Quantification of Proteins by iTRAQ - LC-MS/MS in Proteomics: Methods and Applications - page 210

3.3.DataAnalysis

1. To analyse the data, a number of platforms can be used,
but currently the most advanced is the ProteinPilot software
from AB Sciex. To set up a search select 'Identify Proteins'
from the workflow toolbar. Under the 'Data Sets to Process'
window select 'Add' and enter all of the files for a partic-
ular run. Peptide data from all data files/SCX fractions is
summed and averaged in the final results output.
2. In the 'Process Using' window construct a Paragon Method
with all appropriate experimental parameters (alkylation
reagent, enzyme, instrument, etc.) and also any enrichment
used or whether biological modifications need be consid-
ered. These affect the novel PTM discovery tools employed
by the Paragon algorithm ( 18 ) .
3. Run a thorough search for all experiments on complex mix-
tures (a rapid search is more suited to test experiments where
sample is simple and well defined, in our experience). Save
the method.
4. To run a false discovery rate calculation, select the PSPEP
(Proteomics System Performance Evaluation Pipeline)
option.
5. Select where to save the .group file containing the results,
and hit Process. The analysis output gives a ratio for each
reporter vs a 'Reference' (the reference can be changed
under the 'Summary' tab in the results), along with an error
factor and p value. The first check should be a QC for the
run, determining the efficiency of the iTRAQ labelling reac-
tions. All lysine side chains should be labelled, with around
90% of peptide N-termini also labelled.
6. The point at which a change is deemed 'significant' can be
calculated by including replicate samples in the experiment
and determining what settings (ratio/ p value) a user-defined
proportion of the ratios in this comparison (should all be
1:1) exceed ( see Note 6 ). Typical values in our lab for almost
all experiments are ratios greater than 1.25 or less than 0.8
( see Note 7 ).

4. Notes

1. All solutions should be made in HPLC-grade water.
2. The iTRAQ reagent tags are extremely unstable in water.
It is important to prevent condensation forming in unused
tubes, so ensure that a kit is removed from the freezer,
reagents taken, and the kit returned as quickly as possible.
The reaction is left for 1 h essentially to ensure that all