P2: SFK 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 this does not present a problem However, when investigating, for example, regulatory cascades, the proteins of interest are likely to be present in very low abundance and may at times be undetectable because of the dominance of high-abundance ones Simply increasing the amount of sample is usually not an option, as it will give rise to overloading artifacts in the gels (O’Farrell 1975) In transcriptomic studies, where a similar disparity can be seen in the abundance of RNA transcripts present, this problem can be overcome by amplifying the lowabundance transcripts using the polymerase chain-reaction, but no such technique is available for proteins The remaining option, then, is fractionation of the protein sample in order to weed out the high-abundance proteins, allowing a larger sample of the remaining proteins to be analyzed A large number of fractionation protocols, both specific and general, are available Thus, Østergaard and coworkers used acetone precipitation to reduce the abundance of hordeins present in barley (Hordeum vulgare) extracts (Østergaard et al 2002) whereas Locke and coworkers used preparative isoelectrofocussing to fractionate Chinese snow pea (Pisum sativum macrocarpon) lysates into fractions covering three pH regions (Locke et al 2002) The fractionation method of choice will depend on the specific requirements of the study and on the tissue being studied Discussion of some fractionation methods can be found in Ahmed (2009), BodzonKulakowska et al (2007), Butt et al (2001), Canas et al (2007), Corthals et al (1997), Dreger (2003), Fortis et al (2008), Issaq 411 et al (2002), Lee and Pi (2009), Lopez et al (2000), Millea and Krull (2003), Pieper et al (2003), Righetti et al (2005a, b) Rothemund et al (2003), von Horsten (2006) Identification by Peptide Mass Fingerprinting Identification of proteins on 2DE gels is most commonly achieved via mass spectrometry (MS) of trypsin digests Briefly, the spot of interest is excised from the gel, digested with trypsin (or another protease), and the resulting peptide mixture is analyzed by MS The most popular MS method is matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS (Courchesne and Patterson 1999), where peptides are suspended in a matrix of small, organic, ultraviolet-absorbing molecules (such as 2,5-dihydroxybenzoic acid) followed by ionization by a laser at the excitation wavelength of the matrix molecules and acceleration of the ionized peptides in an electrostatic field into a flight tube where the time of flight of each peptide is measured, giving its expected mass The resulting spectrum of peptide masses (Fig 22.5) is then used for protein identification by searching against expected peptide masses calculated from data in protein sequence databases, such as SwissProt or the National Centre for Biotechnology Information (NCBI) nonredundant protein sequences data base, using the appropriate software Several programs are available, many with a web-based open-access interface The ExPASy Intensity P1: SFK/UKS BLBS102-c22 Mass (m/z) Figure 22.5 A trypsin digest mass spectrometry fingerprint of a rainbow trout liver protein spot, identified as apolipoprotein A I-1 (S Martin, unpublished) The open arrows indicate mass peaks corresponding to trypsin self-digestion products and were, therefore, excluded from the analysis The solid arrows indicate the peaks that were found to correspond to expected apolipoprotein A I-1 peptides P1: SFK/UKS BLBS102-c22 P2: SFK BLBS102-Simpson 412 March 21, 2012 13:41 Trim: 276mm X 219mm Printer Name: Yet to Come Part 3: Meat, Poultry and Seafoods Tools website (http://www.expasy.org/tools) contains links to most of the available software for protein identification and several other tools Attaining a high identification rate is problematic in fish and seafood proteomics due to the relative paucity of available protein sequence data for these animals As can be seen in Table 22.1, this problem is surprisingly acute for species of commercial 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 available, 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 obtaining protein identities is by direct sequence comparison Until recently, this was accomplished by N-terminal or internal (after proteolysis) sequencing