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410 Part 3: Meat, Poultry and Seafoodsradiolabeling, such as with [^35 S]methionine, and staining with
fluorescent dyes such as the SYPRO or Cy series of dyes. Multi-
ple staining with dyes fluorescing at different wavelengths offers
the possibility of differential display, allowing more than one
proteome to be compared on the same gel, such as in difference
gel electrophoresis (DIGE). Patton published a detailed review
of visualization techniques for proteomics (Patton 2002).AnalysisAlthough commercial 2DE image analysis software, such as
ImageMaster (Amersham), PDQuest (BioRad), or Progenesis
(Nonlinear Dynamics), has improved by leaps and bounds in
recent years, analysis of the 2DE gel image, including protein
spot definition, matching, and individual protein quantification,
remains the bottleneck of 2DE-based proteome analysis and still
requires a substantial amount of subjective input by the investi-
gator (Barrett et al. 2005). In particular, spot matching between
gels tends to be time consuming and has proved difficult to
automate (Wheelock and Goto 2006). These difficulties arise
from several sources of variation among individual gels, such
as protein load variability due to varying IPG strip reswelling
or protein transfer from strip to slab gel. Also, gene expression
in several tissues varies considerably among individuals of the
same species, and therefore individual variation is a major con-
cern and needs to be accounted for in any statistical treatment
of the data. Pooling samples may also be an option, depending
on the type of experiment. These multiple sources of varia-
tion has led some investigators (Barrett et al. 2005, Karp et al.
2005, Wheelock and Goto 2006) to cast doubt on the suitabil-
ity of univariate tests such as the Student’st-test, commonly
used to assess the significance of observed protein expression
differences. Multivariate analysis has been successfully used by
several investigators in recent years (Gustafson et al. 2004, Karp
et al. 2005, Kjaersgard et al. 2006b).Some Problems and Their SolutionsThe high resolution and good sensitivity of 2DE are what make
it the method of choice for most proteomics work, but the
method nevertheless has several drawbacks. The most signif-
icant of these have to do with the diversity of proteins and
their expression levels. For example, hydrophobic proteins do
not readily dissolve in the buffers used for isoelectrofocussing.
This problem can be overcome, though, using nonionic or
zwitterionic detergents, allowing for 2DE of membrane- and
membrane-associated proteins (Chevallet et al. 1998, Herbert
1999, Henningsen et al. 2002, Babu et al. 2004). Vilhelmsson
and Miller (2002), for example, were able to use “membrane pro-
tein proteomics” to demonstrate the involvement of membrane-
associated metabolic enzymes in the osmoadaptive response of
the foodborne pathogenStaphylococcus aureus.A2DEgelim-
age ofS. aureusmembrane-associated gels is shown in Figure
22.4.
Similarly, resolving alkaline proteins, particularly those with
pI above 10, on two-dimensional gels has been problematic in the24 kDa36 kDa45 kDa55 kDa66 kDa84 kDa97 kDa310 pIFigure 22.4.A two-dimensional electrophoresis membrane
proteome map fromStaphylococcus aureus,showing proteins with
pI between 3 and 10 and molecular mass about 15–100
(O. Vilhelmsson and K. Miller, unpublished). Isoelectrofocussing was
in the presence of a mixture of pH 5–7 and pH 3–10 carrier
ampholytes and the second dimension was in a 10%
polyacrylamide slab gel with a 4% polyacrylamide stacker.past. Although the development of highly alkaline, narrow-range
IPGs (Bossi et al. 1994) allowed reproducible two-dimensional
resolution of alkaline proteins (Gorg et al. 1997), their repre- ̈
sentation on wide-range 2DE of complex mixtures such as cell
extracts remained poor. Improvements in resolution and repre-
sentation of alkaline proteins on wide-range gels have been made
(Gorg et al. 1999), but nevertheless an approach that involves ̈
several gels, each of a different pH range, from the same sam-
ple is advocated for representative inclusion of alkaline proteins
when studying entire proteomes (Cordwell et al. 2000). Indeed,
Cordwell and coworkers were able to significantly improve the
representation of alkaline proteins in their study on the rela-
tively highly alkalineHelicobacter pyloriproteome using both
pH 6–11 and pH 9–12 IPGs (Bae et al. 2003).
A second drawback of 2DE has to do with the extreme differ-
ence in expression levels of the cell’s various proteins, which
can be as much as 10,000-fold. This leads to swamping of
low-abundance proteins by high-abundance ones on the two-
dimensional map, rendering analysis of low-abundance proteins
difficult or impossible. For applications such as species iden-
tification or study of the major biochemical pathways, where
the proteins of interest are present in relatively high abundance,