The determination of amino acid sequences presentation autumne 2015
-
Upload
richard-trinh -
Category
Education
-
view
840 -
download
14
Transcript of The determination of amino acid sequences presentation autumne 2015
![Page 1: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/1.jpg)
LOGO
TRINH MAI DUY LUU
Class: Advanced Bioscience and Biotechnology Frontiers
Professor: Dr. Yuji SAITO 1
![Page 2: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/2.jpg)
OUTLINE
� Common strategy in protein sequencing –
Edman degradation
� Other strategies for protein sequencing
� Conclusion
2
![Page 3: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/3.jpg)
0
3
![Page 4: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/4.jpg)
Releasing target protein from cells0.i
�Homogenate: Disrupting the
cell membrane
�Differntial Centrifugation:
Fractionating the mixture
Incase of intracellular proteins
4
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
![Page 5: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/5.jpg)
Purifying target protein0.ii
Figure 3. Gel-filtration chromatography
5
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”
![Page 6: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/6.jpg)
Purifying target protein0.iii
Figure 6. Polyacrylamide gel
6
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
![Page 7: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/7.jpg)
I
7
![Page 8: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/8.jpg)
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
8
6. Repeat steps 4 and 5 to determine sub-sequences and
create “overlapping”
7. Reconstruct the original protein
8. Locate the disulfide bonds.
![Page 9: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/9.jpg)
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
9
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
![Page 10: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/10.jpg)
Break all disulfide bondsI.2
“Copyright 2007 from Biochemistry.
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
![Page 11: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/11.jpg)
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?
11
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”
![Page 12: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/12.jpg)
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
12
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
![Page 13: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/13.jpg)
N-Terminal and C-Terminal residue
identificationI.3
13
![Page 14: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/14.jpg)
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
14
CHNH C NH CH
ORn-2 Rn-1
C
O
H3N CH O
Rn
C
O
O
2 Carboxypeptidase
![Page 15: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/15.jpg)
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
15
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)
![Page 16: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/16.jpg)
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)
16
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
![Page 17: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/17.jpg)
Edman DegradationI.4
17
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.
![Page 18: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/18.jpg)
Divide and conquerI.5
Table 1. Specific cleavage of polypeptide
18
In practice, the peptides cannot be much longer than about 50 residues
� Specific cleaving protein into smaller peptides by chemical or
enzymatic methods
![Page 19: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/19.jpg)
Divide and conquerI.5
How can we order the peptides to obtain the primary structure of the original protein?
19
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.
![Page 20: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/20.jpg)
The positions of the original dissulfide bondsI.6
20
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).
![Page 21: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/21.jpg)
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
21
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
![Page 22: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/22.jpg)
What is your strategy? ???^_^
22
![Page 23: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/23.jpg)
II
23
![Page 24: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/24.jpg)
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
• ?
24
(ESI)
![Page 25: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/25.jpg)
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
25
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)
![Page 26: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/26.jpg)
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.
26
![Page 27: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/27.jpg)
Protein Identification by MS
Spot removed from gel
Fragmented using trypsin
Spectrum of fragments generated
Lib
rary
II.4
27
Artificial spectra built
Artificially trypsinated
Database of sequences
(i.e. SwissProt)
MATCH
Lib
rary
![Page 28: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/28.jpg)
OTHER STRATEGIESII.5
•De novo methods
28
![Page 29: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/29.jpg)
OTHER STRATEGIESII.6
29
![Page 30: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/30.jpg)
IV
30
![Page 31: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/31.jpg)
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
31
![Page 32: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/32.jpg)
32
![Page 33: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/33.jpg)
Database search for protein identification
33
![Page 34: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/34.jpg)
34
![Page 35: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/35.jpg)
How Does a Peptide Fragment?
35
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)
![Page 36: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/36.jpg)
Matching Sequence with Spectrum
36
![Page 37: The determination of amino acid sequences presentation autumne 2015](https://reader034.fdocuments.in/reader034/viewer/2022051709/58753f271a28abb8208b4909/html5/thumbnails/37.jpg)
37