1. Lecture 2 Techniques in proteomics By Ms. Shumaila Azam

2. Proteomics Proteome is the entire collection of protein in a cell.
Proteome gives an additional information i.e obtained only by transcriptome.
Transcriptome gives the information which genes are active in the cell.
Gives less information about the protein in the cell.
This is because the factor that influence the protein contents contains not only the amount of each mRNA that is availabale but also the rate which mRNA is translated into proteins and the rate at which the protein is degraded.

3. Protein Profiling
The method by which protein constituents are identified is called protein profiling.

4. Techniques Protein profiling is based on two techniques.
2D-gel electrophoresis
Mass spectrometry

5. What is 2-DE? Digest to peptide fragment MS analysis First dimension:
denaturing isoelectric focusing
separation according to the pI
2. Second dimension:
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Digest to peptide fragment
MS analysis

6. Separating protein in a proteome
In order to characterize a proteome it is necessary to prepare pure sample of constituent proteins.
A mammalian cell may contain 10,000-20,000 proteins.
A highly discriminating system is needed.

7. PAGE is the standard method for separating the protein in a mixture.
Depending upon the condition and under which the electrophoresis is carried out, different chemical and physical properties of proteins can be used as the basis for their separation.
The technique make use of the detergent called SDS(sodium dodecyle sulphate)
SDS:
Denatures protein
And confers negative charge that is roughly equivalent to the length of the unfolded polypeptide.

8. Iso- Electric Focusing
Under these conditions proteins separate according to their molecular masses
The smaller proteins migrating more quickly to the positive electrode.
Iso- Electric Focusing
Proteins can be separated by iso electric focusing in the gel that contains chemical which establish pH gradient when the electric charge is applied.
Protein migrates to its iso-electric point, the position in the gradient where its net charge is zero.

9. 2D-gel electrophoresis
These methods are combined in two dimensional gel electrophoresis.
In the first dimension the proteins are separated by their iso electric focusing.
The gel is soaked in the dodecyl sulphate.
Rotated by 90o.
2nd electrophoresis separating the proteins according to their sizes, carried out at right angles to the first.
This can separate several thousand proteins in single gel.

10. Staining
Staining the gel reveals a complex pattern of spots containing different proteins.

11. Run 2-DE, step by step

12. Run 2-DE step by step

13. Run 2-DE step by step

14. Run 2-DE step by step