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Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
How To Sequence A Protein
W. Robert Midden
Department of Chemistry
Bowling Green State University
2
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Protein SequencingPreliminary Steps
For multisubunit proteins, the individual protein chains must first be separated
Break interchain disulfide bonds, if necessary
Two reagents are commonly used: performic acid mercaptoethanol
3
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
2-Mercaptoethanol
2-mercaptoethanol reduces disulfides to sulfhydryls But the sulfhydryls are easily oxidized back to the disulfide
S
S
C
H
2
C
H
2
C
H
C
H
N
H
C
C
N
H
C
C
O
O
O
O
S
H
S
H
C
H
2
C
H
2
C
H
C
H
N
H
C
C
N
H
C
C
O
O
O
O
S
H
C
H
2
C
H
2
O
H
S
C
H
2
C
H
2
O
H
S
C
H
2
C
H
2
O
H
S
H
C
H
2
C
H
2
O
H
S
S
C
H
2
C
H
2
C
H
C
H
N
H
C
C
N
H
C
C
O
O
O
O
S
H
S
H
C
H
2
C
H
2
C
H
C
H
N
H
C
C
N
H
C
C
O
O
O
O
S
H
C
H
2
C
H
2
O
H
S
C
H
2
C
H
2
O
H
S
C
H
2
C
H
2
O
H
S
H
C
H
2
C
H
2
O
H
4
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Preventing Reversal
to prevent oxidation the suflhydryls are alkylated with iodoacetic acid or acrylonitrile
S
H
C
H
2
C
H
N
H
C
C
O
O
S
C
H
2
C
H
N
H
C
C
O
O
C
H
2
C
H
C
N
C
H
2
C
H
2
C
N
S
H
C
H
2
C
H
N
H
C
C
O
O
S
C
H
2
C
H
N
H
C
C
O
O
C
H
2
C
H
C
N
C
H
2
C
H
2
C
N
S
H
C
H
2
C
H
N
H
C
C
O
O
I
C
H
2
C
O
O
S
C
H
2
C
H
N
H
C
C
O
O
C
H
2
C
O
O
S
H
C
H
2
C
H
N
H
C
C
O
O
I
C
H
2
C
O
O
S
C
H
2
C
H
N
H
C
C
O
O
C
H
2
C
O
O
5
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Perfomic Acid
H
C
O
O
O
H
S
S
C
H
2
C
H
2
C
H
C
H
N
H
C
C
N
H
C
C
O
O
O
O
S
O
3
-
S
O
3
-
C
H
2
C
H
2
C
H
C
H
N
H
C
C
N
H
C
C
O
O
O
O
H
C
O
O
O
H
S
S
C
H
2
C
H
2
C
H
C
H
N
H
C
C
N
H
C
C
O
O
O
O
S
O
3
-
S
O
3
-
C
H
2
C
H
2
C
H
C
H
N
H
C
C
N
H
C
C
O
O
O
O
Performic acid oxidizes cysteine to negatively charged cysteic acid
6
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Reversal Prevented
The repulsion of the negatively charged SO3-
groups prevents reformation of the disulfide bond Therefore alkylation is not necessary with
performic acid
S
O
3
-
S
O
3
-
C
H
2
C
H
2
C
H
C
H
N
H
C
C
N
H
C
C
O
O
O
O
S
O
3
-
S
O
3
-
C
H
2
C
H
2
C
H
C
H
N
H
C
C
N
H
C
C
O
O
O
O
7
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Protein SequencingPreliminary Steps
After breaking disulfide bonds, the chains are separated by disrupting noncovalent interchain interactions with pH extremes, 8 M urea, 6 M guanidium hydrochloride, or high salt
Then the individual protein chains are separated by electrophoresis or chromatography on the basis of size or charge
8
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Determining AminoAcid Sequence
Once each protein is purified the amino acid sequence is determined by:
1) determining the amino acid composition (how many of each amino acid are in the protein)
2) identifying the amino and carboxyl terminal amino acids
3) cleaving the protein into two or more sets of peptides using specific enzymatic or chemical reagents such as trypsin or cyanogen bromide
9
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
DeterminingProtein Sequence
4) determining the amino acid sequence of each of the peptide fragments
5) determining the entire protein sequence from the sequences of overlapping peptide fragments
6) locating the position of disulfide bridges between cysteines
10
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Determining Amino Acid Composition
The amino acid composition is determined by:
Hydrolysis with 6N HCl for one to three days
Separating and quantifying individual amino acids by ion exchange HPLC using an amino acid analyzer
11
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Determining the N-Terminal Amino Acid
