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 H´olmfr´ıður Sveinsd´ottir, Samuel A M Martin, and Oddur T Vilhelmsson Proteomics Methodology Two-Dimensional Electrophoresis Basic 2DE Methods Overview Sample Extraction and Cleanup First-Dimension Electrophoresis Equilibration Second-Dimension Electrophoresis Staining Analysis Some Problems and Their Solutions Identification by Peptide Mass Fingerprinting Seafood Proteomics and Their Relevance to Processing and Quality Early Development and Proteomics of Fish Changes in the Proteome of Early Cod Larvae in Response to Environmental Factors Tracking Quality Changes Using Proteomics Antemortem Effects on Quality and Processability Species Authentication Identification and Characterization of Allergens Impacts of High Throughput Genomic and Proteomic Technologies References Abstract: Proteomics involves the study of proteins, with regards to proteins, their expression by genomes, their structures and functions The entire set of proteins or proteome expressed by a genome display variations in tissues and organisms, and can be used as the basis for evaluating the status and changes in the proteins in living organisms including fish and shellfish This feature can be useful for developing standards for fish and other food materials and assessing their quality and/or safety This chapter discusses current uses of proteomics for establishing the attributes of fish and fish products Proteomics is most succinctly defined as “the study of the entire proteome or a subset thereof,” the proteome being the expressed protein complement of the genome Unlike the genome, the proteome varies among tissues, as well as with time in reflection of the organism’s environment and its adaptation thereto Proteomics can, therefore, give a snapshot of the organism’s state of being and, in principle at least, map the entirety of its adaptive potential and mechanisms As with all living matter, foodstuffs are in large part made up of proteins This is especially true of fish and meat, where the bulk of the food matrix is constructed from proteins Furthermore, the construction of the food matrix, both on the cellular and tissue-wide levels, is regulated and brought about by proteins It stands to reason, then, that proteomics is a tool that can be of great value to the food scientist, giving valuable insight into the composition of the raw materials, quality involution within the product before, during, and after processing or storage, the interactions of proteins with one another or with other food components, or with the human immune system after consumption In this chapter, a brief overview of “classical” proteomics methodology is presented, and their present and future application in relation to fish and seafood processing and quality is discussed PROTEOMICS METHODOLOGY Unlike nucleic acids, proteins are an extremely variegated group of compounds in terms of their chemical and physical properties It is not surprising, then, that a field that concerns itself with “the systematic identification and characterization of proteins for their structure, function, activity and molecular interactions” (Peng et al 2003) should possess a toolkit containing a wide spectrum of methods that continue to be developed at a brisk pace While high-throughput, gel-free methods, for example, based on liquid chromatography tandem mass spectrometry (LC-MS/MS) (Peng et al 2003), surface-enhanced laser desorption/ionization (Hogstrand et al 2002), or protein arrays (Lee and Nagamune 2004), hold great promise and are deserving of discussion in their own right, the “classic” process of Food Biochemistry and Food Processing, Second Edition Edited by Benjamin K Simpson, Leo M.L Nollet, Fidel Toldr´a, Soottawat Benjakul, Gopinadhan Paliyath and Y.H Hui C 2012 John Wiley & Sons, Inc Published 2012 by John Wiley & Sons, Inc 406 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 407 Figure 22.1 An overview over the “classic approach” in proteomics First, a protein extract (crude or fractionated) from the tissue of choice is subjected to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) Once a protein of interest has been identified, it is excised from the gel, subjected to degradation by trypsin (or other suitable protease) and the resulting peptides analyzed by mass spectrometry (MS), yielding a peptide mass fingerprint In many cases, this is sufficient for identification purposes, but if needed, peptides can be dissociated into smaller fragments and small partial sequences obtained by tandem mass spectrometry (MS/MS) See text for further details two-dimensional electrophoresis (2DE) followed by protein identification via peptide mass fingerprinting of trypsin digests (Fig 22.1) 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: Andersen and Mann (2006), Balestrieri et al (2008), Beretta (2009), Bogyo and Cravatt (2007), Drabik et al (2007), Ikonomou et al (2009), Issaq and Veenstra (2008), Jorrin-Novo et al (2009), Latterich et al (2008), L´opez (2007), Mamone et al (2009), Malmstrom et al (2007), Premsler et al (2009), Smith et al (2009), Wang et al (2006), Wilm (2009), Yates et al (2009) Two-Dimensional Electrophoresis 2DE, the cornerstone of most proteomics research, is the simultaneous separation of hundreds, or even thousands, of proteins on a two-dimensional polyacrylamide slab gel The potential of a two-dimensional protein separation technique was realized early on, with considerable development efforts taking place in the 1960s (Margolis and Kenrick 1969, Kaltschmidt and Wittmann 1970) The method most commonly used today