1. The document outlines common strategies used for protein sequencing, including Edman degradation. Edman degradation involves breaking the peptide bonds of a protein one by one to determine the amino acid sequence. 2. Other strategies discussed include peptide mass fingerprinting, tandem mass spectrometry, and de novo sequencing methods. Peptide mass fingerprinting involves breaking a protein into peptides and using mass spectrometry to determine the peptide masses and compare them to databases. Tandem mass spectrometry further breaks peptides into fragment ions for sequencing. 3. The strategies each have advantages and limitations. While Edman degradation was previously standard, mass spectrometry techniques are increasingly used due to their high-throughput capabilities and ability to sequence smaller protein amounts. The

1. LOGO
TRINH MAI DUY LUU
Class: Advanced Bioscience and Biotechnology Frontiers
Professor: Dr. Yuji SAITO
1

2. OUTLINE
Common strategy in protein sequencing –
Edman degradation
Other strategies for protein sequencing
Conclusion
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3. 0
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4. Releasing target protein from cells0.i
Homogenate: Disrupting the
cell membrane
Differntial Centrifugation:
Fractionating the mixture
Incase of intracellular proteins
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Figure 1. Differential
centrifugation
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Fractionating the mixture
Each fraction will be the
source of material to
isolating target protein

5. Purifying target protein0.ii
Figure 3. Gel-filtration chromatography
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Figure 2. Dialysis
Figure 4. Antibody-affinity chromatography
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Figure 5. High-Pressure
Liquid Chromatography
(HPLC)
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6. Purifying target protein0.iii
Figure 6. Polyacrylamide gel
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Figure 7. Electrophoretic analysis of
a protein purification
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Figure 6. Polyacrylamide gel
electrophoresis

7. I
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8. Common strategyI.0
1. Determine the amino acid composition
2. Break all disulfide bonds
3. N-terminal and C-terminal residue identification
4. Edman degradation (N-terminal sequence determination)
5. Divide and conquer
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6. Repeat steps 4 and 5 to determine sub-sequences and
create “overlapping”
7. Reconstruct the original protein
8. Locate the disulfide bonds.

9. Amino acid compositionI.1
Hydrolysis peptide
(HCl 6 M, 110 oC, 24 h)
Seperate amino acids
(HPLC, ion-exchange
chromatography, etc)
Analysis results
Ala-Gly-Asp-Phe-Arg-Gly
Ala,Gly,Asp,Phe,Arg,Gly
ion-exchange chromatography,
ninhydrin reaction
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Analysis results
(comperation of the chromatographic
pattern of our sample with that of a
standard mixture of amino acids)
Amino acid composition
(elements and
concentration)
Ala, Arg, Asp, 2Gly, Phe

10. Break all disulfide bondsI.2
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Published by W.H. Freeman and Company -
Newyork”
10
Ure and Guadinium chloride effectly disrupt a protein’s non-convalent bond. The
disulfide bond can be cleaved reversibly by reducing them with a reagent such as
β-mercaptoethanol or Dithiothreitol
Figure 8. Reduction and denaturation of Ribonuclease

11. N-Terminal and C-Terminal residue
identification
I.3
How many peptides constructing the target protein? Which of amino acid forms the N-
terminus and C-terminus of a peptide chain?
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Figure 9. Amino acid
sequence of bovine
nuclease
Figure 10. Amino acid
sequence of bovine
insuline
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01bd-4ac5-a641-de285fdac0f1@7/proteins
‘
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Published by W.H. Freeman and Company -
Newyork”

12. N-Terminal and C-Terminal residue
identification
I.3
1. The free unprotonated α-amino groups are labeled
using a reagent (2,4-dinitroflorobenzene – DFNB,
Sanger’s reagent, dansyl chloride, phenylisothiocianate –
Edman’s reagent) that will label the terminal amino acid.
2. The labeled peptide is hydrolyzed with acid which yield
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2. The labeled peptide is hydrolyzed with acid which yield
the labeled N-terminal residue and other free amino acids
3. Each of these labeled N-terminal residues can be
separated and identified using chromatography

13. N-Terminal and C-Terminal residue
identification
I.3
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14. N-Terminal and C-Terminal residue
identification
I.3
CHNH C NH CH NH CH
O
O
Rn-2 Rn-1 Rn
C
O
C
O
H2O Carboxypeptidase
Carboxypeptidase cleavage at the C-terminus
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CHNH C NH CH
ORn-2 Rn-1
C
O
H3N CH O
Rn
C
O
O
2 Carboxypeptidase

15. Edman DegradationI.4
Coupling: Phenyl isothiocyanate (PITC) reacts with
an α-amino acid group at N-terminal end to form a
phenylthiocarbamyl derivative of the terminal residue
(pH 8.6 controled by pyridine/’Quarol’/trimethylamine/
N-methylpiperidine)
Cleavage: Strong acid (Triflouroacetic acid) breaks
the peptide at the first peptide bond giving the peptide
(minus the first residue) and the liberated first residue
as the anilinothiazolinone (ATZ) form (be as
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as the anilinothiazolinone (ATZ) form (be as
anhydrous as is practically posible)
Conversion: The ATZ residue is separated from
the peptide by extraction in organic solvent
(ethylacetate/chlorobutane) , and is then converted
to phenylthiohydantoin (PTH) form (a more stable
form) (25% TFA v/v in water)
Analysis of PTH residue by using chromatography
(thin-layer chromatography/ reversed-phase high-
performance liquid choromatography)

