Biology Reference
In-Depth Information
are isolated as detergent-resistant membrane (DRM) fractions,
have been studied because of their involvement in important
cellular process in PM areas: microdomains are even more intrac-
table to comprehensive proteomics study [
10
-
15
]. These factors
together have challenged researchers to obtain a comprehensive
view of the PM and microdomain proteomic profi les. Liquid chro-
matography and mass spectrometry technologies have advanced
rapidly, providing higher resolution and reliable results for a huge
amount of peptides [
16
,
17
]. In particular, nano-fl ow reverse-
phase liquid chromatography allows the separation of proteins
without pre-separation using 1-DE or 2-DE [
17
]. Here, we
describe PM and DRM protein preparation methods that are
adapted to nano-LC-MS/MS-based shotgun proteomics using
two sample preparation methods, “in-gel” and “in-solution” pep-
tide digestion. We used the aerial parts of oat plants as an example
and the methods described are applicable to any other plants such
as rye [
18
],
Arabidopsis
[
19
], and
Brachypodium distachyon
(data
not shown). In the “in-gel digestion protocol,” solubilized PM and
DRM proteins were applied to sodium dodecyl sulfate (SDS) in a
polyacrylamide gel to remove non-proteinaceous materials and sub-
sequently subjected to tryptic digestion. In the “in-solution diges-
tion protocol,” we used an MPEX PTS reagents kit (GL science
Inc., Tokyo, Japan), which has been widely used for solubilization
of proteins in mammalian and bacteria PMs, such as HeLa cells and
Escherichia coli
cells, respectively [
20
]. Using these methods, thou-
sands of PM and DRM proteins were consistently identifi ed, includ-
ing highly hydrophobic proteins with 20+ transmembrane domains.
Furthermore, these data can be used for protein quantifi cation.
2
Materials
Prepare all solutions using ultrapure water (prepared by purifying
deionized water to attain a sensitivity of 18.2 M
cm at 24 °C) and
analytical grade reagents. Prepare and store all reagents at room
temperature (unless indicated otherwise). Diligently follow all
waste disposal regulations when disposing of waste materials.
Ω
2.1 Plasma
Membrane Purifi cation
Components
Several items, including 2 L of ultrapure water, a Polytron homog-
enizer, centrifuge rotors, and ultracentrifuge rotors, should be
chilled at 4 °C.
1. Homogenizing medium: 0.5 M sorbitol, 50 mM Mops-KOH,
pH 7.6, 5 mM EGTA (pH 8.0), 5 mM EDTA (pH 8.0), 5 %
(w/v) polyvinylpyrrolidone-40 (molecular weight 40,000),
0.5 % (w/v) BSA, 2.5 mM phenylmethanesulfonyl fl uoride
(PMSF), 4 mM salicylhydroxamic acid (SHAM), 2.5 mM
1,4-dithiothreitol (DTT). Store at 4 °C (
see
Note 1
).
