Food Biochemistry and Food Processing

18 Proteomics in Fish Processing 403

Nagamune 2004), hold great promise and are de-
serving of discussion in their own right, the classic
process of two-dimensional electrophoresis (2DE)
followed by protein identification via peptide mass
fingerprinting of trypsin digests (Fig. 18.1) remains
the workhorse of most proteomics work, largely
because of its high resolution, simplicity, and mass
accuracy. This “classic approach” will therefore be
the main focus of this chapter. A number of reviews
on the advances and prospects of proteomics within
various fields of study are available. Some recent
ones include Aebersold and Mann (2003), Cash
(2002), Cash and Kroll (2003), Graves and Haystead
(2003), Huber et al. (2003), Kvasnicka (2003), Phi-
zicky et al. (2003), Piñeiro et al. (2003), Pusch et al.
(2003), Takahashi et al. (2003), and Tyers and Mann
(2003).

TWO-DIMENSIONALELECTROPHORESIS

Two-dimensional electrophoresis (2DE), the corner-
stone of most proteomics research, is the simultane-
ous separation of hundreds, or even thousands, of
proteins on a 2D polyacrylamide slab gel. The po-
tential of a 2D protein separation technique was
realized early on, and considerable development
efforts took place in the 1960s (Kaltschmidt and
Wittmann 1970, Margolis and Kenrick 1969). The
method most commonly used today was developed
by Patrick O’Farrell and is described in his seminal
and thorough 1975 paper (O’Farrell 1975). O’Far-
rell’s method first applies a process called isoelectric
focusing, where an electric field is applied to a tube
gel on which the protein sample and carrier am-
pholytes have been deposited. This separates the
proteins according to their molecular charge. The
tube gel is then transferred onto a polyacrylamide
slab gel, and the isoelectrically focused proteins are
further separated according to their molecular mass
by conventional sodium dodecyl sulfate–polyacry-
lamide gel electrophoresis (SDS-PAGE), yielding a
two-dimensional map (Fig. 18.2) rather than the fa-
miliar banding pattern observed in one-dimensional
SDS-PAGE. The map can be visualized and individ-
ual proteins quantified by radiolabeling or by using
any of a host of protein dyes and stains, such as
Coomassie blue, silver stains, or fluorescent dyes.
By comparing the abundance of individual proteins
on a number of gels (Fig. 18.3), up- or downregula-
tion of these proteins can be inferred. It is worth

emphasizing that great care must be taken that the

proteome under investigation is reproducibly repre-

sented on the 2DE gels, and that individual variation

in specific protein abundance is taken into consider-

ation by running gels from a sufficient number of

samples and performing the appropriate statistics.

Pooling samples may also be an option, depending

on the type of experiment. Although a number of

refinements have been made to 2DE since O’Far-

rell’s paper was published, most notably the intro-

duction of immobilized pH gradients (IPGs) for iso-

electrofocusing (Görg et al. 1988), the procedure

remains essentially as outlined above. For more de-

tailed, up-to-date descriptions of methods, the read-

er is referred to any of a number of excellent books

and laboratory manuals, such as Berkelman and

Stenstedt (1998), Link (1999), Walker (2002), and

Westermeier and Naven (2002).

Some Problems and Their Solutions

The high resolution and good sensitivity of 2DE

are what make it the method of choice for most pro-

teomics work, but the method nevertheless has

Figure 18.2.A 2DE protein map of rainbow trout

(Oncorhynchus mykiss)liver proteins with pH between

4 and 7 and molecular mass about 10–100 (S. Martin,

unpublished). The proteins are separated according to

their pH in the horizontal dimension and according to

their mass in the vertical dimension. Isoelectrofocusing

was by pH 4–7 immobilized pH gradient (IPG) strip, and

the second dimension was in a 10–15% gradient poly-

acrylamide slab gel.