2D-Gel Analysis Jennifer Wagner Image retrieved from

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.,

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.,

2DGE Load sample Isoelectric SDS-PAGE focusing Images retrieved from

Visualization of proteins Coomassie blue staining Detect 36-47ng Silver staining Detect ng Fluorescent staining Detect 1-2 ng From Jefferies, et al., Images from

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., 2001 Images from and Expression-and-Analysis/Protein-Gel-Electrophoresis/2D-Gel-Electrophoresis.html?cid=invggl s&

Alternatives to 2DGE Large scale peptide or protein arrays Fey, et al., 2001 Image from

Alternatives to 2DGE Capillary isoelectric focusing Fey, et al., 2001 Image from

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 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., 2005 Image retrieved from detected-through-saliva-and-nano-bio-chip/

Proteomic analysis “Typical” method: 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 “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 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 with typical 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

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., 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 2D-gel websites GELBANK: GelScape: NCI Flicker: World-2D PAGE repository:

World-2DPAGE Repository

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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?