Agriculture Reference
In-Depth Information
Table 2.6
(continued)
Database Name
Plant Species/Purpose
Uniform Resource Locator
(URL)
SILAC—Stable
isotope labeling by
amino acids in cell
culture
Stable isotope labeling by amino
acid in cell culturing for MS
quantitative proteomics
Super-SILAC Technol-
ogy for Quantita-
tive Proteomics in
Neoplasms
Peptide fingerprint analysis method
used in conjunction with MS
ionization
Duke Proteomics Core
Facility—Protein
Quantitation—IGSP
Facilities and methodology for pro-
tein quantification using methods
described in Table 8
Sample Preparation
Sample preparation is perhaps one of the most important steps in proteomics re-
search. Methods that use trichloroacetic acid (TCA) and acetone are still the most
commonly used procedures for protein precipitation and separation from other me-
tabolites in plant mixtures (Song et al.
2006
; Wang et al.
2006
). An alternate method
using phenol and ammonium acetate/methanol is also popular for plant separation.
Sample purification effectively improves protein detection and increases proteome
separation in subsequent steps by reducing interference in samples. The use of dif-
ferent reagents to separate proteins by their different solubilities and membrane as-
sociations can be effective in reducing the complexity of proteins in fractions, and
to enrich rare proteins in samples. Modifications of these basic procedures can be
used to separate membrane associated proteins in large amounts from the remainder
of the soluble protein fraction (Agrawal et al.
2005
; Barjaktarovic et al.
2007
).
Chromatography (SDS-PAGE)
Sequential treatment of the isolated proteins on various inert substrates is a common
method for separating protein samples based on a combination of solubility, molec-
ular mass and isoelectric point. At one time only one-dimensional SDS-PAGE had
wide use in separating complex proteins, based only on their molecular mass, but
now this approach has limited application. In contrast, high-resolution separation
of proteins by two-dimensional gel electrophoresis (2-DE), which uses isoelectric
focusing (IEF) in the first dimension and SDS-PAGE in the second dimension, is
a more effective and exact technique. The recent development of the immobilized
pH gradient (IPG)-IEF strips for use in the first dimension has improved reproduc-
ibility and resolution. The 2-DE method has been widely accepted in proteomics
for a range of important crop and bacterial species containing massive cell walls
(Chen and Harmon
2006
; Herbert et al.
2006
; Berkelman and Stenstedt
2002
). Da-
tabases housing 2-DE information have been developed and released to the public.
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