1. Proteome Analysis July 1, 2009 Learning objectives. Understand the theoretical basis of 2D-SDS Gel Electrophoresis. Understand the latest technique for identifying proteins using mass spectrometry. Become familiar with the SWISS-2DPAGE Database Workshop on identifying proteins with the pI, MW and a few peptide masses.

2. What is the proteome? genome transcriptome proteome metabolome

3. 2D-SDS Gel Electrophoresis What is it? This is a method for the separation and identification of proteins in a sample by displacement in 2 dimensions oriented at right angles to one another. Proteins separate over a large area, having a greater chance of resolution.

4. First dimension Separation by isoelectric point Isoelectric point-pH at which the net charge on the molecule is zero

5. Visuals http://www.aber.ac.uk/parasitology/Proteome /Tut_2D.html#Section%203

6. Figure 4-44. Alberts et al. Molecular Biology of the Cell, 3rd edn.

7. 2nd Dimension-to separate by size Five steps: 1. Isolate proteins 2. Denature proteins 3. Load onto gel 4. Apply voltage gradient 5. Stain

8. http://www.davidson.edu/academic/biology /courses/Molbio/SDSPAGE/SDSPAGE.html

9. http://www.davidson.edu/academic/biology /courses/Molbio/SDSPAGE/SDSPAGE.html

10. http://www.davidson.edu/academic/biology /courses/Molbio/SDSPAGE/SDSPAGE.html

12. Figure 4-44. Alberts et al. Molecular Biology of the Cell, 3rd edn.

14. Figure 3-42. Lodish et al. Molecular Cell Biology.

16. Databases of 2D-Gels http://www.umh.ac.be/~biochim/balhf.htm http://www-lmmb.ncifcrf.gov/EP/table2Ddatabases.html

17. http://www.weihenstephan.de/blm/deg/ manual/manualwork2html02testp9.htm

20. Laser Detector m/z Ab % 975.43 Vacuum Plate Linear Mode Reflection Mode

24. http://www.matrixscience.com/cgi/search_form.pl?FORMVER=2&SEARCH=MIS

25. Protein Prospector Another Web-based program is Protein Prospector (UCSF) http://prospector.ucsf.edu/