BLBS102-c22 BLBS102-Simpson March 21, 2012 13:41 Trim: 276mm X 219mm Printer Name: Yet to Come
22 Application of Proteomics to Fish Processing and Quality 417the effects on the proteasome are particularly noteworthy. The
proteasome is a multisubunit enzyme complex that catalyzes
proteolysis via the ATP-dependent ubiquitin–proteasome path-
way, which, in mammals, is thought to be responsible for a
large fraction of cellular proteolysis (Rock et al. 1994, Craiu
et al. 1997). In rainbow trout, the ubiquitin–proteasome pathway
has been shown to be downregulated in response to starvation
(Martin et al. 2002) and to have a role in regulating protein
deposition efficiency (Dobly et al. 2004). It, therefore, seems
likely that the observed difference in texture is affected by ante-
mortem proteasome activity, although further studies are needed
to verify that statement.
In addition to having a hand in controlling autolysis determi-
nants, protein turnover is a major regulatory engine of cellular
structure, function, and biochemistry. Cellular protein turnover
involves at least two major systems: (1) the lysosomal system and
(2) the ubiquitin–proteasome system (Hershko and Ciechanover
1986, Mortimore et al. 1989). The 20S proteasome has been
found to have a role in regulating the efficiency with which rain-
bow trout deposit protein (Dobly et al. 2004). It seems likely
that the manner in which protein deposition is regulated, par-
ticularly in muscle tissue, has profound implications for quality
and processability of the fish flesh.
Protein turnover systems, such as the ubiquitin–proteasome or
the lysosome systems, are suitable for rigorous investigation us-
ing proteomic methods. For example, lysosomes can be isolated
and the lysosome subproteome queried to answer the question
whether and to what extent lysosome composition varies among
fish expected to yield flesh of different quality characteristics.
Proteomic analysis on lysosomes has been successfully per-
formed in mammalian (human) systems (Journet et al. 2000,
Journet et al. 2002).
An exploitable property of proteasome-mediated protein
degradation is the phenomenon of polyubiquitination, whereby
proteins are targeted for destruction by the proteasome by
covalent binding to multiple copies of ubiquitin (Hershko
and Ciechanover 1986, Ciechanover 1994). By targeting these
ubiquitin-labeled proteins, it is possible to observe the ubiquitin–
proteasome “degradome,” that is, which proteins are being
degraded by the proteasome at a given time or under given
conditions. Gygi and coworkers have developed methods to
study the ubiquitin–proteasome degradome in the yeastSaccha-
romyces cerevisiaeusing multidimensional LC-MS/MS (Peng
et al. 2003).
Some proteolysis systems, such as that of the matrix metal-
loproteases, may be less directly amenable to proteomic study.
Activity of matrix metalloproteases is regulated via a complex
network of specific proteases (Brown et al. 1993, Okumura et al.
1997, Wang and Lakatta 2002). Monitoring of the expression
levels of these regulatory enzymes, and how they vary with
environmental or dietary variables, may be more conveniently
carried out using transcriptomic methods.
A well-known quality issue when farmed fish are compared
with wild catch is that of gaping, a phenomenon caused by
cleavage of myocommatal collagen cross-links that results in
weakening and rupturing of connective tissue (Børresen 1992,
Foegeding et al. 1996). Gaping can be a serious quality issue inthe fish processing industry as, apart from the obvious visual de-
fect, it causes difficulties in mechanical skinning and slicing of
the fish (Love 1992). Weakening of collagen and, hence, gaping
is facilitated by low pH. Well-fed fish, such as those reared in
aquaculture, tend to yield flesh of comparatively low pH, which,
thus, tends to gape (Foegeding et al. 1996, Einen et al. 1999).
Gaping is, therefore, a cause for concern with aquaculture-reared
fish, particularly of species with high natural gaping tendency,
such as the Atlantic cod. Gaping tendency varies considerably
among wild fish caught in different areas (Love et al. 1974)
and, thus, it is conceivable that gaping tendency can be con-
trolled with dietary or other environmental manipulations. Pro-
teomics and transcriptomics, with their capacity to monitor mul-
tiple biochemical processes simultaneously, are methodologies
eminently suitable to finding biochemical or metabolic markers
that can be used for predicting features such as gaping tendency
of different stocks reared under different dietary or environmen-
tal conditions.Species AuthenticationFood authentication is an area of increasing importance, both
economically and from a public health standpoint. Taking into
account the large difference in market value of different fish
species and the increased prevalence of processed product on the
market, it is perhaps not surprising that species authentication
is fast becoming an issue of supreme commercial importance.
Along with other molecular techniques, such as DNA-based
species identification (Sotelo et al. 1993, Mackie et al. 1999,
Martinez et al. 2001b, Pascoal et al. 2008) and nuclear magnetic
resonance-based techniques for determining geographical origin
(Campana and Thorrold 2001), proteomics are proving to be a
powerful tool in this area, particularly for addressing questions
on health status of the organism, stresses or contamination lev-
els at place of breeding, and postmortem treatment (Martinez
et al. 2003, Martinez and Jakobsen Friis 2004). Since, unlike
the genome, the proteome is not a static entity, but changes
between tissues and with environmental conditions, proteomics
can potentially yield more information than genomic ones, pos-
sibly indicating freshness and tissue information in addition to
species. Therefore, although it is likely that DNA-based methods
will remain the methods of choice for species authentication in
the near term, proteomic methods are likely to develop rapidly
and find commercial uses within this field. In many cases, the
proteomes of even closely related fish species can be easily dis-
tinguishable by eye from one another on two-dimensional gels
(Fig. 22.6), indicating that diagnostic protein spots may be used
to distinguish closely related species.
From early on, proteomic methods have been recognized as
a potential way of fish species identification. During the 1960s,
one-dimensional electrophoretic techniques were developed to
identify the raw flesh of various species (Tsuyuki et al. 1966,
Cowie 1968, Mackie 1969), which was soon followed by meth-
ods to identify species in processed or cooked products (Mackie
1972, Mackie and Taylor 1972). These early efforts were re-
viewed in 1980 (Mackie 1980, Hume and Mackie 1980).