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 *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., http://www.aber.ac.uk/parasitology/Proteome/Tut_2D.html#Section%201

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

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

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 Discuss how pH gradient is created: see note sheet 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 Discuss pros and cons of each method: see note sheet How does this create bias? 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

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

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 Sample prefractionation: uses affinity columns Better visualization: fluorescent dyes or radioisotopic detection 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&

Alternatives to 2DGE Large scale peptide or protein arrays Offer powerful, high-throughput answers to specific questions about particular proteins Image from http://microarray.swmed.edu/p_protein.html Fey, et al., 2001

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 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

“Typical” proteomics methods, using 2DGE 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” How does this create bias? What if….? Hu, et al., 2005 Image retrieved from http://www.healthjockey.com/2008/04/17/heart-attack-detected-through-saliva-and-nano-bio-chip/

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

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

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

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 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

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

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

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

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

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 No results were found http://world-2dpage.expasy.org/repository/

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? To be added