Protein sequencing

Separation and quantitation
The amino acids can be separated by
ion-exchange chromatography then
derivatized to facilitate their detection.
More commonly, the amino acids are
derivatized then resolved by reversed
phase HPLC.
An example of the ion-exchange
chromatography is given by the NTRC
using sulfonated polystyrene as a matrix,
adding the amino acids in acid solution
and passing a buffer of steadily

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increasing pH through the column.
Amino acids are eluted when the pH
reaches their respective isoelectric
points. Once the amino acids have been
separated, their respective quantities are
determined by adding a reagent that will
form a coloured derivative. If the
amounts of amino acids are in excess of
10 nmol, ninhydrin can be used for this; it
gives a yellow colour when reacted with
proline, and a vivid purple with other
amino acids. The concentration of amino
acid is proportional to the absorbance of
the resulting solution. With very small
quantities, down to 10 pmol, fluorescent
derivatives can be formed using reagents

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such as ortho-phthaldehyde (OPA) or
fluorescamine.
Pre-column derivatization may use the
Edman reagent to produce a derivative
that is detected by UV light. Greater
sensitivity is achieved using a reagent
that generates a fluorescent derivative.
The derivatized amino acids are
subjected to reversed phase
chromatography, typically using a C8 or
C18 silica column and an optimised
elution gradient. The eluting amino acids
are detected using a UV or fluorescence
detector and the peak areas compared
with those for derivatised standards in

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order to quantify each amino acid in the
sample.
Determining which amino acid forms the
N-terminus of a peptide chain is useful
for two reasons: to aid the ordering of
individual peptide fragments' sequences
N-terminal amino acid
analysis
Sanger's method of peptide end-group analysis: A derivatization of N-terminal end with 
Sanger's reagent
 (DNFB), B
total acid hydrolysis of the dinitrophenyl peptide

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into a whole chain, and because the first
round of Edman degradation is often
contaminated by impurities and therefore
does not give an accurate determination
of the N-terminal amino acid. A
generalised method for N-terminal amino
acid analysis follows:
1. React the peptide with a reagent
that will selectively label the
terminal amino acid.
2. Hydrolyse the protein.
3. Determine the amino acid by
chromatography and comparison
with standards.

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There are many different reagents which
can be used to label terminal amino
acids. They all react with amine groups
and will therefore also bind to amine
groups in the side chains of amino acids
such as lysine - for this reason it is
necessary to be careful in interpreting
chromatograms to ensure that the right
spot is chosen. Two of the more
common reagents are Sanger's reagent
(1-fluoro-2,4-dinitrobenzene) and dansyl
derivatives such as dansyl chloride.
Phenylisothiocyanate, the reagent for the
Edman degradation, can also be used.
The same questions apply here as in the
determination of amino acid
composition, with the exception that no

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stain is needed, as the reagents produce
coloured derivatives and only qualitative
analysis is required. So the amino acid
does not have to be eluted from the
chromatography column, just compared
with a standard. Another consideration to
take into account is that, since any amine
groups will have reacted with the
labelling reagent, ion exchange
chromatography cannot be used, and
thin-layer chromatography or high-
pressure liquid chromatography should
be used instead.

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The number of methods available for C-
terminal amino acid analysis is much
smaller than the number of available
methods of N-terminal analysis. The
most common method is to add
carboxypeptidases to a solution of the
protein, take samples at regular intervals,
and determine the terminal amino acid
by analysing a plot of amino acid
concentrations against time. This
method will be very useful in the case of
polypeptides and protein-blocked N
termini. C-terminal sequencing would
greatly help in verifying the primary
C-terminal amino acid
analysis

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structures of proteins predicted from
DNA sequences and to detect any
posttranslational processing of gene
products from known codon sequences.
The Edman degradation is a very
important reaction for protein
sequencing, because it allows the
ordered amino acid composition of a
protein to be discovered. Automated
Edman sequencers are now in
widespread use, and are able to
sequence peptides up to approximately
50 amino acids long. A reaction scheme
for sequencing a protein by the Edman
Edman degradation

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degradation follows; some of the steps
are elaborated on subsequently.
1. Break any disulfide bridges in the
protein with a reducing agent like 2-
mercaptoethanol. A protecting
group such as iodoacetic acid may
be necessary to prevent the bonds
from re-forming.
2. Separate and purify the individual
chains of the protein complex, if
there are more than one.
3. Determine the amino acid
composition of each chain.
4. Determine the terminal amino acids
of each chain.

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5. Break each chain into fragments
under 50 amino acids long.
. Separate and purify the fragments.
7. Determine the sequence of each
fragment.
. Repeat with a different pattern of
cleavage.
9. Construct the sequence of the
overall protein.