Protein sequencing

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Protein sequencing
Protein sequencing is the practical
process of determining the amino acid
sequence of all or part of a protein or
peptide. This may serve to identify the
protein or characterize its post-
translational modifications. Typically,
partial sequencing of a protein provides
sufficient information (one or more
sequence tags) to identify it with
reference to databases of protein

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sequences derived from the conceptual
translation of genes.
The two major direct methods of protein
sequencing are mass spectrometry and
Edman degradation using a protein
sequenator (sequencer). Mass
spectrometry methods are now the most
widely used for protein sequencing and
identification but Edman degradation
Using a Beckman-Spinco Protein-Peptide Sequencer, 1970

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remains a valuable tool for characterizing
a protein's N-terminus.
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. The misincorporation of low
levels of non-standard amino acids (e.g.
Determining amino acid
composition

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norleucine) into proteins may also be
determined.
[1]
A generalized method
often referred to as amino acid analysis
[2]
for determining amino acid frequency is
as follows:
1. Hydrolyse a known quantity of
protein into its constituent amino
acids.
2. 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

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groups may require longer heating
periods. However, these conditions are
so vigorous that some amino acids
(serine, threonine, tyrosine, tryptophan,
glutamine, and cysteine) 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

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the quantity of ammonia evolved to
determine the extent of amide hydrolysis.