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412 Part 3: Meat, Poultry and SeafoodsTools website (http://www.expasy.org/tools) contains links to
most of the available software for protein identification and sev-
eral other tools. Attaining a high identification rate is problematic
in fish and seafood proteomics due to the relative paucity of avail-
able protein sequence data for these animals. As can be seen in
Table 22.1, this problem is surprisingly acute for species of com-
mercial importance. To circumvent this problem, it is possible to
take advantage of the available nucleotide sequences, which in
many cases is more extensive than the protein sequences avail-
able, to obtain a tentative identity. How useful this method is
will depend on the length and quality of the available nucleotide
sequences. It is important to realize, however, that an identity
obtained in this manner is less reliable than that obtained through
protein sequences and should be regarded only as tentative in
the absence of corroborating evidence (such as two-dimensional
immunoblots, correlated activity measurements, or transcript
abundance). In their work on the rainbow trout (Oncorhynchus
mykiss) liver proteome, Martin et al. (2003b) and Vilhelmsson
et al. (2004) were able to attain an identification rate of about
80% using a combination of search algorithms that included
the open-access Mascot program (Perkins et al. 1999) and a
licensed version of Protein Prospector MS-Fit (Clauser et al.
1999), searching against both protein databases and a database
containing all salmonid nucleotide sequences. In those cases
where both the protein and nucleotide databases yielded results,
a 100% agreement was observed between the two methods.
A more direct, if rather more time-consuming, way of obtain-
ing protein identities is by direct sequence comparison. Until
recently, this was accomplished byN-terminal or internal (after
proteolysis) sequencing by Edman degradation of eluted or elec-
troblotted protein spots (Kamo and Tsugita 1999, Erdjument-
Bromage et al. 1999). Today, the method of choice is tandem
mass spectrometry (MS/MS). In the peptide mass fingerprinting
discussed earlier, each peptide mass can potentially represent
any of a large number of possible amino acid sequence com-
binations. The larger the mass (and longer the sequence), the
higher the number of possible combinations. In MS/MS, one
or several peptides are separated from the mixture and dissoci-
ated into fragments that then are subjected to a second round
of MS, yielding a second layer of information. Correlating this
spectrum with the candidate peptides identified in the first round
narrows down the number of candidates. Furthermore, several
short stretches of amino acid sequence will be obtained for each
peptide, which, when combined with the peptide and fragment
masses obtained, enhances the specificity of the method even
further (Wilm et al. 1996, Chelius et al. 2003, Yu et al. 2003b, ).
MS methods in proteomics are reviewed in Yates (1998, 2004)
and Yates et al. (2009).SEAFOOD PROTEOMICS AND
THEIR RELEVANCE TO PROCESSING
AND QUALITY2DE-based proteomics have found a number of applications
in food science. Among early examples are such applications
as characterization of bovine caseins (Zeece et al. 1989), wheatflour baking quality factors (Dougherty et al. 1990), and soybean
protein bodies (Lei and Reeck 1987). In recent years, proteomic
investigations on fish and seafood products, as well as in fish
physiology, have gained considerable momentum, as can be seen
in recent reviews (Parrington and Coward 2002, Pineiro et al. ̃
2003). Herein, we consider recent and future developments in
fish and seafood proteomics as related to issues of concern in
fish processing or other quality considerations.Early Development and Proteomics of FishFishes go through different developmental stages (embryo, larva,
and adult) during their lifespan that coincide with changes in the
morphology, physiology, and behavior of the fish (O’Connell
1981, Govoni et al. 1986, Skiftesvik 1992, Osse and van den
Boogaart 1995). The morphological and physiological changes
that occur during these developmental stages are characterized
by differential cellular and organelle functions (Einarsdottir et al. ́
2006). This is reflected in variations of global protein expres-
sion and posttranslational modifications of the proteins that may
cause alterations of protein function (Campinho et al. 2006).
Proteome analysis provides valuable information on the vari-
ations that occur within the proteome of organisms. These vari-
ations may, for example, reflect a response to biological per-
turbations or external stimuli (Anderson and Anderson 1998,
Martin et al. 2001, Martin et al. 2003b, Vilhelmsson et al. 2004)
resulting in different expression of proteins, posttranslational
modifications, or redistribution of specific proteins within cells
(Tyers and Mann 2003). To date, few studies on fish development
exist in which proteome analysis techniques have been applied.
Recent studies on global protein expression during early devel-
opmental stages of zebrafish (Tay et al. 2006) and Atlantic cod
(Sveinsdottir et al. 2008) revealed that distinctive protein pro- ́
files characterize the developmental stages of these fishes even
though abundant proteins are largely conserved during the ex-
perimental period. In both these studies, the identified proteins
consisted mainly of proteins located in the cytosol, cytoskeleton,
and nucleus. Proteome analyses in developing organisms have
shown that many of the identified proteins have multiple iso-
forms (Paz et al. 2006), reflecting either different gene products
(Guðmundsdottir et al. 1993) or posttranslationally modified ́
forms of these proteins (Jensen 2004). Different isoforms gen-
erated by posttranslational modifications are largely overlooked
by studies based on RNA expression. This fact further indicates
the importance of the proteome approach to understand cellular
mechanisms underlying fish development. Studies on various
proteins have shown that during fish development sequential
synthesis of different isoforms appear successively (Huriaux
et al. 1996, Galloway et al. 1998, Focant et al. 1999, Galloway
et al. 1999, Huriaux et al. 1999, Focant et al. 2000, Huriaux et al.
2003, Focant et al. 2003, Hall et al. 2003, Galloway et al. 2006,
Campinho et al. 2006, Campinho et al. 2007). In this context, de-
velopmental stage-specific muscle protein isoforms have gained
a special attention (Huriaux et al. 1996, Galloway et al. 1998,
Focant et al. 1999, Galloway et al. 1999, Huriaux et al. 1999,
Focant et al. 2000, Focant et al. 2003, Hall et al. 2003, Huriaux
et al. 2003, Galloway et al. 2006, Campinho et al. 2007).