2 D-Gel Analysis Jennifer Wagner Image retrieved from

2 D-Gel Analysis Jennifer Wagner Image retrieved from http: //en. wikipedia. org/wiki/File: Coomassie-2 D-Gels. jpg

2 D-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

2 D-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_2 D. html#Section%201

2 D-gel analysis “Typical” steps: 1) Isolate sample 2) Separate proteins by 2 DGE 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

2 D-gel analysis “Typical” steps: 1) Isolate sample 2) Separate proteins by 2 DGE 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

2 DGE What is it?

2 DGE 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_2 D. html#Section%201

2 DGE Load sample Isoelectric focusing SDS-PAGE Images retrieved from http: //genome. wellcome. ac. uk/doc_wtd 021045. html

Visualization of proteins Coomassie blue staining Detect 36 -47 ng Silver staining Detect 0. 5 -1. 2 ng Fluorescent staining Detect 1 -2 ng From Jefferies, et al. , http: //www. aber. ac. uk/parasitology/Proteome/Tut_2 D. html#Section%201 Images from http: //www. kendricklabs. com/2 d+Coomassie. Blue. htm http: //www. unil. ch/dbcm/page 48211_fr. html

Advantages of 2 D-gel analysis 1) Very sensitive 2) High resolution >10, 000 different proteins 3) Unbiased search Fey, et al. , 2001

Limitations of 2 D-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 p. H gradients, standardized conditions 3) Better visualization Fey, et al. , 2001 Images from http: //www. kendricklabs. com/2 d+autorad. htm and http: //www. invitrogen. com/site/us/en/home/Products-and-Services/Applications/Protein. Expression-and-Analysis/Protein-Gel-Electrophoresis/2 D-Gel-Electrophoresis. html? cid=invggl 12300000704 s&

Alternatives to 2 DGE Large scale peptide or protein arrays Image from http: //microarray. swmed. edu/p_protein. html Fey, et al. , 2001

Alternatives to 2 DGE Capillary isoelectric focusing Image from http: //www. convergentbiosci. com/revolution. html Fey, et al. , 2001

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 2 DGE 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” Image retrieved from http: //www. healthjockey. com/2008/04/17/heart-attackdetected-through-saliva-and-nano-bio-chip/ Hu, et al. , 2005

Proteomic analysis “Typical” method: 1) Isolate sample 2) Separate proteins by 2 DGE 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 MALDIMS/MS to obtain sequence information “Shotgun” method: 1) Isolate sample 2) 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 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 2 D 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 Hu, et al. , 2005

2 D-gel vs. shotgun “Typical” method: “Shotgun” method: Visualized 300 protein spots 105 were characterized 64 proteins identified 600 candidate sequence tags generated 266 proteins identified <10 k. Da – ~100 k. Da 2. 9 k. Da – 590 k. Da Wider range of isoelectric points Hu, et al. , 2005

Hu, et al. , 2005 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

Future directions from Hu, et al. , 2005 2 D 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

Useful 2 D-gel websites GELBANK: http: //www. gelbank. anl. gov Gel. Scape: http: //www. gelscape. ualberta. ca: 8080/htm/index. html NCI Flicker: http: //www. lecb. ncifcrf. gov/flicker/ World-2 D PAGE repository: http: //world-2 dpage. expasy. org/repository/

World-2 DPAGE Repository http: //world-2 dpage. expasy. org/repository/

Search by gene name No results were found http: //world-2 dpage. expasy. org/repository/

Search by p. I/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?