Biology Reference
In-Depth Information
3.4 Protein Sample
Preparation for LC-MS
One hundred
g of proteins corresponding to the different fractions
were subjected to one-dimensional, denaturing SDS-PAGE accord-
ing to Laemmli [
20
], with minor modifi cations. We used 7 cm
stacking (5 % polyacrylamide) and 3 cm resolving (12.5 % polyacryl-
amide) gels, these run at 80 V in a Mini PROTEAN II cell (
see
Note
17
) (Fig.
2b
). Gel was stained by colloidal Coomassie [
21
].
Bands were excised using a clean scalpel, transferred to
Multiwell 96 plates and digested with modifi ed porcine trypsin by
using a ProGest (Genomics Solution) digestion station. The diges-
tion protocol used is that of Schevchenko et al. [
22
], with minor
variations. Gel plugs were distained by incubation (twice for
30 min) with 200 mM ammonium bicarbonate in 40 % ACN at
37 °C, then subjected to three consecutive dehydration-rehydra-
tion cycles with pure acetonitrile and 25 mM ammonium bicarbon-
ate in 40 % ACN, respectively; and, fi nally, dried at room temperature
for 10 min. Trypsin, at a fi nal concentration of 12.5 ng/
μ
L in
25 mM ammonium bicarbonate, was added to the dry gel pieces
and the digestion proceeded at 37 °C for 12 h. Peptides were
extracted from gel plugs with 0.5 % TFA, 50 % ACN (15 min incu-
bation), dried under vacuum and resuspended in 0.1 % TFA.
μ
Tryptic peptides (obtained according Subheading
3.4
) were
injected into the LC system (i.e., a Finnigan Surveyor HPLC sys-
tem). Peptides were detected in a LTQ-Orbitrap equipped with a
nanoelectrospray ion source (nESI). The acquired data can be
analyzed with Proteome Discoverer v1.3 software (Thermo Fisher
Scientific, USA) and MASCOT (
http:// www.matrixscience.com
)
or SEQUEST (
http:// fi elds.scripps.edu/sequest/
) algorithms.
Protein identifi cation was conducted by combining search (MS
plus MS/MS) to the entries of a nonredundant protein Viridiplantae
database downloaded from the National Center for Biotechnology
Information. Search parameters were the enzyme trypsin, taxon-
omy restrictions to Viridiplantae, ±50 ppm peptide mass tolerance
in MS, ±0.8 Da for MS/MS data and, carbamidomethyl (C) as a
fi xed modifi cation, whereas methionine oxidation as a variable
modifi cation (
see
Note 18
).
3.5 LC-MS and
Protein Identifi cation
Considering the particular composition of the acorn fl our, rich in
non-protein material [
23
,
24
], the main challenge was to obtain
protein extracts of enough quality for the subsequent analyses and
identifi cation of proteins by MS. In Holm oak acorns, glutelin was
the major fraction, corresponding approximately to 80 % of the
total protein extracted with this protocol.
The analysis by LC coupled with MS/MS allowed the identifi -
cation of 509 protein species, belonging to the following functional
categories: storage, energy production and metabolism, defense,
antioxidant system, signaling and gene expression regulation,
3.6 Main Results
