Protein sequencing

Proteolytic digests
A general scheme for protein
identification is described.
[4][5]
1. The POI is isolated, typically by SDS-
PAGE or chromatography.
2. The isolated POI may be chemically
modified to stabilise Cysteine

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residues (e.g. S-amidomethylation
or S-carboxymethylation).
3. The POI is digested with a specific
protease to generate peptides.
Trypsin, which cleaves selectively
on the C-terminal side of Lysine or
Arginine residues, is the most
commonly used protease. Its
advantages include i) the frequency
of Lys and Arg residues in proteins,
ii) the high specificity of the
enzyme, iii) the stability of the
enzyme and iv) the suitability of
tryptic peptides for mass
spectrometry.

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4. The peptides may be desalted to
remove ionizable contaminants and
subjected to MALDI-TOF mass
spectrometry. Direct measurement
of the masses of the peptides may
provide sufficient information to
identify the protein (see Peptide
mass fingerprinting) but further
fragmentation of the peptides inside
the mass spectrometer is often
used to gain information about the
peptides’ sequences. Alternatively,
peptides may be desalted and
separated by reversed phase HPLC
and introduced into a mass
spectrometer via an ESI source. LC-
ESI-MS may provide more

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information than MALDI-MS for
protein identification but uses more
instrument time.
5. Depending on the type of mass
spectrometer, fragmentation of
peptide ions may occur via a variety
of mechanisms such as collision-
induced dissociation (CID) or post-
source decay (PSD). In each case,
the pattern of fragment ions of a
peptide provides information about
its sequence.
. Information including the measured
mass of the putative peptide ions
and those of their fragment ions is
then matched against calculated

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mass values from the conceptual
(in-silico) proteolysis and
fragmentation of databases of
protein sequences. A successful
match will be found if its score
exceeds a threshold based on the
analysis parameters. Even if the
actual protein is not represented in
the database, error-tolerant
matching allows for the putative
identification of a protein based on
similarity to homologous proteins. A
variety of software packages are
available to perform this analysis.
7. Software packages usually generate
a report showing the identity
(accession code) of each identified

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protein, its matching score, and
provide a measure of the relative
strength of the matching where
multiple proteins are identified.
. A diagram of the matched peptides
on the sequence of the identified
protein is often used to show the
sequence coverage (% of the protein
detected as peptides). Where the
POI is thought to be significantly
smaller than the matched protein,
the diagram may suggest whether
the POI is an N- or C-terminal
fragment of the identified protein.

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