Biology Reference
In-Depth Information
taken storing the kit inside a bag containing silica gel in the
freezer.
2. The success for any proteomic experiment including a gel-free
approach such as iTRAQ strongly depends on the quality of
the protein extracts and on the quality of the solutions.
Accordingly, special attention should be paid to optimize a
high-quality protein extraction protocol. In this regard, pro-
tein extraction from plant material is well suited in a phenol-
based protocol [
28
,
29
]. All the chemicals and buffers should
be prepared using HPLC-grade water, reducing the potential
occurrence of contaminants and favoring peptide detection
by mass spectrometry.
3. Protein pellets could be diffi cult to resuspend in TEAB buffer
alone, so alternatively we use this buffer to solubilize protein
extracts although having in mind that higher concentration
of urea (>1 M) or SDS (>0.05 %) should be avoided during
trypsin digestion.
4. The protein pellet should be resuspended in the minimal
volume of iTRAQ dissolution buffer. The protocol described
is optimized up to 50
L although for higher volumes it has
to be scaled. Keep a ratio of 10
μ
μ
L iTRAQ dissolution buffer
L SDS. Incubate sample for solubilization overnight at
4 °C and/or incubate at 60 °C for 30 min. From this step
onwards do not use a sonication probe to resuspend the pro-
tein sample or any instrument implying sample losses.
5. It is important to use a trypsin of high quality, a mass spectro-
metric grade trypsin. The reason is that modifi ed trypsin is
treated to avoid auto-proteolysis, a process generating frag-
ments that can interfere with protein sequencing. In addition,
auto-proteolysis can result in the generation of pseudotrypsin,
which has been shown to exhibit chymotrypsin-like specifi city.
The vials containing trypsin are resuspended in 100 mM of
iTRAQ dissolution buffer mixing with a pipette, and the con-
tent is pooled in order to add the same volume of trypsin solu-
tion to each sample. For 20
per 1
μ
μ
g vials, resuspend in 250
μ
L and
L to ensure that the same volume is
added to each sample. Trypsin digestion is a critical step in the
iTRAQ protocol and should be performed under equal condi-
tions for all the samples in the same experiment.
6. A critical step for the iTRAQ protocol success is trypsin diges-
tion. So, a double digestion is performed to ensure a complete
and exhaustive protein digestion.
7. Digested protein is evaporated frozen under vacuum to avoid
sample losses.
8. Add iTRAQ reagents to one sample at a time, and vortex
immediately. The iTRAQ reagent tags are extremely unstable
add to each sample 120
μ
