1. http://creativecommons.org/licens es/by-sa/2.0/

2. Large Scale Approaches to the Study of Protein Levels and Activity Prof:Rui Alves ralves@cmb.udl.es 973702406 Dept Ciencies Mediques Basiques, 1st Floor, Room 1.08 Website of the Course:http://web.udl.es/usuaris/pg193845/Courses/Bioinformatics_2007/ Course: http://10.100.14.36/Student_Server/

3. The proteome –Protein complement of a genome Variable –In different cell and tissue types in same organism –In different growth and developmental stages of organism Dynamic –Depends on response of genome to environmental factors »Disease state »Drug challenge »Growth conditions »Stress

4. Why Studying Proteins Directly? Just because the gene is regulated, is it translated? If so, under what circumstances ? Are there qualitative and quantitative aspects to the regulation of the translation under different conditions?

5. Proteome Wide Studies With fully sequenced genomes available one can finally study how gene expression is regulated in the whole genome simultaneously The same has become true of the (almost) entire protein complement of a cell: PROTEOMICS

6. What can we study with proteomics? Regulation of Translation –Comparing Protein Levels to corresponding gene levels allows study of how translation is regulated Regulation of Protein Modification (activity) –E.g. comparing phosphorylation state of proteins Protein Interaction Networks –E.g. finding out how proteins cooperate to achieve an effect Protein-DNA Interaction Networks ChIP-chip or PBM arrays

7. How Can We Measure Protein Levels? Core technology –2D-PAGE High resolution protein separation and display –Mass spectrometry (NMR/IRS) Protein identification

8. 2D-PAGE Cell Protein solubilization Separation by size/charge (IEF) Iso-Electric Focusing PAGE Staining –Silver –Coomassie blue –Fluorescent dyes Sypro Ruby –Radioisotopic labelling

9. Creation of Master Gel Image Gel under basal conditions Compare Gels generated under other conditions to Master Gel and determine what has changed http://www.expasy.org/tools/

10. Identification of Spots At this stage, accumulated images of gels in which spots have been identified and/or knowledge of isoelectric point/size can help identify proteins. http://www.expasy.org/tools/ What about unidentifed/confusing spots

11. Mass Spectroscopy Extraction/Proteolysis MALDI/TOF-MS –Matrix-Assisted –Laser Desorption / Ionisation –Time-Of-Flight analysis

12. Mass Spectrometry (MS) Introduce sample to the instrument Generate ions in the gas phase Separate ions on the basis of differences in m(ass)/z(charge) with a mass analyzer Detect ions

13. Mass Spectrometry (MS)

14. Identifying The Spectra ACTGHRSKAASKAASRLLMN… Trypsin ACTGHRS KAAS RLLMN… … Time m/z From Protein sequence/other knowledge we can know how a given protein is to be hydrolized From knowledge of the smaller peptides we can calculate their predicted mass/charge ratio and their migration time Comparing the real spectra to theoretical/pre-existing spectra can allow for the identification of proteins Spectrum comparison can be made using for example Fourier Transforms NMR and other forms of spectroscopy can also be used

15. Algorithmic approaches to “tag” identification Cross Correlation (Eng et al. - SEQUEST): comparison between observed and theoretically generated spectra. Peptide sequence tags (Mann): extract an unambiguous sequence tag for ID. De novov probability-based matching (Perkins, and the proprietary Mascot by Matrix Science): takes statistical significance of fragmentation into account.

16. Sequence tagging Algorithms

17. De novo algorithms

18. Advantages vs. Disadvantages of proteomics Determination of MW and aa. Sequence Detection of posttranslational modifications High-throughput capability High capital costs Requires sequence databases for accurate analysis De novo methods not very mature yet

19. Other Proteomic Tools FYR

20. Array-based Proteomics Employ two-hybrid assays Use GFP, FRET, and GST –GFP = green florescent protein –FRET = florescence resonance energy transfer –GST = glutathione S-transferase, a well characterized protein used as a marker protein.

21. Two-Hybrid Assay Figure 12- 35. Griffiths et. al. Modern Genetic Analysis.

22. Array-based Proteomics Phosphoproteomics

23. Array-based Proteomics Offer a high-throughput technique for proteome analysis. These small plates are able to hold many different samples at a time. Current research is ongoing in an attempt to interface array methodologies with Mass Spectrometry.

24. Structural Proteomics Pioneering work is undergoing by Baumeister et al, which can significantly reduce the amount of painstaking labor in the crystallization of proteins. Current techniques are not considered “high throughput” within the structural realm. Novel solutions combine current technologies, such as NMR and XRC.

25. Conclusions Proteomics –Enables global screening of complex samples –Provides qualitative / quantitative evidence for changes in protein expression in different biological situations –Identifies targets for further investigation / validation

26. To Do Look for Proteomics experiments on M. xanthus. If found, look for HKs and RR and see what you can find.