by Edman degradation of eluted or electroblotted protein spots (Kamo and Tsugita 1999, ErdjumentBromage 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 combinations 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 dissociated 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 QUALITY 2DE-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), wheat flour 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, Pi˜neiro 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 Fish Fishes 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 (Einarsd´ottir et al 2006) This is reflected in variations of global protein expression and posttranslational modifications of the proteins that may cause alterations of protein function (Campinho et al 2006) Proteome analysis provides valuable information on the variations that occur within the proteome of organisms These variations may, for example, reflect a response to biological perturbations 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 developmental stages of zebrafish (Tay et al 2006) and Atlantic cod (Sveinsd´ottir et al 2008) revealed that distinctive protein profiles characterize the developmental stages of these fishes even though abundant proteins are largely conserved during the experimental 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 isoforms (Paz et al 2006), reflecting either different gene products (Guðmundsd´ottir et al 1993) or posttranslationally modified forms of these proteins (Jensen 2004) Different isoforms generated 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, developmental 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) P1: SFK/UKS BLBS102-c22 P2: SFK BLBS102-Simpson March 21, 2012 13:41 Trim: 276mm X 219mm Printer Name: Yet to Come 413 22 Application of Proteomics to Fish Processing and Quality Table 22.1 Some Commercially or Scientifically Important Fish and Seafood Species and the Availability of Protein and Nucleotide Sequence Data as of February 18, 2010, According to the NCBI TaxBrowser(a) Protein Sequences Nucleotide Sequences Actinopterygii (ray-finned fishes) 257,843 1,586,862 Elopomorpha Anguilliformes (eels and morays) 3,218 2,898 4,459 4,144 European eel (Anguilla anguilla) Clupeomorpha Clupeiformes (herrings) 227 2,333 2,333 404 4,697 4,697 Atlantic herring (Clupea harengus) European pilchard (Sardina pilchardus) Ostariophysii Cypriniformes (carps) 105 79 166 401 104,088 94,392 211,491 196,415 70,151 7,149 163,995 10,472 1,890 1,996 32,187 31,244 42,475 42,278 16,230 6,216 19,107 8,361 257 5,396 4,517 2,106 382 465 6,397 5,573 1,964 303 Zebrafish (Danio rerio) Siluriformes (catfishes) Channel catfish (Ictalurus punctatus) Protacanthopterygii Salmoniformes (salmons) Atlantic salmon (Salmo salar) Rainbow trout (Oncorhynchus mykiss) Arctic charr (Salvelinus alpinus) Paracanthopterygii Gadiformes (cods) Atlantic cod (Gadus morhua) Alaska pollock (Theragra chalcogramma) Saithe (Pollachius virens) Haddock (Melanogrammus aeglefinus) Lophiiformes (anglerfishes) Monkfish (Lophius piscatorius) Protein Sequences Nucleotide Sequences 34,032 114,496 2,593 28,483 16,406 126,200 138 102 4,858 126 100 7,896 13 20 17 30 6,407 692,476 1,812 261 2,234 197 48 52 Lamniformes (mackrel sharks) Basking shark (Cetorhinus maximus) Rajiformes (skates) Thorny skate (Raja radiata) 554 66 676 72 848 41 1,050 60 Blue skate (Raja batis) Little skate (Raja erinacea) 201 185 Mollusca (mollusks) Bivalvia 52,622 14,572 154,100 25,149 Tetraodontiformes (puffers and filefishes) Pufferfish (Takifugu rubripes) Green pufferfish (Tetraodon nigroviridis) Zeiformes (dories) John Dory (Zeus faber) Scorpaeniformes (scorpionfishes/flatheads) Redfish (Sebastes marines) Lumpsucker (Cyclopterus lumpus) Chondrichthyes (cartilagenous fishes) Carcharhiniformes (ground sharks) Lesser spotted dogfish (Scyliorhinus canicula) Blue shark (Prionace glauca) 51 189 53 151 Blue mussel (Mytilus edulis) Bay scallop (Argopecten irradians) 1,307 194 1,266 380 412 40 471 104 32,489 121,832 11 Acanthopterygii Perciformes (perch like) Gilthead sea bream (Sparus aurata) 104,101 44,027 516 1,369,306 619,647 956 133 3,964 34 279 5,045 49 European sea bass (Dicentrachus labrax) Atlantic mackrel (Scomber scombrus) Albacore (Thunnus alalunga) Bluefin tuna (Thunnus thynnus) Spotted wolffish (Anarhichas