Protein sequencing

De novo sequencing
The pattern of fragmentation of a peptide
allows for direct determination of its
sequence by de novo sequencing. This
sequence may be used to match
databases of protein sequences or to
investigate post-translational or chemical
modifications. It may provide additional
evidence for protein identifications
performed as above.
N- and C-termini
The peptides matched during protein
identification do not necessarily include
the N- or C-termini predicted for the

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matched protein. This may result from
the N- or C-terminal peptides being
difficult to identify by MS (e.g. being
either too short or too long), being post-
translationally modified (e.g. N-terminal
acetylation) or genuinely differing from
the prediction. Post-translational
modifications or truncated termini may
be identified by closer examination of the
data (i.e. de novo sequencing). A repeat
digest using a protease of different
specificity may also be useful.
Post-translational modifications
Whilst detailed comparison of the MS
data with predictions based on the

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known protein sequence may be used to
define post-translational modifications,
targeted approaches to data acquisition
may also be used. For instance, specific
enrichment of phosphopeptides may
assist in identifying phosphorylation
sites in a protein. Alternative methods of
peptide fragmentation in the mass
spectrometer, such as ETD or ECD, may
give complementary sequence
information.
Whole-mass determination
The protein’s whole mass is the sum of
the masses of its amino-acid residues
plus the mass of a water molecule and

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adjusted for any post-translational
modifications. Although proteins ionize
less well than the peptides derived from
them, a protein in solution may be able to
be subjected to ESI-MS and its mass
measured to an accuracy of 1 part in
20,000 or better. This is often sufficient
to confirm the termini (thus that the
protein’s measured mass matches that
predicted from its sequence) and infer
the presence or absence of many post-
translational modifications.