2. Protein
sequencin
g

3. Introduction:
Protein:
 polymer of amino acid
 protein’s st. and function depends
upon the amino acid sequence.
Protein Sequencing:
technique to find out the amino
acid sequence in protein
 Imp for understanding cellular
process
 Imp in targetting drugs to
specific metabolic pathways.

4. SEQUENCING METHODS
1-N-terminal sequencing
2-C-terminal sequencing
3-Prediction from DNA
sequence

5. The N-terminal sequencing is
done through
1. Sanger’s method
2. Dansyl chloride method
3. Edman’s degradation
method
N-TERMINAL
SEQUENCING:

6. Sanger’s method
• Treat with DNFB to form a
derivative of the amino-terminal
amino acid
• Acid hydrolysis
• Extraction of DNP-derivative
with organic solvent
• Identification of DNP-derivative
by chromatography and
comparison with standards

8. • Forms a highly fluorescent
derivative of the amino-
terminal amino acid
• Identified by
chromatography &
fluorescence detection after
acid hydrolysis.
• Highly sensitive.
Dansyl chloride
method

9. Edman’s degradation method
 Used to sequence the
peptides
 It removes one amino acid
from the N-terminal end of
the peptide
 under ideal conditions
the sequence of 30-60
amino acids can be
determined

10. • Protein purification
• Protein denaturation
• Protein digestion
• N-terminal labeling
• Separation by
chromatography
• Detection by mass
spectrometry
Steps

11. Protein purification
Source Material
Homogenization &Extraction
Precipitation
Chromatography
Concentration
Purity

13. C-TERMINAL
SEQUENCIN
G• Add carboxypeptidases to
a solution of the protein
• Take samples at regular
intervals
• Determine the terminal
amino acid by analyzing a
plot of amino acid
concentrations against time.

14. From DNA sequencing
 Protein sequence can also be
determined indirectly from the
mRNA
 Design primers from the amino
acid sequence and amplify the
gene
 Sequence the gene and
determine the amino acid
sequence of protein

15. Peptide Mass Fingerprinting
(PMF)
• Proteolytic enzyme digest protein
• 2-D gel electrophoresis is used for
protein separation.
• Separated spots are then identified by
PMF.
• Trypsin cleaves lysine and arginine sites.
• After digestion a set of diff masses of
peptides.
Mass of each peptide = amino acids present

17. Mass spectrometry

18.  Recombinant protein synthesis
 Drugs production
 Functional genomics
 Determine the protein folding
patterns
Applications of Protein Sequenci