How To Sequence A Protein: W. Robert Midden Department of Chemistry Bowling Green State University

How To Sequence
A Protein
W. Robert Midden
Department of Chemistry
Bowling Green State University
Copyright1998W.R.Midden
BowlingGreenStateUniversity ProteinSequencing
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Protein Sequencing
Preliminary 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
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2-Mercaptoethanol
OO OO OO OO
HH HH
CC NN CC CC CC NN CC CC
HH HH
CH
CH22 CH
CH
2
2
SS HH
SS CH2 CH
CH 2 OH
2 CH2 OH SH
SH SS CH2 CH
CH 2 OH
2 CH2 OH
SS HS CH SH SS CH2 CH2 OH
HS 2 CH
CH 2 OH SH CH2 CH2 OH
2 CH2 OH
OO CH
CH22 OO OO CH2 O
CH2 O
HH HH HH HH
2-mercaptoethanol reduces disulfides to sulfhydryls

But the sulfhydryls are easily oxidized back to the disulfide
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Preventing Reversal
OO OO OO OO
HH HH
HH HH
CH OO CH
CH OO
CH
2
2
2
2
SH II CH2 CC
SS CH2 CC
CH
SH CH2 2
OO OO
OO OO OO OO
HH HH
HH HH
CH CH
CH
CH
2
2
2
2
SH HH
2CC CH SS HH
2CC CH2 CC NN
SH 2 CH CC NN
2 CH2
to prevent oxidation the suflhydryls are alkylated with

iodoacetic acid or acrylonitrile
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Perfomic Acid
OO OO OO OO
HH HH
HH HH
CH CH
CH22
CH22
OO
SS SO
SO3-3-
HH CC OO OO HH
SS SO
SO3-3-
OO CH OO CH
CH22 OO
CH22 OO
HH HH HH HH
Performic acid oxidizes cysteine to negatively charged cysteic

acid
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Reversal Prevented
OO OO
HH
CC NN CC CC
HH
CH
CH22 SO
SO3-3-
SO
SO3-3- OO CH
CH22 OO
CC NN CC CC
HH HH
The repulsion of the negatively charged SO3- groups

prevents reformation of the disulfide bond
Therefore alkylation is not necessary with performic acid
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Protein Sequencing
Preliminary 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
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Determining Amino
Acid 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
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Determining
Protein 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
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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
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Determining the
N-Terminal Amino Acid
The N-terminal amino acid is determined using
either chemical reagents or enzymes
Chemical reagents include:
Sangers reagent
dansyl chloride
Edman Degradation
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Determining the
Sangers reagent
Treat with dinitrofluorobenzene to
OO
+
OO form a dinitrophenyl (DNP)
N+
N derivative of the amino-terminal
amino acid
Acid hydrolysis
OO
Extract the DNP-derivative from the
+
NN+ acid hydrolysate with organic
solvent
FF OO Identify the DNP-derivative by
chromatography and comparison
with standards
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Determining the
HH3CC CH
CH33
Dansyl chloride
3
NN (dimethylaminonaphthylenesulfonyl
chloride)
Forms a highly fluorescent derivative
of the amino-terminal amino acid
Identified by chromatography and
fluorescence detection after acid
OO SS OO hydrolysis
Highly senstive
Cl
Cl
Best choice when the amount of
protein is limited
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Determining the
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
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Determining the
C-Terminal Amino Acid
Hydrazinolysis
hydrazine at 100C 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
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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
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Why Use Fragments?
Why is the protein broken into fragments? Why

isnt 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
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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?
Theres 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
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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
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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
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Cyanogen Bromide
CH
CH33 Br
Br CH
CH33 HHC
3 C
3
+
SS CC SS+ CC NN SS CC NN
CH NN CH
CH22 CH
CH22 CH22
CH CH
CH22 OO
CH22 OO HH2CC
2
OO
NN CC CC NN NN CC CC NN NN CC CC
OO
HH HH HH HH HH HH HH HH
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
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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
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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?
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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
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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
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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)
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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
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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
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Edman Degradation
NN
NH
NH
CC NN
OO SS
CC SS
HH
SS
HH
NN NN
HH
2N
2N HH
3CC HH
HH CC CH
CH
3 3
3
HH CC CH
CH
3
3
CC OO
CC OO HH
2N
2N
NH
NH
NH
NH
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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
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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
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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
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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)
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How To Sequence

2-mercaptoethanol reduces disulfides to sulfhydryls

to prevent oxidation the suflhydryls are alkylated with

Performic acid oxidizes cysteine to negatively charged cysteic

The repulsion of the negatively charged SO3- groups

After determining the amino acid composition

Why is the protein broken into fragments? Why

At which amino acid in the protein sequence does the

Where in the protein sequence do the enzymes,

Where in the protein sequence does the

Where in the protein sequence does the

The following endopeptidases are less specific

Edman degradation is most often used to

Pheylisothiocyanate attaches to the N-terminal

The location of disulfide bridges can be

Used for sequencing peptides

International databases of protein sequences

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