Biology Reference
In-Depth Information
9. Create a pivot table and fi lter the proteins with minimum one
peptide with ladder score >50. Discard the rest of the proteins
and create a new fi le, i.e., database fi le.
10. Import the new Excel fi le in Cytoscape, i.e., database network
(Fig.
2
).
11. Sort from the original fi le the data according to
m
/
z
(ascending)
and ladder score (descending) and keep the best hit per
m
/
z
.
Remove the redundancy in the peptides by fi ltering for
unique
m
/
z
.
12. Filter all the peptides with a ladder score greater than 50 and
discard peptides assigned to keratin or trypsin.
13. Submit the high quality peptide characterizations of mixed ori-
gin (database, automod, de novo) to MS homology taxonomy
Green plants (freely available at the Web site of the University
of California (
http://www.ucsf.edu/
)). Set the number of
allowed amino acid substitutions for the MS Homology search
as “the length of the peptide divided by 5” (
see
Note 10
).
14. Save the results as a new Excel fi le, i.e., MShomology fi le.
15. Sort from the MShomology fi le the data according to sequence
(ascending) and peptide score (descending) and protein score
(descending), keep the best hit per sequence. Remove the
redundancy in the peptides by fi ltering for unique sequences.
16. Import the new nonredundant Excel fi le in Cytoscape, i.e., MS
homology network.
17. Merge both networks in Cytoscape to create a fi nal network,
i.e., union network.
18. Give a different layout to the nodes of peptides and proteins.
19. Give a different color to the different interactions between
the peptides and proteins correlated to the confi dence level
of identifi cation (ladder score, MS homology score) (
see
Figs.
2
,
3
and
4
).
6
Notes
1. Phenol is toxic and needs to be handled with great care. Wear
gloves, work in a dedicated room. Use only under the hood and
make sure that the waste (tips, pipettes, tubes) is disposed safely.
2. The grinding process is very important. Do not stop grinding
until the power is very fi ne and homogeneous.
3. Depending on the protein concentration of the particular tis-
sue, more or less starting material is needed.
4. For a quantitative peptide based approach, the quantifi cation of
the proteins and peptides is very important but challenging. Each
quantifi cation method has its limitations since the concentration
