Medicago truncatula Proteomics for Systems Biology: Novel Rapid Shotgun LC-MS Approach for Relative Quantification Based on Full-Scan Selective Peptide Extraction (Selpex) - Plant Proteomics: Methods and Protocols - page 295

corresponding peptide sequences, the number of spectral counts, as
well as the retention time and the mass-to-charge ratio ( m/z ) ( see
Note 1 ). The Qual Browser (Xcalibur v2.1, Thermo) software was
used for peptide peak extraction, visualization, and evaluation.
Doubly charged peptides with robust and reproducible high-qual-
ity peak shape, reproducible found in all replicates, were selected for
the target peptide list for further selective peptide extraction
(Selpex) analysis (Table 1 ) ( see Note 2 ). All peptide sequences have
been tested and marked for protein specifi city (proteotypic), as far
as the present Medicago protein annotation information allows for
( see Note 3 ). Retention times for these peptides were adapted to a
very short 30-min LC-MS gradient analysis.

3.4 Full-Scan Selpex
for Rapid Relative
Quantifi cation Based
on Ion Intensity Count
Using ProtMAX

For a Selpex check, DDP LC-MS/MS analyses were compared
with the FS-based LC-MS detection (Fig. 1 ). A fi le (tab-delimited
text) with the target list of the selected m/z peptides and corre-
sponding retention times was generated (Table 1 ) and imported
into the ProtMAX tool ( http://www.univie.ac.at/mosys/software.
html ) . The converted RAW chromatogram fi les in mzXML format
(MassMatrix MS Data File Conversion, v3.9) were imported into
ProtMAX. The selected preference settings used to perform the
quantitation were as follows: (1) target list was chosen as method;
(2) intensity for quantifi cation; (3) cut to two decimals; (4) +2
charge state and retention time (RT) [min] environment was set to
3. A data matrix was retrieved from the software including the tar-
get m/z values with corresponding ion intensity counts (cumulative
ion intensity), scan number, and retention time for each sample
analyzed.

4 Anticipated Results

The Selpex approach is useful in order to minimize MS analysis
time enabling high-throughput measurements of huge sample sets
that often occur in systems biology experiments. Selpex subse-
quently allows for the database-independent protein (peptide)
identifi cation and relative quantifi cation. Due to peptide-level res-
olution, Selpex also enables the recognition of modifi cation-
induced changes as previously shown for the MAPA approach [ 8 ].
Here, we demonstrate the possibility to quantify around 40
identifi ed proteins using a selected peptide list even from an
extremely short 30-min LC gradient using FS MS only (Fig. 1 )
( see Note 4 ). Target 738.40 m/z shows most abundant ion inten-
sity count (peak intensity). The lower ratio of that target in the
30-min gradient compared to the 120-min gradient might be due
to ion suppression effects ( see Note 5 ). Thus, the dynamic range of
the 30-min gradient seems to be the limitation for high-abundance
peptides ( see Note 6 ). As a consequence, quantitative changes of