1. AH Biology: Unit 1 Proteomics and Protein Structure 1
THERE'S LITTLE DOUBT THAT PROTEOMICS -- THE STUDY OF AN ORGANISM'S COMPLETE COMPLEMENT OF PROTEINS -- WILL HAVE GREAT IMPACT IN ALL AREAS OF THE LIFE SCIENCES IN THE YEARS TO COME. AND THE REASON IS CLEAR.
"TO REALLY UNDERSTAND BIOLOGICAL PROCESSES, WE NEED TO UNDERSTAND HOW PROTEINS FUNCTION IN AND AROUND CELLS SINCE THEY ARE THE FUNCTIONING UNITS,"
HANNO STEEN, DIRECTOR OF THE PROTEOMICS CENTER AT BOSTON CHILDREN'S HOSPITAL.

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?
Think about these questions as you study proteins in this section. Knowing the genome of an organism forms only the known words of the genetic language, but does not show you the sentences and paragraphs that correspond to the function of the proteins produced and how they interact.
Also see the PowerPoint presentation in Scientific Method 1 for more information on the human genome and proteomics.

3. Proteomics
The proteome is the entire set of proteins expressed by a genome. Activation and inactivation of genes Transcription animation Translation animation
To understand proteomics you need to understand transcription and translation first. You then need to understand the function of the rough endoplasmic reticulum and golgi apparatus in relation to protein modification.

4. RNA splicing

5. RNA splicing
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. Exons are the coding sequence and will be expressed in the protein molecule. RNA splicing in detail.
Transcription still happens in the same way using RNA polymerase.

6. Post-translational modification
Post-translational modification is the alteration of the protein after translation
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, e.g. methylation)
glycosylation (addition of a sugar group, e.g. glucose or oligosaccharides)
oxidation.
2. the covalent cleavage of sections of the polypeptide
proteases (trypsinogen to trypsin)
autocatalytic cleavage (the zymogen pepsinogen to pepsin).

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.
Intracellular, e.g. lysosymes found in lysosomes and proteins required for organelles such as mitochondria.
Membrane bound, e.g. intrinsic and extrinsic proteins.
Extracellular, e.g. 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 approximately. 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. The Jacob Monod hypothesis or lac Operon is an example of this process. 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 Sanger sequencing in detail gel electrophoresis
While DNA sequencing and microarray technology allow the routine analysis of the genome and transcriptome, the analysis of the proteome is far more complex.
Genome analysis involves the following techniques:
Sanger sequencing in detail
gel electrophoresis
cycle sequencing
microarray in detail.

15. Analysis of the proteome
Proteome analysis involves:
Isolation of proteins expressed by an active cell at a given time.
The functional interaction between the proteins active in the cell.
See Proteomics tutorial 2 about the parasite Trypanosoma brucei and Trypanosoma evansi and the proteins identified in its life cycle.

16. Analysis of the proteome
Techniques used to identify expressed proteins:
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).
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.
Mass spectrometry to separate out proteins and identify specific fragments.
See Proteomics tutorial 2 about the parasite Trypanosoma brucei and Trypanosoma evansi and the proteins identified in its life cycle.
Note that for the third link you will have to click through to the relevant animation.

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.
See Proteomics tutorial 2 about the parasite Trypanosoma brucei and Trypanosoma evansi and the proteins identified in its life cycle.

18. SDS PAGE
A very common method for separating proteins by electrophoresis uses a discontinuous polyacrylamide gel as a support medium and sodium dodecyl sulfate (SDS) to denature the proteins.
The method is called sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

19. Banding represents proteins. Smaller proteins travel further
SDS PAGE
Banding represents proteins.
Smaller proteins travel further
The banding represents the distance the different protein fragments have travelled in the gel. Smaller proteins travel further.

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

21. WESTERN BLOTTING
Used to identify specific amino-acid sequences in proteins

22. Mass spectrometry
For more detail on mass spectrometry click the following link to Leeds University.
Samples ionised first (given charge).
Samples separated by mass to charge ratio in mass analyser.
Detector identifies ionised protein fragments in order and in relation to their size and abundance.
This information is then compared against a database of known proteins to determine whether new unidentified or previously identified proteins are present.

23. 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. For example enzymes and the induced fit to their substrate Also binding of oxygen to haemoglobin
This will be discussed in detail in Proteomics and Protein Structure 3.

24. Enzymes induced fit
This will be discussed in detail in Proteomics and Protein Structure 3.

25. Haemoglobin
This will be discussed in detail in Proteomics and Protein Structure 3.

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 inhibition
Sodium potassium pump
Cell 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.
This will be discussed in detail in Proteomics and Protein Structure 3 and 4.

27. PROTEOMICS: FURTHER READING
Boston Children’s Hospital: 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: /journal.pone
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: /journal.pone

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?
Think about these questions again and answer them as part of a discussion in class based on what you have learned in this section.
Also see the PowerPoint presentation in Scientific Method 1 for more information on the human genome and proteomics.