The N-terminal amino acid is determined using either chemical reagents or enzymes
Chemical reagents include: Sanger’s reagent dansyl chloride Edman Degradation
12
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Determining the N-Terminal Amino Acid
Sanger’s reagent Treat with
dinitrofluorobenzene to form a dinitrophenyl (DNP) derivative of the amino-terminal amino acid
Acid hydrolysis Extract the DNP-derivative
from the acid hydrolysate with organic solvent
Identify the DNP-derivative by chromatography and comparison with standards
N
+
N
+
F
O
O
O
O
N
+
N
+
F
O
O
O
O
13
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Determining the N-Terminal Amino Acid
Dansyl chloride (dimethylaminonaphthylenesulfonyl chloride)
Forms a highly fluorescent derivative of the amino-terminal amino acid
Identified by chromatography and fluorescence detection after acid hydrolysis
Highly senstive Best choice when the amount
of protein is limited
N
C
H
3
C
H
3
S
O
O
C
l
N
C
H
3
C
H
3
S
O
O
C
l
14
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Determining the N-Terminal Amino Acid
Edman degradation phenylisothiocyanate (phenyl-N=C=S) adds to
N-terminus then acid treatment cleaves the N-terminal amino acid as a PTH derivative
the remaining protein chain is intact and the cycle can be repeated
under ideal conditions the sequence of 30-60 amino acids can be determined
Leucine aminopeptidase enzyme from hog kidney hydrolyzes the N-
terminal peptide bond best with nonpolar amino acids
15
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Determining theC-Terminal Amino Acid
Hydrazinolysis hydrazine at 100°C cleaves all peptide bonds
forming hydrazides except for the carboxyl terminal
C-terminus reduced with LiAlH4 forms amino alcohol at C-terminus
Carboxypeptidases enzymatic removal of C-terminus Carboxypeptidase A all except proline, arginine and
lysine Carboxypeptidase B only arginine and lysine Carboxypeptidase C any amino acid care required since rate of removal varies with the
type of amino acid
16
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Peptide Fragments
After determining the amino acid composition and the N & C-terminal amino acids, at least two different sets of protein fragments are needed for sequencing
17
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Why Use Fragments?
Why is the protein broken into fragments? Why isn’t the protein sequenced directly?
The sequencing methods currently available are only accurate for peptides up to about 20-30 amino acids, 60 under ideal conditions
18
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Why 2 Sets of Fragments?
Why can't the entire protein amino acid sequence be determined from a single set of peptide fragments obtained by cleavage with a single reagent?
There’s no way to determine how the fragments are connected with just one set
A second or third set of fragments are used to deduce how the fragments are connected by identification and comparison of overlapping sequnces
19
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Protein Cleavage Reagents
What types of reagents are best suited for preparing these sets of fragments?
Reagents that cleave the protein chain only at a few specific sites forming fragments that are less than 20-30 amino acids in length
20
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Protein Cleavage Reagents
Chemical or enzymatic reagents can be used to prepare protein fragments
The most commonly used reagents are:
cyanogen bromide various enzymes including
trypsin chymotrypsin clostripain Staphylococcal protease various endopeptidases
21
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Cyanogen Bromide
At which amino acid in the protein sequence does the reagent, cyanogen bromide, cleave protein chains?
