2. PROTEIN
• Biomolecules
• Polymers of amino acids
• Variation in protein structure and function is
due to the difference in amino acid sequence
in peptide chains
3. Protein sequencing
• Technique to find out the sequence of amino
acids in a protein
Sequencing methods
1-N-terminal sequencing
(Edman degradation)
2-C-terminal sequencing
3-Prediction from DNA sequence
5. STEPS
• Protein purification
• Protein denaturation
• Protein digestion
• N-terminal labeling
• Separation of labeled amino acid by
chromatography
• Detection through mass spectrometry
• Data analysis
6. Protein isolation(purification)
• 1-SDS-PAGE
(sodium dodecyl sulfate-poly
acryl amide gel)
2-Two dimensional gels
Protein of interest is
immobilized by being
absorbed onto a chemically
modified glass or by electro
blotting onto a porous
polyvinylidene fluoride
(PVDF) membrane.
7. Protein hydrolysis(denaturation)
by heating a sample of the
protein in 6 Molar HCL up
to 100-110 degrees Celsius
for 24 hours or longer
It may degrade some amino
acids
To avoid this
Thiol reagents or phenol are
used
- Performic acid for intra
chain or inter chain S-S
bonds
8. Protein digestion
• Use Endoproteinase Lys-C, CNBr, Pepsin or
trypsin to digest proteins into a population of
peptides
• Other enzymes include Glu-C and
chymotrypsin
• Add enzyme at 1:20 enzyme: protein ratio
• incubate at room temperature for 6-9hrs
• For better results use mixture of enzymes
9.
10. N-terminal labeling
• The Edman reagent, phenylisothiocyanate (PTC), is
added to the adsorbed peptide, together with a mildly
basic buffer solution of 12% trimethylamine
• This reacts with the amine group of the N-terminal
amino acid
• The terminal amino acid can then be selectively
detached by the addition of anhydrous acid
• The derivative then isomerises to give a substituted
phenylthiohydantoin which can be washed off and
identified by chromatography, and the cycle can be
repeated
11.
12. CHROMATOGRAPHY
• Chromatography is a
technique in which
molecules are separated
based on volatility and bond
characteristics when
subjected to a carrier
• Derivatives of amino acid
can be separated by
• 1-HPLC
• 2-Gas chromatography
• In gas chromatography
(GC), the mobile phase is an
inert gas such as helium
13. MASS SPECTROMETERY
• Mass spectrometry (MS) is an analytical
technique that measures the mass-to-charge
ratio of charged particles
• The MS principle consists of ionizing chemical
compounds to generate charged molecules or
molecule fragments and measuring their
mass-to-charge ratios
• Separated amino acid derivatives are analyzed
by mass spectrometer
14. MS procedure
• A sample is loaded onto the MS instrument, and
undergoes vaporization
• The components of the sample are ionized by one of a
variety of methods (e.g., by impacting them with an
electron beam), which results in the formation of
charged particles (ions)
• The ions are separated according to their mass-to-
charge ratio in an analyzer by electromagnetic fields
• The ions are detected, usually by a quantitative
method
• The ion signal is processed into mass spectra
16. MS data analysis
• first strategy for
identifying an unknown
compound is to compare
its experimental mass
spectrum against a library
of mass spectra
• Standard solutions of
amino acids are also used
and the resulting pattern
is compared with
standard spectrum.
17. Limitations of Edman degradation
• Need Pure Samples of Peptides
• Requires 40-60 min / Amino Acid
• Can’t Analyze N-Terminally Modified Peptides
• Advantages
• Most Reliable Sequencing Technique
18. C-terminal sequencing
• The number of methods available for C-
terminal amino acid analysis is much smaller
than the number of available methods of N-
terminal analysis. The most common method
is to add carboxypeptidases to a solution of
the protein, take samples at regular
intervals, and determine the terminal amino
acid by analyzing a plot of amino acid
concentrations against time.
19. Prediction from DNA sequence
• Protein sequence can also be determined
indirectly from the mRNA or, in organisms that
do not have introns (e.g. prokaryotes)
• Sequence a short section, perhaps 15 amino
acids long, of the protein
• Design primers from the amino acid sequence
and amplify the gene, sequence the gene and
determine the amino acid sequence of protein
20. Automatic protein sequencers
• Automatic protein
sequencers are
designed that perform
the 3
steps(labeling, separatio
n and analysis of amino
acids) at a time and
analyze data and give
results automatically
21. Applications of protein sequencing
• Recombinant protein synthesis
• Drugs production
• Antibiotic production
• Functional genomics
• Determine the protein folding patterns
• In bioinformatics
• It plays vital role in proteomics
• Used for the prediction of final structure, function and
location of protein
• To find out the location of gene coding for that protein
