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.
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
2)To identify mechanisms linking the genotype and environment together into the phenotype
“a snapshot in time”
Fey, et al., 2001
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
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
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
2DGE
What is it?
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
2DGE
Load sample Isoelectric SDS-PAGE
focusing
Images retrieved from http://genome.wellcome.ac.uk/doc_wtd021045.html
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
Advantages of 2D-gel analysis
1) Very sensitive
2) High resolution
>10,000 different proteins
3) Unbiased search
Fey, et al., 2001
Limitations of 2D-gel analysis
1) Lack of resolution of all proteins present
2) Irreproducibility of results
3) Biased
Fey, et al., 2001
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&
Alternatives to 2DGE
Large scale peptide or protein arrays
Fey, et al., 2001
Image from http://microarray.swmed.edu/p_protein.html
Alternatives to 2DGE
Capillary isoelectric focusing
Fey, et al., 2001Image from http://www.convergentbiosci.com/revolution.html
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
Hu, et al., 2005
“Typical” proteomics methods, using 2DGE
vs.
“shotgun” proteomics
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/
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
Shotgun proteomics
Figure 1 from Hu, et al., 2005
Shotgun proteomics
LC-ESI
mass spectrum
MS/MS
Figures 3 and 4 from Hu, et al., 2005
Mass/charge ratio used to identify proteins
Proteins identified with shotgun proteomics
Hu, et al., 2005
Typical proteomics
2D gel
Proteins were visualized with SYPRO Ruby
Figure 5 from Hu, et al., 2005
Typical proteomics
MALDI-MS analysis
mass/charge ratio used to identify proteins
Figure 6 from Hu, et al., 2005
Proteins identified withtypical proteomics
Hu, et al., 2005
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
Hu, et al., 2005
Figure 7 from Hu, et al., 2005
Conclusionsfrom Hu, et al., 2005
Shotgun proteomics was successful!
Combination of “typical” and “shotgun” approaches most effective
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
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/
World-2DPAGE Repository
http://world-2dpage.expasy.org/repository/
Search by gene name
http://world-2dpage.expasy.org/repository/
No results were found
Search by pI/Mw range
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?