protein sequence determination methods.

2. • Proteins central dogma
2

3. proteins- amino acids( hair and skin)
Amino Acids: Amine, Carboxylic Acid, R-group
carbohydrates- monosaccharide's, polysaccharides and
disaccharides
Nucleic acids- nucleotides (make up the DNA)
Nucleic Acids: Sugar, Phosphate, Base
lipids or fatty acids- glycerol
biochemical compounds consisting of one or
more polypeptides typically folded into
a globular or fibrous form in a biologically functional way.
3

4. Protein structures
4

5. There are 22 protein building amino acids
5

6. 6

7. first described by the Dutch chemist Gerardus Johannes Mulder and
named by the Swedish chemist Jöns Jacob Berzelius in 1838
• 1926, when James B. Sumner showed that the enzyme urease was in fact a protein
Pehr Edman
Edman reagent(phenylisothiocyanate (PITC))
Frederick sanger, 1953
first protein to be sequenced was insulin.
Sanger's reagent (1-fluoro-2,4-dinitrobenzene)
The first protein structures to be solved
were Hemoglobin and Myoglobin, by Max Perutz and Sir John
Cowdery Kendrew, 1958.
X ray diffraction method
7

8. preliminary
• Denature protein
• Reduce and alkylate disulfides
• Determine subunits
Analyze
segments
• Cleavage into segments
• Edman degradation
Reconstruct
sequences
• Align sequences
• Assign disulfides
8

9. •unfolded from its compact globular shape into an extended linear form to make
it more accessible to attack by chemical and enzymatic reagents.
•e.g. → urea.
•2-mercaptoethanol(common reducing agent) or dithiothreitol (DTT)
•disulfide bond is transferred from the polypeptide to the mercaptoethanols
•In polypeptide sulfhydryl groups are formed & are treated with iodoacetic
acid ( alkalising agent) →cysteine residues→ S—carboxymethylcysteine
•protein consists of only one type of polypeptide chain.
•two different polypeptides can have the same N-terminal amino acid
Reduce Disulfides
Denature Protein
Determine Subunits
(Edman degradation )
9

10. ♦ Polypeptide sequences longer than 40 to 100 residues cannot be directly
sequenced
Certain enzymes cleave peptide bonds with great specificity
e.g.→ Trypsin, will only hydrolyze peptide bonds after lysine or arginine
residues if the preceeding residue is not proline
♦ In the case of ribonuclease A, trypsin digestion generates a total of 14 fragments.
Chemical Fragmentation Methods:Cyanogen bromide (CNBr) specifically
cleaves Met residues at the C-end forming a homoserine lactone
10

11. 11

12. N-terminal sequencing
SANGER’S METHOD
 Treat with DNFB to form a derivative of the amino-terminal amino acid
hydrolysis
 Sanger's reagent (1-fluoro-2,4-dinitrobenzene
 Extraction of DNP-derivative with organic solvent
 Identification of DNP-derivative by chromatography and comparison with
standards
DANSYL CHLORIDE METHOD
 Forms a highly fluorescent derivative of the amino-terminal amino acid
 Identified by chromatography & fluorescence detection after acid
hydrolysis.
 Highly sensitive.
 Best for small amounts.
EDMAN DEGRADATION
 Treat with phenylisothiocyanate(PITC)
 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
12

13. ♦ Edman degradation.
phenylisothiocyanate o(PITC ) + N-terminal amino acid ----→ PTC
polypeptide.
1
2
stable PTH derivative thiazolinone(derivative)
1 →PTC polypeptide is exposed to anhydrous trifluoroacetic acid
2→ under acidic conditions, is converted to the more stable PTH derivative,
which can then be chromatographically identified
• Acid catalyzed hydrolysis
• ♦6M HCl/ 100-120°C/ 24 h (in oxygen free environment to
prevent
oxidation of SH groups)
♦Some residues are degraded under these harsh conditions
• Base catalyzed hydrolysis
- 4 M NaOH /100°C/ 4-8 hours
-Arg, Cys, Ser and Thr are decomposed and other amino acids are
deaminated and racemized.
-Used mainly to determine Trp which is extensively degraded
under acid catalyzed hydrolysis
13

14. 14

15. •The Edman degradation proceeds from the N-terminus of the protein it will
not work it the N-terminal amino acid has been chemically modified.
•It also required the use of either guess work or a separate- procedure to
determine the position of disulfide bridges.
• The great advantage of the Edman method is that the rest of the peptide
chain after removal of the N-terminal amino acid is left intact for further
cycles of this procedure, thus the Edman method can be used in a
sequential fashion to identify several or many consecutive amino acid
residues starting from the N-terminal end
15

