Biology Reference
In-Depth Information
Separate digested and purifi ed peptide solutions with a C18 col-
umn by nano-fl ow LC. Make a linear gradient of acetonitrile (from
5 % [v/v] to 45 % [v/v]) at a fl ow rate of 500 nL/min for 100 min.
Detect and analyze the separated and ionized peptides in a mass
spectrometer. Examples of analyzed results using 5
3.6 Nano-LC-MS/MS
Analysis
g of oat PM
and DRM proteins are shown in Figs.
4
,
5
, and
6
. You can see
detailed results in fi gure legends.
μ
a
160
In-gel digestion (PM)
140
120
Experiment 1
Experiment 2
Experiment 3
Experiment 4
100
80
60
40
20
0
b
160
In-solution digestion (PM)
140
120
100
Experiment 1
Experiment 2
Experiment 3
Experiment 4
80
60
40
20
0
1 2 3 4 5 6 7 8 9 101112131415161718192021222324
The number of transmembrane domains
Fig.
4
The number of transmembrane domains in oat PM proteins separated and identified following in-
gel or in-solution tryptic digestion. Peptide sequences were searched against the NCBI database (version
20120216, comprising 17,282,984 sequences), taxonomy viridiplantae. Transmembrane domains were
estimated by SOSUI engine ver. 1.10 (
http://bp.nuap.nagoya-u.ac.jp/sosui/
)
. (
a
) Proteins with up to 24
transmembrane domains were identified in oat PM by in-gel digestion in four biological replicates. On
average, 700 proteins with transmembrane domains were identified in the four replicates. (
b
) Proteins
with up to 24 transmembrane domains were identified in oat PM by in-solution digestion in four biologi-
cal replicates. On average, 397 proteins with transmembrane domains were identified in the four
replicates
