Biology Reference
In-Depth Information
peptide charge +4 (>8, >70 %, >2), peptide charge +5 (>12,
>70 %, >2), and peptide charge +2 (>6, >90 %, >1). Validation
is edited to fi lter out those peptides with equal scoring in the
direct and reversed search and
mass (|observed − expected|)
>15 ppm. Validated fi les are summarized in the protein mode
to assemble peptides into proteins in order to generate the
minimal protein list that best explains the matched peptides.
The false-discovery rate (FDR) for automated interpretation of
the MS/MS spectra using the above criteria is estimated by
searching all spectra passing the quality fi lter against the
same database with all of the protein sequences reversed (
see
Note 15
).
Δ
1. After protein identifi cation searches, a list for all peptides and
proteins with the corresponding quantitative data and
p
-value
is obtained. The log average ratio (log AR) in each iTRAQ
ratio (i.e., in a 4-plex experiment the ratios are 115/114,
116/114, and 117/114) for all the peptides identifi ed in the
experiment should be zero. Otherwise, a correction factor of
−log AR determined for each couple of labels has to be applied
to the ratio data (
see
Note 16
).
2. The confi dent iTRAQ ratios should be selected according to a
good
p
-value, at least a
p
-value <0.05 for at least one ratio of
the pair tags.
3. It is useful to build a list with all the proteins passing a restricted
ratio considered as “signifi cant” change. This list of proteins
can be used for global bioinformatics analyses, e.g., pathway
analysis (Blas2GO, Cytoscape, MapMan). Another list of pro-
teins can be performed by inclusion of possible quantifi ed
proteins such as those having a nonsignifi cant ratio, a single
peptide quantifi ed, or higher quantifi cation ratios but elevated
p
-values. This second list typically helps to interpret the data
into a biological context. Those proteins with relevant changes
for the biological system under study can be validated by other
approaches such as western blot (
see
Note 17
).
3.5 Data Analysis
4
Notes
1. iTRAQ kit provides all the chemicals needed for iTRAQ label-
ing protocol just in enough amount. When TEAB buffer is
needed in larger amounts to perform this protocol, a TEAB
buffer from different commercial supplier apart from iTRAQ
kit can be used. Extreme caution should be taken to manipulate
iTRAQ kit in order to prevent hydrolysis of the tags. For this
purpose, remove the tubes needed form the kit and return to
the freezer immediately. Also, additional precaution can be
