Biology Reference
In-Depth Information
2. In SEPro's control panel, indicate the location of the directory
containing the SQTs from one biological replicate and the FASTA
format database used in SEQUEST searches (
see
Note 1
).
3. In SEPro's parameters, select the features that you want to be
used for calculation of the Bayesian score. We strongly advise
to use all parameters for data processing. If data was acquired
in a low-resolution mass spectrometer, uncheck “Delta Mass”.
4. Preprocessing filters will be used to speed up the process, leaving
fewer PSMs for processing. We recommend removing peptides
with a plus one charge state, PSMs with primary score lower
than 1, and consider only peptides longer than six amino acids.
If MS data was acquired with a high-resolution mass spectrom-
eter, “Delta mass” option equal to 10 ppm is advisable.
5. In the “Acceptable False Discovery Rate” box, type percentage
values that obey the logic of the three-tier approach in which a
large number of spectra correlates with a lower number of pep-
tides that in turn map to fewer proteins (
see
Notes 2
and
3
).
6. Post-processing filters are used after the three-tier filtering is
applied. Although being a strong-quality filter, for general
spectral counting projects we recommend to consider only
proteins having a minimum of two spectral counts and two
peptides (
see
Note 4
). As mentioned before, “DeltaMass” fea-
ture should only be used if high-resolution instrument was
used for MS data acquisition.
7. The “Similar Proteins” panel should enable the elimination of
internal proteins.
8. In the “General Panel” choose the enzyme used for in vivo
and in silico protein digestion. Check the radio buttons for
grouping PSMs by charge state and number of enzymatic ter-
mini (
see
Note 5
).
9. For classical forward-decoy databases, indicate the tag used to
identify decoy protein sequences in the FASTA database. This
tag should be typed in the “Labeled Decoy Tag” box (
see
Note 6
).
10. After all parameters are adjusted, we recommend saving the
parameters as default for future processing.
11. To start the filtering process, go back to “Control” panel and
click “GO!”. A real-time follow-up can be achieved by clicking
on “Followup” panel.
12. Upon completion, go to “ResultBrowser” panel and check the
number of retained spectra, peptides, and protein. Also, keep
track of the post-processing spectra, peptide, and protein FDR
for future comparisons within biological replicates.
13. Save SEPro result.
