Biology Reference
In-Depth Information
and MOC had the serious handicap of contamination with
non-phosphorylated acidic peptides that also have affi nity for the
metals [
5
-
7
]. Therefore, strong cation exchange chromatography
(SCX) was often used in combination with IMAC or MOC,
because SCX provides a crude separation of phosphopeptides from
non-phosphopeptides [
8
]. Recently, improved IMAC or MOC
with enhanced specifi city against phosphopeptides allowed for the
effi cient enrichment of phosphopeptides by IMAC or MOC alone,
without the SCX pre-fractionation. The advent of a simple, single-
step method brought phosphoproteomics within reach for people
who are otherwise unfamiliar with shotgun proteomics. Still, pre-
fractionation is very useful strategy for covering the phosphopro-
teome in depth. Selectivity of the different phosphopeptide
enrichment methods differs signifi cantly, and thus some combina-
tion of methodology was effective for improving coverage [
9
]. For
tyrosine phosphoproteomics, antibodies which specifi cally recog-
nize tyrosine-phosphorylated peptides are available [
10
].
Combination approaches are favored for greater coverage, but
more LC-MS runs and larger samples are required for analysis.
Thus, the choice of an appropriate enrichment strategy requires
careful consideration.
Hydroxy acid-modifi ed metal oxide chromatography
(HAMMOC) has been successfully applied for analyzing phospho-
proteomes of various organisms, including plants [
11
-
15
]. The
principle of HAMMOC is to reduce non-phosphorylated acidic
peptide binding, and is similar to the blocking step of Western blot
analysis in that it reduces nonspecifi c antibody binding [
14
].
Hydroxy acids binding to metal oxides is weaker than to a phos-
phate group, but stronger than to the carboxylic groups of non-
phosphorylated acidic peptides. Therefore, addition of the hydroxy
acids during affi nity binding steps can suppress binding of non-
phosphorylated acidic peptides, but not phosphopeptides, to metal
oxides. Titania and zirconia with lactic acid and
-hydroxypropanoic
acid, respectively, are effective combinations for selective phospho-
peptide enrichment [
14
].
In general, phosphopeptide enrichment methods are very sim-
ple, and are routinely utilized in laboratories where these methods
were developed. However, very often these methods do not work
well in other laboratories for unknown reasons. Here, a protocol
for simple and robust phosphopeptide enrichment from plant
materials based on titania utilized HAMMOC is described in detail
so that it can be reproduced in any laboratory. The handling of
titania beads is the key consideration for successful phosphopep-
tide enrichment. The use of self-producible stop-and-go-extraction
tips (StageTip) makes phosphopeptide sample preparation for
LC-MS/MS analysis economically affordable [
16
-
18
].
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