Biology Reference
In-Depth Information
initial processing and assembly while still within the ER [
51
,
52
].
In addition to the N-terminal signal sequence, the canonical SSP-
precursor primary-sequence includes a pro-sequence containing
the PSV-targeting information, plus at least two linker/protease
cleavage sites. It is not uncommon for the SSP pro-proteins to
display the canonical Asn-X-Ser/Thr glycosylation sequon, and
become N-glycosylated during passage through the ER. In some
instances the N-glycosylation sequons are within the pro-sequence
or linker regions, and are subsequently removed during proteolytic
processing. In other instances, however, they persist and are pres-
ent in the fi nal fully processed SSP. Proteolytic processing and
assembly continue during transit through the secretory pathway,
and are completed within the PSV [
53
].
After proteolytic processing has been completed, the fi nal poly-
peptide associations are stabilized by formation of at least one disul-
fi de-bond. The fi nal stable SSP structures can be as simple as a
“
3-heterohexamer. It is
noteworthy that the incisive SSP-based research of a generation of
plant cell biologists has been mechanistically verifi ed and extended
by recent results based upon availability of genome sequence infor-
mation and application of tandem mass spectrometry (MS/MS)
[
4
]. It is equally important to note that the understanding of SSP
processing can greatly simplify interpretation of MS results. This is
especially true of gel-based results, where it could be diffi cult to
interpret the position of a protein that is very different from the
MW and pI of the primary translation product unless one has prior
knowledge of both proteolytic and glycolytic processing events [
3
].
αβ
-heterodimer” or as complex as a
α
3
β
3.3 Addressing
the Dynamic Range
Problem
The abundance of the SSP can be a great benefi t … if you are
studying SSP. If not, especially if a gel-based strategy is employed,
then the SSP can substantially interfere with analysis of total pro-
teins. Even if the seeds of a hypothetical plant have only a 2S albu-
min and an 11S globulin as SSP, the complexity in 2D gel
spot-patterns can be nearly overwhelming because of the contribu-
tions of extended multigene families plus heterogeneity in both
proteolytic and glycolytic processing. Advances in high perfor-
mance ion trap mass spectrometry (MS) employing electrospray
ionization (ESI) and nanofl ow liquid chromatography (nLC) have
signifi cantly improved the ability to analyze proteins [
14
]. Despite
these advances there remain serious inherent limitations. Simply
put, the range of protein concentrations in biological samples is
very large (as much as 10
12
) [
10
] and the dynamic range of the
analytical methods used is small (less than 10
3
in most cases) [
54
].
The only practical way to circumvent this problem is by including
a prefractionation/depletion step to the work-fl ow (Table
1
).
There are several different strategies by which this can be achieved
[
55
]. With the exception of the prolamins (Fig.
4a
), fractional
solubility has not proved to be generally useful in SSP-depletion.
