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
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
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”