Biology Reference
In-Depth Information
in an aqueous environment. In order to minimize hydrolysis
and optimize effi ciency of peptide labeling, maintain ratio
iTRAQ dissolution buffer:ethanol at least in 4:6 during label-
ing reaction. The addition of iTRAQ dissolution buffer at high
concentration (0.5 M) ensures around 100 mM concentration
during labeling reaction (pH 7.0). The addition of ethanol
precipitates pectins, which act as contaminants and may inter-
fere with the analyses. Pectin polymers precipitate out of solu-
tion, converting samples in a gelatinous state unsuitable for
further analysis [
30
]. Thus, it is strongly advisable to run a
previous experiment without labeling exactly performed in the
same way as the experiment with samples labeled with iTRAQ
in order to guarantee a good condition for the sample under
analysis.
9. Addition of SCX-A buffer hydrolyzes free tags and stops label-
ing reaction.
10. Wash SCX column between runs with SCX-C buffer to avoid
cross-contamination among experiments.
11. A desalting step is important for removing salts prior to MS/
MS.
12. There are available software solutions not limited to compati-
bility constraints. A thorough review of different freely avail-
able software for analysis of mass spectrometry data can be
found in Mueller et al. [
31
].
13. Database searching can be speeded up by using a restricted
database. This database can be created with a subset of
National Center for Biotechnology Information non-redundant
(NCBInr) protein database (
http://www.ncbi.nlm.nih.gov/
)
.
Sequences are retrieved using the keyword search enquiry
“organism,” adding potentially contaminating proteins
retrieved using the keyword search enquiry “trypsin” or
“keratin.”
14. In spite of the exponential availability of sequences by the
recent plant genome sequencing projects and the public release
of ESTs for plants within databases, there is still a diffi culty
for the protein identifi cation for most plant species. Database
searching for unsequenced organisms can be helped using dif-
ferent strategies as de novo sequencing [
32
,
33
] or as
described by Grossman et al. [
34
] by a high-throughput analy-
sis based on a spectrum quality scoring, de novo sequencing,
and error-tolerant Blast searches.
15. Apply iTRAQ correction factors in the SMPW and the soft-
ware performs automatically iTRAQ calculations in Protein/
Peptide Summary for the application of the correction factors.
16. Radiometric data conversion to log
2
is recommended. The rea-
son is that in the quantitative data obtained, twofold increase
