Exploring Proteins and Proteomes

1. Exploring Proteins and Proteomes

2. Genomes and Proteomes

3. Obtaining the protein: cell lysis and Separation

4. Protein Purification Techniques • Salting out • Dialysis • Gel Filtration Chromatography • Ion-exchange Chromatography • Affinity Chromatography • HPLC • Gel Electrophoresis • Isoelectric Focusing • 2D Gel Electrophoresis • Ultracentrifugation

5. Salting Out

6. Dialysis

7. Gel Filtration Chromatography

8. Ion-exchange Chromatography

9. Affinity Chromatography

10. ELISA

11. HPLC

12. Gel Electrophoresis

13. Isoelectric Focusing

14. 2D Gel Electrophoresis

15. Ultracentrifugation

16. Structure Determination

17. Amino Acid Sequencing: Edman Degradation

18. Broad Classification of Protease • Serine Proteases • A serine residue acts as a nucleophile in the active site, facilitating the reaction • Operates via the catalytic triad His, Ser- Asp • Cystein Proteases • Cys as the nucleophile and activated by a nearby basic aa • Papain is an example • Aspartate Proteases • Two Asp residues cleaves the peptide bond by activating a water molecule • Metalloprotease • A metallic Zn or Co is involved in the catalytic mechanism

19. Examples of Proteases • Trypsin – carboxyl side of Lys and Arg • Chymotrypsin – carboxyl side of bulky hydrophobic aa • Elastase – carboxyl side of small hydrophobic aa • Clostripain – carboxyl side of Arg • Thrombin – serine protease with specific cleavage site: Leu-Val-Pro-Arg—Gly-Ser • Carboxypeptidase A – amino side of C terminal residues of aromatic or aliphatic aa • An exopeptidase

20. Mass Spectrometry

21. MALDI-TOF MS

22. Electrospray Ionization

23. X-ray Crystallography

24. X-ray Diffraction Pattern of Mevalonate Kinase

25. NMR Spectroscopy