minor) Beryciformes (sawbellies) 466 36,916 351 335 111 129 Gastropoda Common whelk (Buccinum undatum) Abalone (Haliotis tuberculata) Cephalopoda Northern European squid (Loligo forbesi) Common cuttlefish (Sepia officinalis) Common octopus (Octopus vulgaris) 169 204 116 179 38 794 227 935 23 616 Crustacea (crustaceans) Caridea Northern shrimp (Pandalus borealis) 44,586 5,103 19 70,954 7,630 26 P1: SFK/UKS BLBS102-c22 P2: SFK BLBS102-Simpson March 21, 2012 13:41 Trim: 276mm X 219mm 414 Printer Name: Yet to Come Part 3: Meat, Poultry and Seafoods Table 22.1 (Continued ) Orange roughy (Hoplostethus atlanticus) Pleuronectiformes (flatfishes) Protein Sequences Nucleotide Sequences 11 40 4,109 11,851 Atlantic halibut (Hippoglossus hippoglossus) Witch (Glyptocephalus cynoglossus) 218 2,958 22 54 Plaice (Pleuronectes platessa) Winter flounder (Pseudopleuronectes americanus) Turbot (Scophthalmus maximus) 88 139 385 Astacidea (lobsters and crayfishes) Protein Sequences Nucleotide Sequences 1,742 4,466 195 173 28 39 19 35 285 196 American lobster (Homerus americanus) European crayfish (Astacus astacus) Langoustine (Nephrops norvegicus) Brachyura (short-tailed crabs) Edible crab (Cancer pagurus) 5,143 37 8,238 38 1,014 Blue crab (Callinectes sapidus) 123 133 Source: (a) http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html/ The developmental changes in the composition of muscle protein isoforms have been tracked by proteome analysis in African catfish (Heterobranchus longifilis) (Focant et al 1999), common sole (Solea solea) (Focant et al 2003), and dorada (Brycon moorei) (Huriaux et al 2003) These studies demonstrated that the muscle shows the usual sequential synthesis of protein isoforms in the course of development For example, in the common sole, 2DE revealed two isoforms (larval and adult) of myosin light chain 2, and likewise in dorada larval and adult isoforms of troponin I were sequentially expressed during development Proteomic techniques have, thus, been shown to be applicable for investigating cellular and molecular mechanisms involved in the morphological and physiological changes that occur during fish development The major obstacle on the use of proteomics in embryonic fish has been the high proportion of yolk proteins These interfere with any proteomic application that intends to target the cells of the embryo proper In a recent study on the proteome of embryonic zebrafish, the embryos were deyolked to enrich the pool of embryonic proteins and to minimize ions and lipids found in the yolk prior to two-dimensional gel analysis (Tay et al 2006) Despite this undertaking, a large number of yolk proteins remained prominently present in the embryonic protein profiles Link et al (2006) published a method to efficiently remove the yolk from large batches of embryos without losing cellular proteins The success in the removal of yolk proteins by Link et al (2006) is probably due to dechorionation prior to the deyolking of the embryos By dechorionation, the embryos fall out of their chorions facilitating the removal of the yolk (Huriaux et al 1996, Westerfield 2000) Changes in the Proteome of Early Cod Larvae in Response to Environmental Factors The production of good quality larvae is still a challenge in marine fish hatcheries Several environmental factors can interfere with the protein expression of larvae affecting larval quality like growth and survival rate Proteome analysis allows us to examine the effects of environmental factors on larval global protein expression, posttranslational modifications, and redistribution of specific proteins within cells (Tyers and Mann 2003), all important information for controlling factors influencing the aptitude to continue a normal development until adult stages A variety of environmental factors have shown to improve the health and survival of fish larvae, including probiotic bacteria (Tinh et al 2008) and protein hydrolysates (Cahu et al 1999) However, the beneficial effects of these treatments on fish larvae are poorly understood at the molecular level Only a few proteome analysis studies on fish larvae have been published (Focant et al 2003, Tay et al 2006, Guðmundsd´ottir and Sveinsd´ottir 2006, Sveinsd´ottir et al 2008, Sveinsd´ottir and Guðmundsd´ottir 2008, Sveinsd´ottir et al 2009), of which two have focused