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peptides that reflect this unique site distribution (distinct sequence) allow-
ing the matching of the experimental digest with a theoretical digest stored
in databases, thus aiding the identification of a protein (Yates, 1998).
De novo sequencing
Mass fingerprinting is not a very high-resolution method and often fails
when the sample is not pure, protein of interest is not in the database, or
when you need to distinguish related proteins or isoforms. Therefore, it is
still important to get actual sequence information through de novo sequenc-
ing, which can be achieved when powerful mass analyzers, for example,
the quadrupole time of flight (Q-ToF), are used (Fig. 2) (Wood et al .,
2002). The Q-ToF is endowed with the capability to perform tandem MS.
As described previously, this refers to the possibility of using the quadru-
pole as a mass filter allowing the isolation of ions of a desired mass/charge
ratio ( m / z ) and subsequently subjecting these to collisions with inert gas
molecules to cause fragmentation [collision induced dissociation (CID)
or collision activated dissociation (CAD)] (Sleno et al ., 2004). The accel-
erated ions will impact against inert gas molecules (usually argon) result-
ing in an increase in internal energy and inducing the fragmentation of
the ions. The weakest bonds are those along the peptide backbone.
Statistically, the peptide is going to break frequently at the amide bond,
and hence the most abundant fragments that are going to be produced are
the b and y series (Fig. 3), according to the retention of the charge at the
amino or carboxy terminal of the fragments, respectively. Fragments vary
by a discernable mass difference corresponding to known amino acids
from which the sequence can be pieced together.
Post-translational and Chemical Modifications
MS also allows determination of post-translational and chemical modifica-
tions when mass differences not corresponding to any of the essential amino
acids are detected. Typical modifications may include a mass increase of
80 Da for phosphorylation or addition of 16 Da for oxidation. There
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