16. Edman DegradationI.4
1950: Pehr Edman, Method for determination of the amino acid sequence in
peptides, ACTA CHEMICA SCANDINAVICA, 4, pp. 283-293
1967: Edman and Begg, partly automatic instrumenr named “sequenator”
Late 1960s: ‘Spining cup” sequenator marketed by Beckman
1971: Laursen described “solid-phase” sequencing – different automated sequencer. This system
is useful for sequencing of short peptides that were especially easily lost in extraction steps.
Pehr Voctor Edman
(1916 – 1977)
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1982: Commercial peptide sequencer:
Applied Biosystem Model 470A, and then
471A, 473A, 475A, 477A
Figure 11. Applied
Biosystem Model
470A
is useful for sequencing of short peptides that were especially easily lost in extraction steps.
1981: Hewick decribed “gas-phase” peptide sequenator, so-called because some reagents were
delivered as vapour
Longer stretches and
smaller amount of sample

17. Edman DegradationI.4
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Figure 12. The Edman degradation. The labeled amino-terminal residue (PTH-
alanin in the first round) an be released without hydrolyzing the rest of the peptide.
Hence, the amino-terminal residue of the shorterned peptide can be determined in
the second round. Three more rounds of the Edman degradation reveal the
complete sequence of the original peptide.

18. Divide and conquerI.5
Table 1. Specific cleavage of polypeptide
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In practice, the peptides cannot be much longer than about 50 residues
Specific cleaving protein into smaller peptides by chemical or
enzymatic methods

19. Divide and conquerI.5
How can we order the peptides to obtain the primary structure of the original protein?
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The peptides are separated and purified by some type of chromatography The
necessary additional information is obtained from overlap peptides.
Figure 13. Overlap peptides. The peptide obtained by chymotryptic
digestion overlaps two tryptic peptides, establishing their order.

20. The positions of the original dissulfide bondsI.6
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Figure 14. Diagonal electrophoresis. Peptides joined together by disulfide bonds can
be detected by diagonal electrophoresis. The mixture of peptides is subjected to
electrophoresis in a single lane in one direction (horizontal ) and then treated with
performic acid, which cleaves and oxidizes the disulfide bonds. The sample is then
subjected to electrophoresis in the perpendicular direction (vertical).

21. DrawbacksI.6
• tryptophan, cystein residues are overlapped by
background
• be difficult to characterize the trace peptides, the
blocked N-terminal peptides, or a mixture of peptides
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blocked N-terminal peptides, or a mixture of peptides
• Radioactive reagents (improving the sensitivity) are
hazard waste
• limited in the growing high-throughput proteome
research

22. What is your strategy?
???^_^
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23. II
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24. OTHER STRATEGIES
• Peptide mass fingerprinting
• Tandem mass spectrometry
• Protein sequence tags
• De novo methods
• Multi-enzyme digestion coupled with alternate CID/ETD
tandem mass spectrometr
• ?
II.1
• ?
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(ESI)

25. OTHER STRATEGIES
• Peptide mass fingerprinting
II.2
Protein sample are broken up into smaller peptide
fragments by proteolytic enzymes
The resulting fragments are extracted by acetonitril and
dried by vacuum, and then dissolved in distilled water
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The peptide are then insert into the vacuum champer of
a mass spectrometer (e.g. ESI-TOF or MALDI-TOF)
Compare the peak list against databases (e.g.
SwissProt, GeneBank)

26. Protein sample are broken up into smaller peptide
fragments by proteolytic enzymes or chemicals
Fractionation of peptides by HPLC
Resulting fragments fed into mass spectrometer for
analysis
• Tandem mass spectrometry
OTHER STRATEGIESII.3
Protein Database:
GenBank, Swiss-Prot,
dbEST, etc.
Search engines:
MasCot, Prospector,
Sequest, etc.
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27. Protein Identification by MS
Spot removed
from gel
Fragmented
using trypsin
Spectrum of
fragments
generatedLibrary
II.4
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Artificial
spectra built
Artificially
trypsinated
Database of
sequences
(i.e. SwissProt)
MATCH
Library

28. OTHER STRATEGIESII.5
•De novo methods
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29. OTHER STRATEGIESII.6
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30. IV
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31. Conclusion
Edman chemistry is now a standard method for peptide
sequencing
Mass spectrometry will replace the Edman chemistry
approach. However, the combination by both Edman and
Mass spectrometry will provide complementary
information for protein characterization
IV.1
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33. Database search for protein identification
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34. 34

35. How Does a Peptide Fragment?
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m(y1)=19+m(A4)
m(y2)=19+m(A4)+m(A3)
m(y3)=19+m(A4)+m(A3)+m(A2)
m(b1)=1+m(A1)
m(b2)=1+m(A1)+m(A2)
m(b3)=1+m(A1)+m(A2)+m(A3)

36. Matching Sequence with Spectrum
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