on the changes in the whole larval proteome after treatment with probiotic bacteria (Sveinsd´ottir et al 2009) and protein hydrolysate (Sveinsd´ottir and Gudmundsd´ottir 2008) These studies provide protocols for the production of high-resolution two-dimensional gels of whole larval proteome, where peptide mass mapping (MALDI-TOF MS) and peptide fragment mapping (LC-MS/MS) allowed identification of ca 85% of the of the selected cod protein spots (Guðmundsd´ottir and Sveinsd´ottir 2006, Sveinsd´ottir and Gudmundsd´ottir 2008, Sveinsd´ottir et al 2008, Sveinsd´ottir et al 2009) The advantages of working with whole larvae versus distinct tissues is the ease of keeping the sample handling to a minimum in order to avoid loss or modification of the proteins Nevertheless, there are several drawbacks when working with the whole larval proteome, like the overwhelming presence of muscle and skin proteins These proteins may mask subtle changes in proteins expressed in other tissues or systems, such as the gastrointestinal tract or the central nervous system caused by various environmental factors The axial musculature is the largest tissue in larval fishes as it constitutes approximately 40% of their body mass (Osse and van den Boogaart 1995) This is reflected in our studies on whole cod larval proteome, where the majority P1: SFK/UKS BLBS102-c22 P2: SFK 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 of the highly abundant proteins were identified as muscle proteins (Guðmundsd´ottir and Sveinsd´ottir 2006, Sveinsd´ottir and Gudmundsd´ottir 2008, Sveinsd´ottir et al 2008, 2009) Also, cytoskeletal proteins were prominent among the identified proteins Removal of those proteins may increase detection of other proteins present at low concentrations However, it may also result in a loss of other proteins, preventing identification of holistic alterations in the analyzed proteomes Various strategies have been presented for the removal of highly abundant proteins (Ahmed et al 2003) or enrichment of low-abundance proteins (Oda et al 2001, Ahmed and Rice 2005) Tracking Quality Changes Using Proteomics A persistent problem in the seafood industry is postmortem degradation of fish muscle during chilled storage, which has deleterious effects on the fish flesh texture, yielding a tenderized muscle This phenomenon is thought to be primarily due to autolysis of muscle proteins, but the details of this protein degradation are still somewhat in the dark However, degradation of myofibrillar proteins by calpains and cathepsins (Ogata et al 1998, Ladrat et al 2000) and degradation of the extracellular matrix by the matrix metalloproteases and matrix serine proteases, capable of degrading collagens, proteoglycans, and other matrix components (Woessner 1991, Lødemel and Olsen 2003), are thought to be among the main culprits Whatever the mechanism, it is clear that these quality changes are species dependent (Papa et al 1996, Verrez-Bagnis et al 1999) and, furthermore, appear to display seasonal variations (Ing´olfsd´ottir et al 1998, Ladrat et al 2000) For example, whereas desmin is degraded postmortem in sardine and turbot, no desmin degradation was observed in sea bass and brown trout (Verrez-Bagnis et al 1999) Of further concern is the fact that several commercially important fish muscle processing techniques, such as curing, fermentation, and production of surimi and conserves occur under conditions conducive to endogenous proteolysis (P´erez-Borla et al 2002) As with postmortem protein degradation during storage, autolysis during processing seems to be somewhat specific Indeed, the myosin heavy chain of the Atlantic cod was shown to be significantly degraded during processing of “salt fish” (bacalhau) whereas actin was less affected (Thorarinsdottir et al 2002) Problems of this kind, where differences are expected to occur in the number, molecular mass, and pI of the protein present in a tissue, are well suited to investigation using 2DEbased proteomics It is also worth noting that protein isoforms other than proteolytic ones, whether they be encoded in structural genes or brought about by posttranslational modification, usually have different molecular weight or pI and can, therefore, be distinguished on 2DE gels Thus, specific isoforms of myofibrillar proteins, many of which