Biology Reference
In-Depth Information
of a complicated protein mixture is estimated based on the quan-
tifi cation of one reference protein or peptide mixture.
5. The amount
x
is dependent on the volume that has been taken
to have 50
g of proteins in
step 1
.
6. Adding TFA will acidify the sample and will stop the enzymatic
digestion.
7. Depending on the type of mass spectrometer (speed) those
settings can be adjusted to select more or less peaks.
8. The sequences of the different isoforms of keratin and trypsin
are present in our database to avoid that those peptides are
selected to perform de novo analysis, where they could con-
tribute to false positive identifi cations.
9. The sequence specifi c database should not contain duplicate
sequences in order to avoid additional redundancy. The data-
base can be made nonredundant via the program CD hit.
http://cd-hit.org
[
16
].
10. In this step the peptides belonging to different partial protein
sequences of the EST database are compared with known pro-
tein sequences. EST's belonging to the same protein are clus-
tered to a full protein sequence (Fig.
3
). This step is for orphan
species the only way to reconstruct the proteins based on the
measured peptides.
μ
Acknowledgment
Dr. Jan Cordewener is gratefully acknowledged for technical
assistance.
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