LOGO

TRINH MAI DUY LUU

Class: Advanced Bioscience and Biotechnology Frontiers

Professor: Dr. Yuji SAITO 1

OUTLINE

� Common strategy in protein sequencing –

Edman degradation

� Other strategies for protein sequencing

� Conclusion

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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“Copyright 2007 from Biochemistry. Published by W.H. Freeman and Company - Newyork”

Fractionating the mixture

�Each fraction will be the

source of material to

isolating target protein

Purifying target protein0.ii

Figure 3. Gel-filtration chromatography

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Figure 2. Dialysis

Figure 4. Antibody-affinity chromatography

“Copyright 2007 from

http://www.pha.jhu.edu/~ghzheng/old/webct/note1_5.htm”

Figure 5. High-Pressure

Liquid Chromatography

(HPLC)

“Copyright 2007 from Biochemistry. Published by W.H. Freeman and Company - Newyork”

Purifying target protein0.iii

Figure 6. Polyacrylamide gel

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Figure 7. Electrophoretic analysis of

a protein purification

“Copyright 2007 from Biochemistry. Published by W.H. Freeman and Company - Newyork”

Figure 6. Polyacrylamide gel

electrophoresis

I

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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.

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

Break all disulfide bondsI.2

“Copyright 2007 from Biochemistry.

Published by W.H. Freeman and Company -

Newyork”

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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

N-Terminal and C-Terminal residue

identificationI.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

“Copyright 2007 from

http://archive.cnx.org/contents/db3ce6d5-

01bd-4ac5-a641-de285fdac0f1@7/proteins

‘

“Copyright 2007 from Biochemistry.

Published by W.H. Freeman and Company -

Newyork”

N-Terminal and C-Terminal residue

identificationI.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

N-Terminal and C-Terminal residue

identificationI.3

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N-Terminal and C-Terminal residue

identificationI.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

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)

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, 477AFigure 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

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.

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

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.

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).

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

What is your strategy? ???^_^

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II

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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)

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)

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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Protein Identification by MS

Spot removed from gel

Fragmented using trypsin

Spectrum of fragments generated

Lib

rary

II.4

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Artificial spectra built

Artificially trypsinated

Database of sequences

(i.e. SwissProt)

MATCH

Lib

rary

OTHER STRATEGIESII.5

•De novo methods

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OTHER STRATEGIESII.6

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IV

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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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Database search for protein identification

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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)

Matching Sequence with Spectrum

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