was developed by Patrick O’Farrell It is described in his seminal and thorough 1975 paper (O’Farrell 1975) and is outlined briefly later It is worth emphasizing that great care must be taken that the proteome under investigation is reproducibly represented on the 2DE gels, and that individual variation in specific protein P1: SFK/UKS BLBS102-c22 P2: SFK BLBS102-Simpson March 21, 2012 13:41 408 Trim: 276mm X 219mm Printer Name: Yet to Come Part 3: Meat, Poultry and Seafoods abundance is taken into consideration 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 67 kDa Basic 2DE Methods Overview 43 kDa 30 kDa 21 kDa 14 kDa pI Figure 22.2 A two-dimensional electrophoresis protein map of rainbow trout (Oncorhynchus mykiss) liver proteins with pI between and and molecular mass about 10–100 (S Martin, unpublished) The proteins are separated according to their pI in the horizontal dimension and according to their mass in the vertical dimension Isoelectro focussing was by pH 4–7 immobilized pH gradient (IPG) strip and the second dimension was in a 10–15% gradient polyacrylamide slab gel O’Farrell’s original 2DE method first applies a process called isoelectric focusing (IEF), where an electric field is applied to a tube gel on which the protein sample and carrier ampholytes 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–polyacrylamide gel electrophoresis (SDS-PAGE), yielding a two-dimensional map (Fig 22.2) rather than the familiar banding pattern observed in one-dimensional SDS-PAGE The map can be visualized and individual 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 22.3), upregulation or downregulation of these proteins can be inferred Although a number of refinements have been made to 2DE since O’Farrell’s paper, most notably, the introduction of immobilized pH gradients (IPGs) for IEF (Găorg et al 1988), the procedure Figure 22.3 A screenshot from the two-dimensional electrophoresis analysis program Phoretix 2-D (NonLinear Dynamics, Gateshead, Tyne & Wear, UK) showing some steps in the analysis of a two-dimensional protein map Variations in abundance of individual proteins, as compared with a reference gel, can be observed and quantified 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 remains essentially as outlined earlier In the following sections, a general protocol is outlined briefly with some notes of special relevance to the seafood scientist For more detailed, up-to-date protocols, the reader is referred to any of a number of excellent reviews and laboratory manuals such as Berkelman and Stenstedt (1998), Găorg et al (2000, 2004), Kraj and Silberring (2008), Link (1999), Simpson (2003), Walker (2005) and Westermeier and Naven (2002) Sample Extraction and Cleanup For most applications, sample treatment prior to electrophoresis should be minimal in order to minimize in-sample proteolysis and other sources of experimental artifacts We have found direct extraction into the gel reswelling buffer (7-M urea, 2-M thiourea, 4% (w/v) CHAPS [3-(3-chloramidopropyl)dimethylamino1-propanesulfonate], 0.3% (w/v) DTT [dithiothreitol], 0,5% Pharmalyte ampholytes for the appropriate pH range), supplemented with a protease inhibitor cocktail, to give good results for proteome extraction from whole Atlantic cod larvae (Guðmundsd´ottir and Sveinsd´ottir 2006, Sveinsd´ottir et al 2008) and Arctic charr (Salvelinus alpinus) liver (Coe and Vilhelmsson 2008) Thorough homogenization is essential to ensure complete and reproducible extraction of the proteome Cleanup of samples using commercial two-dimensional sample cleanup kits may be beneficial for some sample types First-Dimension Electrophoresis The extracted proteins are first separated by IEF, which is most conveniently performed using commercial dry IPG gel strips These strips consist of a dried IPG-containing polyacrylamide gel on a plastic backing Ready-made IPG strips are currently available in a variety of linear and sigmoidal pH ranges This method is thus suitable for most 2DE applications and has all but completely replaced the older and less reproducible method of IEF by carrier ampholytes in tube gels Broad-range linear strips (e.g., pH 3–10) are commonly used for whole-proteome analysis of tissue samples, but for many applications narrowrange and/or sigmoidal IPG strips may be more appropriate as these will give better resolution of proteins in the fairly crowded pI 4–7 range Narrow-range strips also allow for higher sample loads (since part of the sample will run off the gel) and thus may yield improved detection of low-abundance proteins Before electrophoresis, the dried gel needs to be reswelled to its original volume A recipe for a typical reswelling buffer is presented earlier Reswelling is normally performed overnight at 4◦ C Application of a low-voltage current may speed up the reswelling process Optimal conditions for reswelling are normally provided by the IPG strip manufacturer If the protein sample is to be applied during the reswelling process, extraction directly into the reswelling buffer is recommended IEF is normally performed for several hours at high voltage and low current Typically, the starting voltage is about 150 V, which is then increased step-wise to about 3500 V, usually totaling about 10,000 to 30,000 Vh, although this will depend on the IPG gradient and the length of the strip The appropriate 409 IEF protocol will depend not only on the sample and IPG strip, but also on the equipment used The manufacturers instructions should be followed Găorg et al (2000) reviewed IEF for 2DE applications Equilibration Before the isoelectrofocused