Protein sequencing is a technique to determine the amino acid sequence of a
protein, as well as which conformation the protein adopts and the extent to
which it is complexed with any non-peptide molecules. Discovering the
structures and functions of proteins in living organisms is an important tool
for understanding cellular processes, and allows drugs that target specific
metabolic pathways to be invented more easily.
The two major direct methods of protein sequencing are mass spectrometry and the
Edman degradation reaction. It is also possible to generate an amino acid
sequence from the DNA or mRNA sequence encoding the protein, if this is known.
However, there are many other reactions that can be used to gain more limited
information about protein sequences and can be used as preliminaries to the
aforementioned methods of sequencing or to overcome specific inadequacies within
them. Protein sequencer
A protein sequencer is a machine that is used to determine the sequence of amino
acids in a protein. They work by tagging and removing one
amino acid at a time, which is analysed and identified. This is done
repetitively for the whole polypeptide, until the whole sequence is established.
This method has generally been replaced by nucleic acid technology, and it is
often easier to identify the sequence of a protein by looking at the DNA that
codes for it. Determining amino acid composition
It is often desirable to know the unordered amino acid composition of a
protein prior to attempting to find the ordered sequence, as this knowledge can
be used to facilitate the discovery of errors in the sequencing process or to
distinguish between ambiguous results. Knowledge of the frequency of certain
amino acids may also be used to choose which protease to use for digestion of
the protein. A generalized method often referred to as amino acid analysis for
determining amino acid frequency is as follows:
Hydrolyse a known quantity of protein into its constituent amino acids.
Separate and quantify the amino acids in some way.
= Hydrolysis = Hydrolysis is done by heating a sample
of the protein in 6 M hydrochloric acid to 100–110 °C for 24 hours or longer.
Proteins with many bulky hydrophobic groups may require longer heating
periods. However, these conditions are so vigorous that some amino acids are
degraded. To circumvent this problem, Biochemistry Online suggests heating
separate samples for different times, analysing each resulting solution, and
extrapolating back to zero hydrolysis time. Rastall suggests a variety of
reagents to prevent or reduce degradation, such as thiol reagents or
phenol to protect tryptophan and tyrosine from attack by chlorine, and
pre-oxidising cysteine. He also suggests measuring the quantity of ammonia
evolved to determine the extent of amide hydrolysis.
= Separation = The amino acids can be separated by
ion-exchange chromatography or hydrophobic interaction chromatography.
An example of the former is given by the NTRC using sulfonated polystyrene as a
matrix, adding the amino acids in acid solution and passing a buffer of
steadily increasing pH through the column. Amino acids will be eluted when
the pH reaches their respective isoelectric points. The latter technique
may be employed through the use of reversed phase chromatography. Many
commercially available C8 and C18 silica columns have demonstrated successful
separation of amino acids in solution in less than 40 minutes through the use of
an optimised elution gradient. = Quantitative analysis =
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, fluorescamine can be used as a marker: This forms a fluorescent
derivative on reacting with an amino acid.
N-terminal amino acid analysis 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 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:
React the peptide with a reagent that will selectively label the terminal
amino acid. Hydrolyse the protein.
Determine the amino acid by chromatography and comparison with
standards. 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 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 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.
C-terminal amino acid analysis 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 structures of proteins predicted from
DNA sequences and to detect any postranslational processing of gene
products from known codon sequences. Edman degradation
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 degradation
follows; some of the steps are elaborated on subsequently.
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. Separate and purify the individual
chains of the protein complex, if there are more than one.
Determine the amino acid composition of each chain.
Determine the terminal amino acids of each chain.
Break each chain into fragments under 50 amino acids long.
Separate and purify the fragments. Determine the sequence of each fragment.
Repeat with a different pattern of cleavage.
Construct the sequence of the overall protein.
= Digestion into peptide fragments = Peptides longer than about 50-70 amino
acids long cannot be sequenced reliably by the Edman degradation. Because of
this, long protein chains need to be broken up into small fragments that can
then be sequenced individually. Digestion is done either by
endopeptidases such as trypsin or pepsin or by chemical reagents such as cyanogen
bromide. Different enzymes give different cleavage patterns, and the
overlap between fragments can be used to construct an overall sequence.
= The Edman degradation reaction = The peptide to be sequenced is adsorbed
onto a solid surface. One common substrate is glass fibre coated with
polybrene, a cationic polymer. The Edman reagent, phenylisothiocyanate, is added
to the adsorbed peptide, together with a mildly basic buffer solution of 12%
trimethylamine. This reacts with the amine group of the N-terminal amino
acid. The terminal amino acid can then be
selectively detached by the addition of anhydrous acid. The derivative then
isomerises to give a substituted phenylthiohydantoin, which can be washed
off and identified by chromatography, and the cycle can be repeated. The
efficiency of each step is about 98%, which allows about 50 amino acids to be
reliably determined. = Limitations of the Edman degradation =
Because the Edman degradation proceeds from the N-terminus of the protein, it
will not work if the N-terminal amino acid has been chemically modified or if
it is concealed within the body of the protein. It also requires the use of
either guesswork or a separate procedure to determine the positions of disulfide
bridges. Mass spectrometry
The other major direct method by which the sequence of a protein can be
determined is mass spectrometry. This method has been gaining popularity in
recent years as new techniques and increasing computing power have
facilitated it. Mass spectrometry can, in principle, sequence any size of
protein, but the problem becomes computationally more difficult as the
size increases. Peptides are also easier to prepare for mass spectrometry than
whole proteins, because they are more soluble. One method of delivering the
peptides to the spectrometer is electrospray ionization, for which John
Bennett Fenn won the Nobel Prize in Chemistry in 2002. The protein is
digested by an endoprotease, and the resulting solution is passed through a
high-pressure liquid chromatography column. At the end of this column, the
solution is sprayed out of a narrow nozzle charged to a high positive
potential into the mass spectrometer. The charge on the droplets causes them
to fragment until only single ions remain. The peptides are then fragmented
and the mass-to-charge ratios of the fragments measured.. The mass spectrum
is analysed by computer and often compared against a database of
previously sequenced proteins in order to determine the sequences of the
fragments. This process is then repeated with a different digestion enzyme, and
the overlaps in the sequences are used to construct a sequence for the protein.
Predicting protein sequence from DNA/RNA sequences
In organisms that do not have introns the amino acid sequence of a protein can
also be determined indirectly from the mRNA or the DNA that codes for the
protein. If the sequence of the gene is already known, then this is all very
easy. However, it is rare that the DNA sequence of a newly isolated protein
will be known, and, so, if this method is to be used, it has to be found in
some way. One way that this can be done is to sequence a short section, perhaps
15 amino acids long, of the protein by one of the above methods, and then use
this sequence to generate a complementary marker for the protein's
RNA. This can then be used to isolate the mRNA coding for the protein, which
can then be replicated in a polymerase chain reaction to yield a significant
amount of DNA, which can then be sequenced relatively easily. The amino
acid sequence of the protein can then be deduced from this. However, it is
necessary to take into account the possibility of amino acids being removed
after the mRNA has been translated. Bioinformatics tools for sequencing
Bioinformatics tools exist that translate nucleic acid sequences into
their corresponding polypeptide chain. Such tool takes an input of a nucleic
acid sequence and will produce the corresponding amino acid sequence based
on the selected settings. Another option is to use a
bioinformatics tool that takes an amino acid sequence and back-translates it
into the most likely nucleic acid sequence.
See also Matthias Mann
References Further reading