At internal methionines by reaction with the methionine sulfur as illustrated above
N
C
C
H
2
C
O
N
H
H
H
C
H
2
S
C
H
3
N
C
B
r
N
C
C
H
2
C
O
N
H
H
H
C
H
2
S
+
C
H
3
N
C
N
C
C
H
2
C
O
H
H
S
N
C
C
H
2
O
C
H
3
N
C
C
H
2
C
O
N
H
H
H
C
H
2
S
C
H
3
N
C
B
r
N
C
C
H
2
C
O
N
H
H
H
C
H
2
S
+
C
H
3
N
C
N
C
C
H
2
C
O
H
H
S
N
C
C
H
2
O
C
H
3
22
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Trypsin & Chymotrypsin
Where in the protein sequence do the enzymes, trypsin and chymotrypsin cleave protein chains?
trypsin cleaves at the carboxyl side of amino acids with positively charged side chains such as lysine and arginine
chymotrypsin cleaves at the carboxyl side of amino acids with aromatic side chains such as phenylalanine and tyrosine
23
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Clostripain
Where in the protein sequence does the enzyme, clostripain, cleave?
prefers positively charged amino acids, arginine even more than lysine
narrower specificity than tryptophan which enzyme is likely to produce
larger fragments?
24
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Staphyloccal Protease
Where in the protein sequence does the enzyme, Staphylococcal protease cleave?
carboxyl side of acidic amino acids in phosphate buffer
in acetate or bicarbonate buffer it is more specific and cleaves only glutamic acid
25
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Endopeptidases
The following endopeptidases are less specific than the enzymes metioned above
Pepsin, papain, subtilisin, thermolysin, elastase (papain is the active ingredient in meat
tenderizer, soft contact cleansing solutions, some laundry detergents)
These enzymes are most often used to further reduce the size of large tryptic or chymotryptic fragments
26
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
How are Peptide Fragments Separated?
Usually by column chromatography, often HPLC
Separations are most often based on differences in polarity (reverse phase) or electric charge (ion exchange)
27
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Edman Degradation
Edman degradation is most often used to sequence the peptides
It removes one amino acid from the N-terminal end of the peptide during each cycle of the procedure
The removal of the N-terminal amino acid is accomplished using the reagent, phenylisothiocyanate
28
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Edman Degradation
Pheylisothiocyanate attaches to the N-terminal amino acid
The peptide amino nitrogen atom bonds to the PITC carbon
Sulfur then bonds to the peptide carboxyl carbon breaking the peptide bond
This cyclization forms a pheylthiohydantoin derivative which is removed from the peptide chain by treatment with anhydrous acid
Identified by extraction, treatment with aqueous acid and analysis by chromatography
29
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Edman Degradation
N
C
S
N
H
2
C
C
H
3
C
O
N
H
H
N
H
C
C
H
3
C
O
N
H
H
N
H
C
S
N
H
2
N
N
O
S
C
H
3
H
H
N
C
S
N
H
2
C
C
H
3
C
O
N
H
H
N
H
C
C
H
3
C
O
N
H
H
N
H
C
S
N
H
2
N
N
O
S
C
H
3
H
H
30
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Disulfide Bridges
The location of disulfide bridges can be determined by diagonal electrophoresis
Fragments with intact disulfide bonds are electrophoresed in one dimension
Treated with fumes of performic acid to cleave disulfide bonds
Then electrophoresed in the second dimension
Fragments that had no disulfide bonds will be on the diagonal
Fragments that had disulfide bonds will migrate off diagonal due to altered mobility
31
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Mass Spectroscopy
Used for sequencing peptides Peptides are fragmented in the mass
spectrometer The fragments are identified by their
mass/charge ratio Peptide mixtures can be analyzed
using a temperature gradient The temperature gradient causes
variation in signals corresponding to different peptides
32
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Protein Sequencing by DNA Sequencing
In fact, while you have just learned how to sequece a protein by chemical and enzymatic degradation, protein sequences are now most often determined by translating the corresponding cloned genes
This latter process is usually easier and quicker once the gene corresponding to a given protein has been identified
33
Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing
Sequence Databases
International databases of protein sequences are maintained
Many of these databases are accessible via the internet
Examples: GenBank Protein Identification Resource (PIR) European Molecular Biology Data
Library (EMBL)