Biology Reference
In-Depth Information
3.8
MS Analysis
1. Load iTRAQ-labeled peptides on the column (75
m internal
diameter, 5 cm; HiQ sil C-18 W-3, KYA TECH Corporation,
Tokyo, Japan) using a DiNa-A auto injection system (KYA
TECH).
2. Elute peptides with a linear gradient from 0 to 33 % B for
240 min, 33 to 100 % B for 10 min, and back to 0 % B for
15 min at a fl ow rate of 300 nL/min. Eluting buffers are 0.1 %
FA and 5 % ACN (A) and 0.1 % FA and 80 % ACN (B). Peptides
eluted from the column are introduced directly into an LTQ-
Orbitrap XL mass spectrometer (Thermo Fisher Scientifi c Inc.,
Bremen, Germany) at a spray voltage of 4.5 kV.
3. The mass spectrometer is operated using Xcalibur software
(Thermo Fisher Scientifi c). The mass range selected for MS
scan sets to 350-1,600
m
/
z
and the top three peaks subject to
MS/MS analysis. Perform full MS scan in the Orbitrap, and
MS/MS scans in the linear ion trap and Orbitrap: normalized
collision energy, 35 eV for CID and 45 eV for HCD; resolu-
tion, 60,000.
4. Carry out protein identifi cation by comparing the obtained
spectra against data in
Oryza sativa
proteins downloaded from
NCBI (
http://www.ncbi.nlm.nih.gov/
)
and UniProt (
http://
www.uniprot.org/
)
database using Proteome Discoverer 1.3
software (Thermo Fisher Scientifi c). The MS data analysis
parameters are as follows: enzyme, trypsin; missed cleavages, 2;
MS tolerance, 10 ppm; MS/MS tolerance, 0.8 Da; static mod-
ifi cation, carbamidomethylation; and dynamic modifi cation,
oxidation (Met). False discovery rates for peptide identifi cation
are less than 5 %.
5. Quantitation of iTRAQ reporter ions is performed using
Proteome Discoverer 1.3 software.
μ
3.9
A Case Study
A case study of identifi cation and quantitation of chloroplast
protein is shown in Fig.
3
. The chloroplast proteins isolated from
rice seedlings grown in hot (33 °C, 12-h light/28 °C, 12-h dark)
and control (28 °C, 12-h light/23 °C, 12-h dark) conditions were
subjected to the quantitative shotgun proteomic analysis with
iTRAQ labeling. Identifi cation and quantitation of superoxide
dismutase [Cu-Zn] protein were carried out, indicating that the
expression of chloroplast superoxide dismutase was up-regulated
more than threefold under the hot condition.
4
Notes
1. It is important to determine whether the leaf material to be
used is of the amylophyll (starchy leaf) or saccharophyll
(sugary leaf) type [
7
,
12
]. When handling the starchy leaves of
