1. AH Biology: Unit 1 Proteomics and Protein Structure 1 Proteomics

2. Think What is the proteome? What codes for the proteome? How will we figure out how the proteome works? Why is it important that we understand the proteome? What are the applications of this technology to mankind in the future?

3. Proteomics The proteome is the entire set of proteins expressed by a genome.genome Activation and inactivation of genes Transcription animation Translation animation

4. RNA splicing

5. When mRNA is transcribed in eukaryotic cells it is composed of introns and exons. Introns are the non-coding sequence of the mRNA and will not be expressed in the protein molecule. They are spliced out (removed) from the mRNA.spliced Exons are the coding sequence and will be expressed in the protein molecule. RNA splicing in detail.RNA splicing in detail

6. Post-translational modification Post-translational modification is the alteration of the protein after translationPost-translational modification Post-translational modification occurs in the rough endoplasmic reticulum, Golgi apparatus and target site of the protein. Post-translational modification can involve –1. the addition of chemical groups –2. the covalent cleavage of the polypeptide

7. Post-translational modification 1.the addition of chemical groups that are catalysed by dedicated post-translational modification enzymes: phosphorylation (addition of a phosphate group) acylation (addition of an acyl group RCO –, where R is an alkyl group) alkylation (addition of an alkyl group, eg methylation) glycosylation (addition of a sugar group, eg glucose or oligosaccharides) oligosaccharides oxidation. 2.the covalent cleavage of sections of the polypeptide proteases (trypsinogen to trypsin) autocatalytic cleavage (the zymogen pepsinogen to pepsin).zymogen

8. Post-translational modification These modifications give the proteins specific functions and target the proteins to specific areas within the cell and the whole organism. 1.Intracellular, eg lyzozymes found in lysosomes and proteins required for organelles such as mitochondria.lysosomes mitochondria 2.Membrane bound, eg intrinsic and extrinsic proteins. 3.Extracellular, eg insulin and digestive enzymes.

9. Membrane proteins

10. Extracellular proteins and exocytosis

11. RNA splicing and post-translational modification RNA splicing and post-translational modification results in the proteome being larger than the genome. One gene may code for many proteins. The proteome may be as many as three orders of magnitude larger than the genome. Human genome = 30,000 genes. Human proteome > 100,000 proteins.

12. Regulation of gene expression Because of regulation of gene expression not all genes are expressed as proteins in a particular cell.regulationexpressed The Jacob Monod hypothesis or lac Operon is an example of this process.lac Operon This ensures that the cell is energy efficient and producing proteins only when they are required.

13. the lac Operon and its control

14. Analysis of the genome While DNA sequencing and microarray technology allow the routine analysis of the genome and transcriptome, the analysis of the proteome is far more complex.DNA sequencingmicroarray technologyanalysis of the genome Genome analysis involves the following techniques: 1.Sanger sequencing in detailSanger sequencing in detail 2.gel electrophoresisgel electrophoresis 3.cycle sequencingcycle sequencing 4.microarray in detail.microarray in detail

15. Analysis of the proteome Proteome analysis involves: 1.Isolation of proteins expressed by an active cell at a given time. 2.The functional interaction between the proteins active in the cell.

16. Analysis of the proteome Techniques used to identify expressed proteins: 1.2D electrophoresis to separate out proteins from cell samples according to their charge (isoelectric point: pH at which the protein has no net charge and does not migrate in an electric field) and molecular weight (SDS PAGE).electrophoresisisoelectric 2.Western blotting: Transfer proteins to nitrocellulose paper. Expose proteins to specific antibody coupled to a radioisotope, easily detectable enzyme or fluorescent dye. Identify desired protein/proteins. 3.Mass spectrometry to separate out proteins and identify specific fragments.Mass spectrometry

17. Analysis of the proteome This is a complex process as the proteins expressed differ from cell to cell and within the life cycle of the cell. In a multicellular organism all the different cell types throughout the lifetime of the organism would have to be sampled in order to determine all the possible proteins expressed. Proteomics technologies and cancer.

18. SDS PAGE

20. Isoelectric point Isoelectric point: pH at which the protein has no net charge and does not migrate in an electric field.Isoelectric point

21. Western blotting

22. Protein structure and activity The distinguishing feature of protein molecules is their folded nature and their ability to bind tightly and specifically to other molecules. Enzymes and the induced fit to their substrate is an example of this. The binding of oxygen to haemoglobin also illustrates this principle.

23. Mass spectrometry For more detail on mass spectrometry click the following link to Leeds University.Leeds University

24. Enzymes induced fitinduced fit

25. Haemoglobin

26. Binding and conformational change Binding causes a conformational change in the protein, which may result in an altered function and may be reversible. –Enzyme inhibitionEnzyme inhibition –Sodium potassium pumpSodium potassium pump –Cell proliferation and phosphorylationCell proliferation and phosphorylation Proteins may have one or more stable conformations depending on binding. This allows the property, regulation and activity of the protein to be controlled. The proteasome animation.proteasome

27. Proteomics: further reading Boston Children’s Hospital: Interactive guide to sequencing and identifying proteins.Interactive guide to sequencing and identifying proteins. Read the following journals to see how proteomics is used. These journals will form the basis for Proteomics Tutorials 1 and 2. –Knight JDR, Qian B, Baker D, Kothary R (2007) Conservation, Variability and the Modeling of Active Protein Kinases. PLoS ONE 2(10): e982. doi:10.1371/journal.pone.0000982.Conservation, Variability and the Modeling of Active Protein Kinases –Roy N, Nageshan RK, Pallavi R, Chakravarthy H, Chandran S, et al. (2010) Proteomics of Trypanosoma evansi Infection in Rodents. PLoS ONE 5(3): e9796. doi:10.1371/journal.pone.0009796. Proteomics of Trypanosoma evansi Infection in Rodents

28. Think What is the proteome? What codes for the proteome? How will we figure out how the proteome works? Why is it important that we understand the proteome? What are the applications of this technology to mankind in the future?