gel strip can be applied to the second-dimension slab gel, it needs to be equilibrated for 30–45 minutes in a buffer containing SDS and a reducing agent such as DTT During the equilibration step, the SDS–polypeptide complex that affords protein-size-based separation will form and the reducing agent will preserve the reduced state of the proteins A tracking dye for the second electrophoresis step is also normally added at this point A typical equilibration-buffer recipe is as follows: 50 mM Tris-HCl at pH 8.8, 6-M urea, 30% glycerol, 2% SDS, 1% DTT, and trace amount of bromophenol blue A second equilibration step in the presence of 2.5% iodoacetamide and without DTT (otherwise identical buffer) may be required for some applications This will alkylate thiol groups and prevent their reoxidation during electrophoresis, thus reducing vertical streaking (Găorg et al 1987) Second-Dimension Electrophoresis Once the gel strip has been equilibrated, it is applied to the top edge of an SDS-PAGE slab gel and cemented in place using a molten agarose solution Optimal pore size depends on the size of the target proteins, but for most applications gradient gels or gels of about 10% or 12% polyacrylamide are appropriate Ready-made gels suitable for analytical 2DE are available commercially While some reviewers recommend alternative buffer systems (Walsh and Herbert 1999), the Laemmli method (Laemmli 1970), using glycine as the trailing ion and the same buffer (25-mM Tris, 192-mM glycine, 0.1% SDS) at both electrodes, remains the most popular one The gel is run at a constant current of 25 mA until the bromophenol blue dye front has reached the bottom of the gel Staining Visualization of proteins spots is commonly achieved through staining with colloidal Coomassie Blue G-250 due to its low cost and ease of use A typical staining procedure includes fixing the gel for several hours in 50% ethanol/2% ortho-phosphoric acid, followed by several 30-minute washing steps in water, followed by incubation for hour in 17% ammonium sulfate/34% methanol/2% ortho-phosphoric acid, followed by staining for several days in 0.1% Coomassie Blue G-250/17% ammonium sulfate/34% methanol/2% ortho-phosphoric acid, followed by destaining for several hours in water There are, however, commercially available colloidal Coomassie staining kits that not require fixation or destaining A great many alternative visualization methods are available, many of which are more sensitive than colloidal Coomassie and thus may be more suitable for applications where visualization of low-abundance proteins is important These include P1: SFK/UKS BLBS102-c22 P2: SFK BLBS102-Simpson March 21, 2012 13:41 Trim: 276mm X 219mm 410 Printer Name: Yet to Come Part 3: Meat, Poultry and Seafoods radiolabeling, such as with [35 S]methionine, and staining with fluorescent dyes such as the SYPRO or Cy series of dyes Multiple 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) 97 kDa 84 kDa 66 kDa 55 kDa Analysis Although 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 investigator (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 concern 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 variation has led some investigators (Barrett et al 2005, Karp et al 2005, Wheelock and Goto 2006) to cast doubt on the suitability of univariate tests such as the Student’s t-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 Solutions The 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 significant of these have to with the diversity of proteins and their expression levels For example, hydrophobic proteins 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 protein proteomics” to demonstrate the involvement of membraneassociated metabolic enzymes in the osmoadaptive response of the foodborne pathogen Staphylococcus aureus A 2DE gel image of S aureus membrane-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 the 45 kDa 36 kDa 24 kDa pI 10 Figure 22.4 A two-dimensional electrophoresis membrane proteome map from Staphylococcus aureus, showing proteins with pI between 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 (Găorg et al 1997), their representation on wide-range 2DE of complex mixtures such as cell extracts remained poor Improvements in resolution and representation of alkaline proteins on wide-range gels have been made (Găorg et al 1999), but nevertheless an approach that involves several gels, each of a different pH range, from the same sample 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 relatively highly alkaline Helicobacter pylori proteome using both pH 6–11 and pH 9–12 IPGs (Bae et al 2003) A second drawback of 2DE has to with the extreme difference 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 twodimensional map, rendering analysis of low-abundance proteins difficult or impossible For applications such as species identification or study of the major biochemical pathways, where the proteins of interest are present in relatively high abundance, ... recent ones include: Andersen and Mann (2006), Balestrieri et al (20 08) , Beretta (2009), Bogyo and Cravatt (2007), Drabik et al (2007), Ikonomou et al (2009), Issaq and Veenstra (20 08) , Jorrin-Novo... (Guðmundsd´ottir and Sveinsd´ottir 2006, Sveinsd´ottir et al 20 08) and Arctic charr (Salvelinus alpinus) liver (Coe and Vilhelmsson 20 08) Thorough homogenization is essential to ensure complete and reproducible... (19 98) , Găorg et al (2000, 2004), Kraj and Silberring (20 08) , Link (1999), Simpson (2003), Walker (2005) and Westermeier and Naven (2002) Sample Extraction and Cleanup For most applications, sample