDI-MS/MS to obtain sequence information
Hu, et al., 2005
![Page 6: 2D-Gel Analysis Jennifer Wagner Image retrieved from Coomassie-2D-Gels.jpg.](https://reader035.fdocuments.us/reader035/viewer/2022062314/56649e525503460f94b47e16/html5/thumbnails/6.jpg)
2DGE
What is it?
![Page 7: 2D-Gel Analysis Jennifer Wagner Image retrieved from Coomassie-2D-Gels.jpg.](https://reader035.fdocuments.us/reader035/viewer/2022062314/56649e525503460f94b47e16/html5/thumbnails/7.jpg)
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
![Page 8: 2D-Gel Analysis Jennifer Wagner Image retrieved from Coomassie-2D-Gels.jpg.](https://reader035.fdocuments.us/reader035/viewer/2022062314/56649e525503460f94b47e16/html5/thumbnails/8.jpg)
2DGE
Load sample Isoelectric SDS-PAGE
focusing
Images retrieved from http://genome.wellcome.ac.uk/doc_wtd021045.html
![Page 9: 2D-Gel Analysis Jennifer Wagner Image retrieved from Coomassie-2D-Gels.jpg.](https://reader035.fdocuments.us/reader035/viewer/2022062314/56649e525503460f94b47e16/html5/thumbnails/9.jpg)
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 fromhttp://www.kendricklabs.com/2d+CoomassieBlue.htm
http://www.unil.ch/dbcm/page48211_fr.html
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Advantages of 2D-gel analysis
1) Very sensitive
2) High resolution
>10,000 different proteins
3) Unbiased search
Fey, et al., 2001
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Limitations of 2D-gel analysis
1) Lack of resolution of all proteins present
2) Irreproducibility of results
3) Biased
Fey, et al., 2001
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Possible Solutions
1) narrow range gels, sample prefractionation
2) immobilized pH gradients, standardized conditions
3) Better visualization
Fey, et al., 2001Images 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&
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Alternatives to 2DGE
Large scale peptide or protein arrays
Fey, et al., 2001
Image from http://microarray.swmed.edu/p_protein.html
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Alternatives to 2DGE
Capillary isoelectric focusing
Fey, et al., 2001Image from http://www.convergentbiosci.com/revolution.html
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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
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Hu, et al., 2005
“Typical” proteomics methods, using 2DGE
vs.
“shotgun” proteomics
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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., 2005Image retrieved from http://www.healthjockey.com/2008/04/17/heart-attack-detected-through-saliva-and-nano-bio-chip/
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Proteomic analysis
“Typical” method:1) Isolate sample2) Separate proteins by 2DGE3) Visualize proteins and
excise spots of interest4) Digest proteins with trypsin5) Use MALDI-MS to measure
molecular mass6) Use LC-MS/MS or MALDI-
MS/MS to obtain sequence information
“Shotgun” method:1) Isolate sample2) Prefractionate sample using
microcon filter
3) Digest proteins with trypsin
4) LC-MS/MS to obtain sequence information
Hu, et al., 2005
![Page 19: 2D-Gel Analysis Jennifer Wagner Image retrieved from Coomassie-2D-Gels.jpg.](https://reader035.fdocuments.us/reader035/viewer/2022062314/56649e525503460f94b47e16/html5/thumbnails/19.jpg)
Shotgun proteomics
Figure 1 from Hu, et al., 2005
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Shotgun proteomics
LC-ESI
mass spectrum
MS/MS
Figures 3 and 4 from Hu, et al., 2005
Mass/charge ratio used to identify proteins
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Proteins identified with shotgun proteomics
Hu, et al., 2005
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Typical proteomics
2D gel
Proteins were visualized with SYPRO Ruby
Figure 5 from Hu, et al., 2005
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Typical proteomics
MALDI-MS analysis
mass/charge ratio used to identify proteins
Figure 6 from Hu, et al., 2005
![Page 24: 2D-Gel Analysis Jennifer Wagner Image retrieved from Coomassie-2D-Gels.jpg.](https://reader035.fdocuments.us/reader035/viewer/2022062314/56649e525503460f94b47e16/html5/thumbnails/24.jpg)
Proteins identified withtypical proteomics
Hu, et al., 2005
![Page 25: 2D-Gel Analysis Jennifer Wagner Image retrieved from Coomassie-2D-Gels.jpg.](https://reader035.fdocuments.us/reader035/viewer/2022062314/56649e525503460f94b47e16/html5/thumbnails/25.jpg)
2D-gel vs. shotgun
“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
Hu, et al., 2005
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Hu, et al., 2005
Figure 7 from Hu, et al., 2005
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Conclusionsfrom Hu, et al., 2005
Shotgun proteomics was successful!
Combination of “typical” and “shotgun” approaches most effective
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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 secretionsDevelop proteome fingerprints for diagnosis of oral diseases
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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/
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World-2DPAGE Repository
http://world-2dpage.expasy.org/repository/
![Page 31: 2D-Gel Analysis Jennifer Wagner Image retrieved from Coomassie-2D-Gels.jpg.](https://reader035.fdocuments.us/reader035/viewer/2022062314/56649e525503460f94b47e16/html5/thumbnails/31.jpg)
Search by gene name
http://world-2dpage.expasy.org/repository/
No results were found
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Search by pI/Mw range
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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?