Biology Reference
In-Depth Information
1
Introduction
The model legume
Medicago truncatula
has recently been subject
to proteomic profi ling studies such as of root plastids [
1
], mito-
chondria [
2
], and comparative proteomics of root nodules exposed
to drought stress [
3
,
4
]. For the latter systems biology approach,
gel-free high-throughput shotgun mass spectrometry was used to
analyze 35 samples.
The analysis was based on database-dependent protein identi-
fi cation and the corresponding spectral count (SC) for the detec-
tion of relative changes on protein levels. To maximize peptide
identifi cation rates, data-dependent (DDP) LC-MS/MS analysis
takes several hours [
5
]. To enhance the explanatory power of the
quantitative information extracted, replicate MS measurements are
also required. Thus, MS runtime is limiting to experiments on
large datasets. The DDP triggers a series of tandem MS (MS/MS)
for peptide identifi cation. It is typically a “top 5-” or “top10”-type
MS experiment in which the 5-10 most intense ions of a survey
scan (also called full scan) were selected for MS/MS. This com-
mon type of DDP approach usually takes less than 1 s per cycle
time, however, slowing down data acquisition on full-scan level
which is in the range of a few milliseconds. Thus a data-independent
analysis (DIA) using only the full scan (FS) results in many more
scans per ion (peptide peak) allowing for a strongly reduced gradi-
ent time. In systems biology the sample number can easily increase
up to one hundred and more including many biological replicates
[
6
]. Runtime on a mass spectrometer may therefore exceed weeks
which needs reduction in future.
We demonstrate a method that allows for a signifi cant reduc-
tion in LC-MS analysis time without loss in the number of protein
identifi cation for relative quantifi cation. This approach is based on
prior peptide identifi cation and retention time extraction and the
preparation of selected target peptide lists in combination with
full-scan analysis without further MS/MS data acquisition. Instead
of the common spectral counting for label-free relative quantifi ca-
tion (
see
also Chapter
13
)
, the ion intensity count (the sum of all
ion intensities of the target peptide) is being used. The target pep-
tides have been picked from
M. truncatula
leaf protein extracts.
2
Materials
2.1
Plant Material
Leaves of 7-week-old
M. truncatula
(Jemalong) plants. Growth
conditions as described previously [
3
] with some changes. Plants
were fertilized with 2.5 mM NH
4
NO
3
instead of rhizobia
inoculation.
