Biology Reference
In-Depth Information
differentially expressed proteins between two or more conditions
dominates the rice proteomics fi eld [
14
]. The use of quantitative
shotgun proteomics has increased in prevalence and has been used
in rice to analyze the effects of abiotic stress in comparative studies
[
15
-
18
], and for revealing the molecular mechanisms of rice devel-
opment [
19
-
21
].
Despite a recent surge in shotgun techniques observed in the
literature, many rice proteomics studies continue to be carried out
with the use of 2-DE gels (
see
ref.
14
for a recent review and testa-
ment to the technique). This technique is compatible with analysis
of plant proteins as the denaturing buffers used for protein separa-
tion are well suited to plant protein extraction, proteins can be
highly resolved, and protein identifi cation can be performed with
simple MS instrumentation and software.
In this chapter we present details of a method, known variously
as a “slice and dice” or GeLC-MS/MS experiment, that can be
used to simultaneously identify and quantify thousands of proteins
at a time without laborious sample handling and visual compari-
sons of protein spots. This method is executed by separating pro-
teins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), in-gel digestion of proteins with trypsin, and subse-
quent extraction of peptides, followed by reversed-phase (RP)
separation and direct elution of peptides onto a tandem mass spec-
trometer [
22
,
23
].
2
Materials
All solutions should be prepared using MilliQ water and highest
mass spectrometric grade analytical reagents. Prepared solutions
are to be stored at room temperature, unless otherwise indicated.
Appropriate waste disposal regulations should be strictly followed.
All solutions and samples must be carefully handled to prevent
keratin contamination.
1. Extraction buffer was prepared with 8 M urea, 100 mM Tris-
HCl, pH 8.5, and 1 % Triton X-100.
2. SDS sample buffer (5× stock solution) was prepared with
6.25 mL 1 M Tris-HCl, pH 6.8, 2 g glycerol, 2.3 g SDS,
617.22 mg dithiothreitol (DTT), 1 mL 5 % (w/v) bromophe-
nol blue, and MilliQ water to 20 mL. Sample buffer was diluted
to 2× with MilliQ water.
3. SDS running buffer (10× stock solution) was prepared to 1 L
with 144.135 g glycine, 30.285 g Tris, 10 g SDS, and MilliQ
water. The stock solution was diluted to 1× with MilliQ water
before use.
4. Urea, SDS, Tris, glycine, and Triton X-100 were from Sigma.
5. Bicinchoninic acid (BCA) reagents were from Pierce.
