Biology Reference
In-Depth Information
6. DTT, iodoacetamide (IAA), 10 % Tris-HCl precast gels, and
Coomassie brilliant Blue G-250 were from Bio-Rad.
7. Trypsin was of Promega sequencing grade.
8. Formic acid was of Fluka 98 % mass spectrometry grade.
9. Acetonitrile was of Merck Lichrosolv liquid chromatography
grade.
10. nanoLC-MS/MS was performed using an LTQ-XL linear ion
trap mass spectrometer (Thermo, San Jose, CA).
11. Reversed phase C18 columns were packed in-house to approx-
imately 7 cm (100
μ
m id) using 100 Å, 5
μ
m Zorbax C18 resin
from Agilent Technologies, CA.
12. Spectrum fi les are converted to .mzxmL fi les prior to database
search using Readw.exe, available for free download from
http://sourceforge.net/projects/sashimi/fi les/ReAdW%20
%28Xcalibur%20converter%29/ .
13. XTandem of the Global Proteome Machine software version
2.1.1 is freely available for download from http://www.
thegpm.org/tandem .
14. The Scrappy program is available as a series of R modules which are
available for download from http://www.proteomecommons.org .
3
Methods
The workfl ow detailed below is a simple and effective methodol-
ogy for a shotgun proteomics experiment that can be routinely
used to analyze tissue samples from almost any organism, includ-
ing recalcitrant tissues (Fig. 1 ). SDS-PAGE is a fundamental and
robust method for protein fractionation and is an amenable tech-
nique for sample preparation prior to MS analysis.
This experiment may be performed for both qualitative and
quantitative assessments of the proteome of any organism. In-gel
digestion can be performed over 2 days once proteins have been
separated electrophoretically, and three replicates of 16 fractions
can be analyzed over 2½ days of machine analysis time on the mass
spectrometer. This means that the workfl ow can be completed in
less than a week for three replicates, and just over a week for a
control-versus-treatment experiment of six replicates in total. This
approach has been used in several published quantitative proteomic
experiments in rice, where the results generated have been ana-
lyzed using quantitative tools such as Scrappy [ 15 - 17 , 24 ]. The
workfl ow is fl exible, in that a variety of protein extraction and MS/
MS analysis methods may be applied. The workfl ow below is writ-
ten for rice leaves, but can be readily adapted to other tissues
including cell cultures, roots, and seeds.
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