Biology Reference
In-Depth Information
Tryptic peptides are purifi ed in an automatic ProMS station
(Genomic Solutions) by using a resin C18 microcolumn (ZipTip,
Millipore), and they are eluted directly with a matrix solution
(
-cyano hydroxycinnamic acid at a concentration of 5 mg/mL in
70 % (v/v) ACN/0.1 % (v/v) TFA) on MALDI plaque in 1
α
L of
fi nal volume. After the cocrystallization on plaque, samples are
analyzed by MALDI-TOF/TOF mass spectrometry to obtain the
peptide mass fi ngerprinting (MS) in a 4800 Proteomics Analyzer
(Applied Biosystems). The settings are: 800-4,000
m
/
z
range,
with an accelerating voltage of 20 kV, in refl ection mode, with
delayed extraction set to “on”, and an elapsed time of 120 ns.
Spectra are internally calibrated with peptides from trypsin autoly-
sis (M + H+ = 842.509, M + H+ = 2,211.104) with an
m
/
z
preci-
sion of ± 20 ppm. Most abundant peptide ions are subjected to
MS/MS analysis, providing information that can be used to defi ne
the peptide sequence.
A combined search (PMF and MS/MS) is performed with
GPS ExplorerTM software v3.5 (Applied Biosystems) over nonre-
dundant NCBI databases using the MASCOT search engine
(Matrix Science Ltd., London;
http://www.matrixscience.com
)
.
The database search utilized the following parameters: taxonomy
restrictions to
Viridiplantae
, one missed cleavage sites, 100 ppm
mass tolerance in MS and 0.5 Da for MS/MS data, cysteine carb-
amidomethylation as a fi xed modifi cation, and methionine oxida-
tion as a variable modifi cation. The confi dence in the peptide mass
fi ngerprinting matches (
p
< 0.05) is based on the MOWSE score,
and confi rmed by the accurate overlapping of the matched pep-
tides with the major peaks of the mass spectrum.
μ
4
Final Remarks
Taking into account our own experience, in this chapter we have
shown the usefulness of a basic proteomic approach—based on
SDS-PAGE and 2-DE analyses of protein extracts from mature
seeds and pollen—for variability studies in Holm oak. These analy-
ses have allowed the separation and grouping of the populations
according to its acorn morphometry, location (northern and
southern), and climate conditions (xeric, mesic, and intermediate),
confi rming previous results of our research group by using acorn
morphometry and NIRS chemical composition [
15
,
16
,
35
].
Therefore, gel-based Proteomics (SDS-PAGE and 2-DE) can be
used to detect variability between different populations from dif-
ferent environments, and to correlate it to environmental condi-
tions (Fig.
1
).
