protein sequencing

1. PROTEIN SEQUENCING
Guided By:
Sadhna Gupta
Submitted By:
Prabha Singh (MSc 1sem Microbiology)

2. What is protein?
● Large macromolecule
● Composed of various
polypeptide chain
● Thiol groups of cystein
residues are linked by
disulfide bond

3. Types of Protein

4. What is protein sequencing
● process of determining sequence of amino
acid
1945- Sanger’s method
1950 - Edman degradation method

5. Basic Terminology

6. OVERVIEW OF PROTEIN SEQUENCING
● Destruction of disulfide bond
● Cleavage of polypeptide chain
● Sequencing of protein
* Sanger Method
* Edman degradation sequencing
● Determination of sequence by chromatography

7. 1. Destruction of Sulphide bond

8. 2 . Cleavage of Polypeptide chain
Proteases and other chemical reagents have a
specific recognition sequence and they cleave
after a particular amino acid.
Example:
Tripsin - after Lys
Pepsin - after Leu

9. 3. Sequencing
● Sanger’s Method
He used 1fluro
2,4dinitrobenzene(DNFB)
known ad Sanger’s reagent
worked on Insulin

10. Procedure of
Sanger’s
method

11. Disadvantage of Sanger’s Method
It cannot be used repeatedly on same peptide
sequence bcz the peptide is totally degraded.

12. ● Edman degradation
method
* In 1950 Peher Edman
gave the method for
determining protein
sequencing
* He used
Phenyisothiocynate
reagent

14. Procedure
involved in
Edman
Degradation
Method

15. Limitation of Edman
Sequencing
Edman sequencing is totally based on N- terminal
sequencing, but if N-terminal is chemically modified
then this sequencing will not work.

16. C Terminal analysis
To determine “C” terminal sequence the peptide
chain is treated with “carboxypeptidase”

17. MASS SPECTROMETRY
● measures mass to charge ratio of ions
● used to identify and quantify the molecules in
simple and complex mixtures.
Broadly used in “proteomics”

18. Mechanism of mass
spectrometry

19. APPLICATION OF PROTEIN
SEQUENCING
● important to know the cellular function
● elucidate the history of life on earth