1. Protein Profiling

2. Background Information

3. How do we determine evolutionary relationships between organisms?

4. Proteomics The study of protein structure and function This includes:
Protein-protein interactions
Protein expression
Posttranslational modifications of protein-coding genes
Proteome = all proteins in a particular cell

5. How are proteins made?

6. Posttranscriptional Modifications: the creation of proteins
RNA Editing
Alternative Splicing
mRNA Synthesis & Degradation
Proteolytic Cleavage
Protein Degradation
Protein-Protein Interaction
Carbohydrate Modification (Glycosylation)
Phosphorylation

7. 1. RNA Editing
Evolved eukaryotes can change the sequence of mRNA’s by substituting bases.
Less evolved eukaryotes can delete bases.
This clearly changes the codons and corresponding amino acids.
New stop codons
New ORF’s
New proteins

8. 2. Alternative Splicing
Alternative splicing allows exons to be included or excluded to produce different mRNA’s
This of course leads to the production of different proteins
Example:

9. 3. mRNA Synthesis & Degradation
Level of mRNA synthesis partly determines the level of protein expression
Chemical modifications to mRNA’s can change their stability and therefore, protein expression levels.

10. 4. Proteolytic Cleavage
Most proteins undergo cleavage after translation:
Peptide bonds are broken via protease
Usually ‘met’ is removed
Misfolded proteins can also undergo proteolytic cleavage

11. 5. Protein Degradation Very similar to mRNA degradation
Ubiquitin: The molecular kiss of death!
Proteins degraded by proteasomes

12. 6. Protein-Protein Interactions
Proteins usually function in complexes. If one protein, necessary for the complex to work properly, is not produced then you may have a non-functional structure.
For example:

13. 7. Carbohydrate Modification
Many proteins are covalently bonded to carbohydrates (sugars)
Modifications to these carbs can alter how the protein functions
For example:
Lymphocyte (T, B and NK cells) carbohydrates are essential in determining how they will infiltrate sites of infection

14. 8. Phosphorylation
The addition of a PO4 group to a protein by a kinase
Activates or deactivates by causing a conformational change
For example:
Smooth muscle contracts when phosphorylated

15. Hopefully we can see that it becomes increasingly important to examine protein expression & modification among species…

16. Our Experiment

17. Day 1: Extract Fish muscle proteins

18. What is Laemmli buffer? What does it do?
Contains SDS, Tris buffer, Glycerol, and Bromophenol blue
Denatures and solubilizes proteins

19. What is DTT? What does it do?
DTT stands for Dithiothreitol
It is a reducing agent that breaks the proteins disulfide bonds

20. Day 2: Run an SDS-PAGE Gel

21. What does SDS-PAGE stand for?

22. What is polyacrylamide?
Gel matrix is polyacrylamide – a polysaccharide
Smaller pores to separate small biomolecules
Proteins are much smaller than DNA
Average amino acid = 110 daltons
Average nucleotide pair = 649 daltons

23. What are the two layers of the gel? What is their purpose?

24. Gel Mechanisms
A molecule’s mobility through gel is affected by 2 things:
Mass
Charge

25. What charge do proteins have?
Proteins can have +, - or Ø charge
So how do we fix this problem?
SDS!

26. What is SDS? SDS detergent (sodium dodecyl sulfate)-
CH2
CH3
SDS
SDS detergent (sodium dodecyl sulfate)
Solubilizes and denatures proteins
Adds negative charge to proteins
s-s
SDS, heat
Proteins with SDS + –

27. How is Protein Size Measured?
Size measured in kilodaltons (kD)
Dalton = approximately the mass of one hydrogen atom or 1.66 x gram
Average amino acid = 110 daltons

28. Actin and Myosin Standard
Used as a reference to help ID major conserved muscle proteins & serves as a control

29. Day 3: Analyze Proteins

30. Procedure

31. Tips
These gels are ONE MILLIMETER thick. They are very fragile once out of the casing.
Loading them will be tricky, and you will need to use the “protein only” pipette tips.

32. Results

33. Your Gel Will Show Proteins of Many SizeskD
Function
Titin
3000
Center myosin in sarcomere
Dystrophin
400
Anchoring to plasma membrane
Filamin
270
Cross-link filaments
Myosin
heavy chain
210
Slide filaments
Spectrin
265
Attach filaments to plasma membrane
Nebulin
107
Regulate actin assembly
-actinin
100
Bundle filaments
Gelosin 90
Fragment filaments
Fimbrin 68
Actin 42
Form filaments
Tropomysin 35
Strengthen filaments
light chain
15-25
Troponin (T.I.C.)
30, 19, 17
Mediate contraction
Thymosin 5
Sequester actin monomers 10 15 20 25 37 50 75
100
150
250
Measure distance from base of wells to the base of the bands
Measure fish protein bands between ~30 and 10 kD
Measure prestained standard bands between ~30 and 10 kD
Prestained
Standards A B C D E
Actin & Myosin