Biology Reference
In-Depth Information
1
Introduction
In comparison with other plant organelles, oil bodies (OBs)
possess an unusual structure: they are composed of a neutral lipid
core surrounded by a “half-unit” membrane formed by a phospho-
lipid monolayer with several embedded proteins [
1
]. The OB
proteins stabilize the particles and are expected to participate in
OB formation and degradation, but their precise role in these
processes is still unclear [
2
,
3
]. Recent reports claim for enzymatic
activity of oleosin [
4
]. The OB proteins are present in a number of
homologous isoforms, whose signifi cance has yet to be determined
[
5
]. The proteins are generally very hydrophobic, which compli-
cates matters. This is the main reason why their detailed structure
and particular function still have not been described suffi ciently.
Even some of the protein isoforms have been escaping the identifi -
cation for decades [
6
].
Hydrophobic proteins comprise long regions without cleavage
sites for trypsin (arginine and lysine), which decreases the number
of peptides produced by trypsin digestion and the protein cover-
age. The extensive protein coverage is a fi rst demand of current
challenges in proteomics such as structural analysis and identifi ca-
tion of posttranslational modifi cations.
To overcome the problems connected with hydrophobic pro-
tein digestion, we brought chymotrypsin into the play [
6
]. This
protease still has not been appreciated enough, mainly due to its
low specifi city compared to trypsin. Nevertheless, with the new
generation of MS instruments, providing peptide fragmentation,
high resolution, and accuracy, the chymotryptic digestion products
can be easily “puzzled out” and chymotrypsin becomes a powerful
tool for hydrophobic protein identifi cation. We isolated the OBs
by the method adapted from Tzen [
7
], but the purifi ed OBs were
dissolved directly in the electrophoresis sample buffer in order to
increase the protein concentration in loaded samples. After the
SDS-PAGE, the protein bands were excised and in-gel digested.
Three different proteolytic systems were used: trypsin, as in classical
proteomic protocols, chymotrypsin, and the mixture of both
enzymes (1:1, w/w). Total protease concentration was 12.5 mg/L
in all cases.
Using chymotrypsin, single or in mixture with trypsin, the
coverage of six well-known OB proteins was enhanced from 7.9 to
34.2 [
8
] up to 80 %; and moreover, four new proteins were identi-
fi ed. Chymotrypsin, digesting in the hydrophobic protein parts,
contributed signifi cantly to the protein coverage. The protease
mixture digestion led to a number of peptides distributed over the
protein sequence and, in some cases, revealed peptides unavailable
with single protease digestion.
Our method can be easily applied to extensive identifi cation of
other hydrophobic proteins.
