Biology Reference
In-Depth Information
display versus wild type [
18
] has been reported. iTRAQ technology
has expanded as a universal tool with unlimited possibilities only
restricted by the experimental design needed to resolve specifi c
biological questions. Among all the possible applications of
iTRAQ is its use as a preferential technique to quantify the phos-
phorylation status of cells [
19
-
21
] which is based on the retention
and preservation in the labeled peptides of other important struc-
tural features such as posttranslational modifi cations. A brilliant
application is the use of iTRAQ for topological localization of
organelle proteins which is known as the LOPIT approach [
22
].
Summarizing, iTRAQ is currently one of the most robust tech-
niques applicable to quantitative proteomics, though some
inherent characteristics of this technique may affect the accuracy of
the protein quantitative ratios measured, including technical,
experimental, and biological variability [
23
]. Independent analysis
of at least two biological replicates is advisable in iTRAQ analyses.
In those cases where sample limitation or fi nancial constraints
cause a problem, it is possible to use a sample pooling strategy
[
24
]. However, it should be pointed out that the iTRAQ
technique offers remarkable advantages compared to other quan-
titative proteomic techniques such as cleavable isotope-coded
affi nity tags (cICAT) or differential in-gel electrophoresis (DIGE)
in regard to detection limit [
25
] having a good dynamic range
detecting up to 24-fold changes [
2
] and a great potential to
identify and quantify low-abundance proteins [
26
] which makes
this technique a good promise having a great potential in dis-
covery studies.
2
Materials
1. 0.5 M stock solution of triethylammonium bicarbonate
(TEAB).
2. 2 % (w/v) sodium dodecyl sulfate (SDS) dissolved in 0.5 M
TEAB.
3. iTRAQ reagent labeling kit (Life Technologies). Store at
−20 °C. Extremely susceptible to hydrolysis.
4. 50 mM stock solution of Tris-(2-carboxyethyl) phosphine
(TCEP).
5. 200 mM Methylmethanethiosulfate (MMTS) dissolved in
isopropanol.
6. Trypsin mass spectrometric grade.
2.1 Sample
Preparation and
Labeling ( See Note 1 )
2.2 Sample
Pre-fractionation
1. SCX chromatography buffer A (SCX-A): 10 mM KH
2
PO
4
,
20 % acetonitrile (ACN), pH 2.7, made in Milli-Q water or
HPLC-grade water.