16. Sanger’s
method
16
Edman
degradation
method

17. C-terminal sequencing
• 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.
17

18. • N- and C- terminal Analysis-Exopeptidase Method
• Exopeptidases cleave the terminal residue of a
polypeptide chain.
• Aminopeptidases cleave the N-terminal
residues.
• Carboxypeptidases cleave the C-terminal
residues.
• Further analyzed by amino acid analyzer.
18

19. HPLC:HIGH PERFORMANCE LIQIUD CHROMATOGRAPHY
1. The amino acid derived from each cycle is identified by
comparison to the retention time on reverse phase high
performance liquid chromatography (HPLC/cLC) to
the retention time of the PTH amino acid standards.
The mixture of fragments
must be separated.
An HPLC device will do
this SEPARATION
19

20. Compare amino acid sequence of one set of peptide
fragments with the sequence of a second set of fragments
obtained using different cleavage points.
20

21. 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
AUTOMATED DEVICE IS DEVELPED BY EDMAN and GEOFFREY BEGG
21

22. 1. This process is known as matrix assisted laser desorption /ionization mass
spectrometry(MALDI MS)
2. Successfully used to measure the mass of a wide range of macromolecules.
3. A solution of analyses is passed through a charged needle that is kept at a
high electrical potential, dispersing the solution into a fine charged micro
droplets.
4. This create a spectrum of species with different mass to charge ratios.
5. Each successive peak corresponds to a species that differs from that of its
neighboring peak by a charge difference of 1 and mass difference of 1(1
proton)
6. the mass of the protein can be determined from any two neighboring peaks.
7. The measure of M/Z of one peak is
• M is mass of the protein
• N2 is the number of charges
• X is the mass of the added groups
22

23. Protein sequencing by Mass Spectrometry
•Mass spectrometry is an important emerging method for the
characterization of proteins.
• The two primary methods for ionization of whole proteins are electro spray
ionization (ESI) and matrix assisted laser desorption/ionization (MALDI).
•Mass spectrometer has three main parts for characterizing the protein –
1. An ionization source
2. A mass analyzer
3. An ion detector
23

24. Working of Mass Spectrometry
Working of Mass Spectrometry
Working of Mass Spectrometry
Mass Spectrometry machine
Source: http://chemistry.umeche.maine.edu/CHY251/MassSpec1.
https://www.uni-due.de/imperia/md/images/zmb/lcq-fleet-zmb-ace-ude.jpg
24

25. 25

26. ADVANTAGES –
•Stable isotopic labeling of proteins may be used to
discriminate between contaminants and original partners
using MS (mass spectrometry) techniques.
•MS (mass spectrometry) has also made advantages in the
analysis of membrane proteins.
DIS-ADVANTAGES-
•Miscalibration is one of the main errors of MS
spectrometry.
•MALDI doesn’t favor the identification of hydrophobic
peptides.
26

27. • Determine number of polypeptide chains (subunits).
• If subunits are too large, fragment them into shorter
polypeptide chains.
• Determine number of disulfide bonds (inter-and intra-chain).
• Determine the amino acid composition of each polypeptide
chain.
• Sequence each fragment using the Edman degradation
method.
• Complete the sequence by comparing overlaps of different
sets of fragments.
• For reference sequence , check it out in
BLAST,UNIPORT,NCBI.
protein sequencing method
27

28. 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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29. CLINICAL applications of protein sequencing
a) Identification of the protein family to which a particular protein
belongs and finding the evolutionary history of that protein.
b) Prediction of the cellular localization of the protein based on its target
sequence (sequence of amino acids at the N terminal end of the protein
which determines the location of the protein inside the cell).
c) Prediction of the sequence of the gene encoding the particular protein.
d) Discovering the structure and function of a protein through various
computational methods and experimental methods
29

30. REFERNCES
• Assignment of the disulfide bonds of huwentoxin-II by Edman degradation
sequencing and stepwise thiol modification.. Available from:
https://www.researchgate.net/publication/12035017_Assignment_of_the_disulfide
bonds_of_huwentoxinII_by_Edman_degradation_sequencing_and_stepwise_thiol
_modification [accessed Oct 26, 2015].
• http://www.niaid.nih.gov/LabsAndResources/labs/aboutlabs/rtb/ProtChemSec/pro
teinSequencingUnit/technology/Pages/techEdman.aspx
• WIELY ONLINE LIBRARY
http://higheredbcs.wiley.com/legacy/college/voet/0470129301/guided_exp/guided
_exploration_4/protein_sequence.html
• Biochemistry- voet and voet
• LEHNINGER’S, PRINCIPLES OF BIOCHEMISTRY.
• WILSON &WALKER’S METHODS AND TECHNIQUES IN MOLECULAR BIOLOGY
AND BIOCHEMISTRY.
• NCBI
• BLAST
• UNIPORT
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