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?