This document discusses protein sequencing techniques. There are three main methods for determining a protein's amino acid sequence: N-terminal sequencing, C-terminal sequencing, and predicting the sequence from DNA sequencing. N-terminal sequencing involves using methods like Edman degradation to sequentially remove amino acids from the N-terminus and identify them. C-terminal sequencing uses carboxypeptidases to sequentially remove amino acids from the C-terminus over time. DNA sequencing allows predicting the protein sequence by first sequencing the gene that encodes the protein. Protein sequencing is important for understanding cellular processes and developing targeted drugs.

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