are correlated with specific textural properties in seafood products, can be observed using 2DE or other proteomic methods (Martinez et al 1990, Pi˜neiro et al 2003) Several 2DE studies have been performed on postmortem changes in seafood flesh (Verrez-Bagnis et al 1999, Morzel et al 2000, Martinez et al 2001a, Kjaersgard and Jessen 2003, 2004, Martinez and Jakobsen Friis 2004, Kjaersgard et al 415 2006a, b) and have demonstrated the importance and complexity of proteolysis in seafood during storage and processing For example, Martinez and Jakobsen Friis used a 2DE approach to demonstrate different protein composition of surimi made from prerigor versus postrigor cod (Martinez and Jakobsen Friis 2004) They found that 2DE could be used as a diagnostic tool to indicate the freshness of the raw material used for surimi production, a finding of considerable economic and public health interest Kjaersgard and Jessen, who used 2DE to study changes in abundance of several muscle proteins during storage of the Atlantic cod (Gadus morhua), proposed a general model for postmortem protein degradation in fish flesh where initially calpains were activated due to the increase in calcium levels in the muscle tissue Later, as pH decreases and ATP is depleted with the consequent onset of rigor mortis, cathepsins and the proteasome are activated sequentially (Kjaersgard and Jessen 2003) Antemortem Effects on Quality and Processability Malcolm Love started his 1980 review paper on biological factors affecting fish processing (Love 1980) with a lament for the easy life of poultry processors who, he said, had the good fortune to work on a product reared from hatching under strictly controlled environmental and dietary conditions “so that plastic bundles of almost identical foodstuff for man can be lined up on the shelf of a shop.” Since the time of Love’s review, the advent of aquaculture has made attainable, in theory at least, just such a utopic vision As every food processor knows, the quality of the raw material is among the most crucial variables that affect the quality of the final product In fish processing, therefore, the animal’s own individual physiological status will to a large extent dictate where quality characteristics will fall within the constraints set by the species’ physical and biochemical makeup It is well known that an organism’s phenotype, including quality characteristics, is determined by environmental as well as genetic factors Indeed, Huss noted in his review (Huss 1995) that product quality differences within the same fish species can depend on feeding and rearing conditions, differences wherein can affect postmortem biochemical processes in the product, which, in turn, affect the involution of quality characteristics in the fish product The practice of rearing fish in aquaculture, as opposed to wild-fish catching, therefore raises the tantalizing prospect of managing quality characteristics of the fish flesh antemortem, where individual physiological characteristics, such as those governing gaping tendency, flesh softening during storage, etc., are optimized To achieve that goal, the interplay between these physiological parameters and environmental and dietary variables needs to be understood in detail With the ever-increasing resolving power of molecular techniques, such as proteomics, this is fast becoming feasible In mammals, antemortem protease activities have been shown to affect meat quality and texture (Vaneenaeme et al 1994, Kristensen et al 2002) For example, an antemortem upregulation of calpain activity in swine (Sus scrofa) will affect postmortem proteolysis and, hence, meat tenderization (Kristensen et al 2002) ... Sequences Nucleotide Sequences 34,032 114,496 2,593 28, 483 16,406 126,200 1 38 102 4 ,85 8 126 100 7 ,89 6 13 20 17 30 6,407 692,476 1 ,81 2 261 2,234 197 48 52 Lamniformes (mackrel sharks) Basking shark... (carps) 105 79 166 401 104, 088 94,392 211,491 196,415 70,151 7,149 163,995 10,472 1 ,89 0 1,996 32, 187 31,244 42,475 42,2 78 16,230 6,216 19,107 8, 361 257 5,396 4,517 2,106 382 465 6,397 5,573 1,964... Sequences Nucleotide Sequences Actinopterygii (ray-finned fishes) 257 ,84 3 1, 586 ,86 2 Elopomorpha Anguilliformes (eels and morays) 3,2 18 2 ,89 8 4,459 4,144 European eel (Anguilla anguilla) Clupeomorpha Clupeiformes