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PROTEIN SEQUENCING

BY
MOBIN ASLAM

PROTEIN
Biomolecules
Polymers of amino acids
Variation in protein structure and
function is due to the difference in
amino acid sequence in peptide
chains

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

Edman degradation
N-terminal sequencing

STEPS

Protein purification
Protein denaturation
Protein digestion
N-terminal labeling
Separation of labeled amino acid by
chromatography
Detection through mass spectrometry
Data analysis

Protein
isolation(purification)
1-SDS-PAGE
(sodium dodecyl sulfatepoly 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.

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

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 69hrs
For better results use mixture of

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

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

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

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-tocharge ratio in an analyzer by electromagnetic fields
The ions are detected, usually by a quantitative
method
The ion signal is processed into mass spectra

Mass spectrometer

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.

Limitations of Edman
degradation
Need Pure Samples of Peptides
Requires 40-60 min / Amino Acid
Cant Analyze N-Terminally Modified
Peptides

Advantages
Most Reliable Sequencing
Technique

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

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

Automatic protein
sequencers
Automatic protein
sequencers are
designed that
perform the 3
steps(labeling,
separation and
analysis of amino
acids) at a time
and analyze data
and give results
automatically

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

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THANKS