Biology Reference
In-Depth Information
a protein using peptide mapping requires digest-
ing the protein into peptides prior to MS analysis.
Althoughmost peptide mapping experiments use
trypsin to produce peptides, other enzymes
(e.g., Lys-C, Glu-C, etc.) can be used, depending
on the experimental requirement. Once digested,
the molecular weight of the peptides are acq-
uired.
1
These experimental masses of the peptides
are compared to masses generated from an in sil-
ico digest of proteins or translated nucleic acid
sequences contained within a database. A protein
will be identi
(
Figure 1
). Because the accuracy between the
experimental and in silico masses is critical to
obtaining the correct protein identi
cation, it is
best to acquire the peptide map on a high mass
measurement accuracy instrument,
such as
atime-of-
guration. The greater
the number of matches between the experimental
and database peptide masses, the higher the
con
ight (TOF) con
cation.
Beyond the availability of a database contain-
ing the necessary protein sequences, software is
also required to turn the rawMS data into protein
identi
dence in the protein
'
sidenti
ed if several of the experimental
masses match those for a speci
c protein in the
cations. Fortunately, there are many freely
available
database within a
certain mass
tolerance
software programs
for
analyzing
Experimental
tryptic peptides
Experimental
peptide map
Experimental protein
EDHGILGGK
TYGHLR
AVLEMMFK
LILLVFTYK
YHILR
YECCGIDSQTK
EDDFLQSGILPDR
ETGK
AVVATSL
EDHGILGGKTYGHLRAVLEMMFK
LILLVFTYKYHILRYECCGIDSQTKE
DDFLQSGILPDRETGKAVVATSL
m/z
In silico protein
sequence
In silico
tryptic peptides
Theoretical
peptide map
EDHGILGGK
TYGHLR
AVLEMMFK
LILLVFTYK
YHILR
YECCGIDSQTK
EDDFLQSGILPDR
ETGK
AVVATSL
EDHGILGGKTYGHLRAVLEMMFK
LILLVFTYKYHILRYECCGIDSQTKE
DDFLQSGILPDRETGKAVVATS
FIGURE 1
Peptide mapping for protein identification using mass spectrometry (MS). In the first step, the protein is
proteolytically digested (usually with trypsin) and the experimental masses of the peptides are measured using MS. The
sequences of the proteins within the selected database are digested in silico based on the speci
city of the enzyme used. The
masses of these peptides are calculated and theoretical mass spectra are constructed. The correct protein is identi
ed based on
the closest match between the experimental and theoretical mass spectra.
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