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Proteomics for the authentication of fish species
Proteomics for the authentication of fish species Maria Fiorella Mazzeo, Rosa Anna Siciliano PII: DOI: Reference:
S1874-3919(16)30064-1 doi: 10.1016/j.jprot.2016.03.007 JPROT 2446
To appear in:
Journal of Proteomics
Received date: Revised date: Accepted date:
17 December 2015 15 February 2016 1 March 2016
Please cite this article as: Mazzeo Maria Fiorella, Siciliano Rosa Anna, Proteomics for the authentication of fish species, Journal of Proteomics (2016), doi: 10.1016/j.jprot.2016.03.007
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Proteomics for the authentication of fish species
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Maria Fiorella Mazzeo, Rosa Anna Siciliano*
Centro di Spettrometria di Massa Proteomica e Biomolecolare, Istituto di Scienze dell'Alimentazione, CNR,
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E-mail: [email protected]; [email protected]
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via Roma 64, 83100 Avellino, Italy. Tel +39 0825 299363, Fax +39 0825 781585.
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*corresponding author
Keywords: fish authentication; proteomics; mass spectrometry; molecular profiling; fish frauds
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ACCEPTED MANUSCRIPT ABSTRACT
Assessment of seafood authenticity and origin, mainly in the case of processed products (fillets, sticks, baby
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food) represents the crucial point to prevent fraudulent deceptions thus guaranteeing market transparency and
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consumers health. The most dangerous practice that jeopardies fish safety is intentional or unintentional
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mislabeling, originating from the substitution of valuable fish species with inferior ones. Conventional analytical methods for fish authentication are becoming inadequate to comply with the strict regulations issued by European Union and with the increase of mislabeling due to the introduction on the
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market of new fish species and market globalization.
This evidence prompts the development of high-throughput approaches suitable to identify unambiguous
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biomarkers of authenticity and screen a large number of samples with minimal time consumption. Proteomics provides suitable and powerful tools to investigate main aspects of food quality and safety and has given an important contribution in the field of biomarkers discovery applied to food authentication. .
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This report describes the most relevant methods developed to assess fish identity and offers a perspective on their potential in the evaluation of fish quality and safety thus depicting the key role of proteomics in the
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authentication of fish species and processed products.
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ACCEPTED MANUSCRIPT 1. Introduction
Authentication of fishery products represents a central issue for the assessment of food quality and safety. In
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the fishery market, the most common falsification is the substitution of more valuable fish species with
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inferior ones. In particular the identification of processed products by visual inspection becomes quite
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challenging as anatomical and morphological characteristics (such as the head, fins, skin, or bones), essential for fish species authentication, are lost during processing. These frauds are constantly growing due to market globalization and are leading to an illegal economic gain but, more alarmingly to severe health risks for the consumers. Since 2000, EU issued directives and regulations regarding fishery and aquaculture products
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establishing what must be reported in label (fish species, geographical origin, and production method (wild or cultivated) (EC No 104/2000 and 2065/2001) [1,2]. []. Moreover, the European Food Safety Authority
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(EFSA) defined a detailed system of traceability for food (including fishery and aquaculture products) and feed businesses to control food safety at all stages, as reported in EC regulation no. 178/2002 [3] . More recently, EU introduced a more stringent regulation (EC No 1379/2013) [4] on different aspects of the common organization of the markets in fishery and aquaculture products (e.g. provisions on consumer
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information, recognition of producer organizations, extension of rules, production and marketing planning),
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and the Commission Implementing Regulation No 1420/2013 [5] to define in details the rules for the adoption of EC No 1379/2013 [6,7]. First analytical methods for fish authentication have been based on the
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on the analysis of protein extracts by electrophoretic, chromatographic and immunological methods.
In particular, IEF analysis of sarcoplasmic proteins has been applied to discriminate different fish and shrimp [8-12] and considered the validated method for species identification by the Association of Official
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Analytical Chemistry [13]. IEF profiles of sarcoplasmic proteins from different fish species have been collected by FDA US in the internet library Regulatory Fish Encyclopedia [14]. More recently, DNA-based approaches, centered on the amplification of specific DNA-fragments (cytochrome b gene [15-18], mtDNA control region [19] and 12S rRNA region [20,21]) using PCR have been developed. DNA-barcoding has been also proposed as a reliable method for fish authentication and DNA barcode data has been included in FDA's Regulatory Fish Encyclopedia 22,23]. These methods are sensitive and specific, even in the case of closely related species [24,25], however, processing of fishery products such as heat treatment and acidic conditions could damage DNA integrity, thus leading to non-specific identification. Moreover, the difficulties to standardize protocols for DNA analyses could cause inconsistencies in results from different laboratories that could lead to regulatory or legal implications. The high costs of analysis should also be taken into account [26,27]. Although the efforts to improve the legislation and the analytical methods for seafood identification have reduced the occurrence of fish mislabeling, in some European countries and, to a lesser extent, in US, this phenomenon still represents a severe risk for consumers health [28,29]. In fact, the possibility to get great 3
ACCEPTED MANUSCRIPT economic gains also throughout illegal, unreported and unregulated (IUU) fishing, exploiting the increasing worldwide fish consumption and the market globalization, continuously fed the fraudulent and dangerous practice of fish mislabeling [30,31] In this frame, it is mandatory the development of high-throughput approaches suitable to discover
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unambiguous biomarkers of authenticity and screen a large number of samples with minimal time
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consumption. In the recent years, among the omics approach, proteomics has significantly contributed to
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investigate the main aspects of food quality and safety, including traceability, authenticity, absence of contaminating and/or adulterating agents and impact of the processing/storage conditions [32]. In the field of fish authentication, first results were obtained applying the classical proteomic strategy that integrates 2-DE and mass spectrometry. Afterwards, methods based on the analysis of peptides and intact proteins by high
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resolution and tandem mass spectrometry opened the way to a new phase in biomarkers discovery, aimed to assess identity of fish-based food [33-35]. On the other hand, molecular profiling strategies based on
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MALDI-TOF-MS emerged as an alternative tool to define biomarkers of fish authenticity [36-38]. The topic of this report is the role that proteomics held in the discovery of biomarkers suitable for fish authentication. A special emphasis is given to the ability of advanced proteomics-based approaches to
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on the protection of consumer health.
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provide reliable quality control methods to detect deceptive practices and frauds, thus fulfilling EU directives
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2. The first proteomic approach integrating 2-DE and MS
Electrophoretic methods have been widely applied to achieve fish authentication. Noteworthy, IEF analyses of the sarcoplasmic proteins extracted from fish muscle tissues led to define species-specific profiles suitable to discriminate several fish species. Differences in protein patterns were originated by a class of proteins, the
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parvalbumins (PRVBs) that showed a high variability in their primary structure [10]. These proteins are calcium-binding proteins with molecular weight in the 11-12 kDa range, abundant in muscle tissue and highly soluble in aqueous buffers. They are also the major allergy-eliciting proteins in fish. Moreover, due to their structural features, PRVBs showed a remarkable thermal stability [39,40]. More recently, the stability of PRVBs to high pressure processing has also been assessed [41]. Therefore, PRVBs hold all the required features to be used as biomarkers to evaluate the identity of raw and processed fish products. The advent of proteomics in the 1994 and its relative rapid and widespread application in each field of protein science provided innovative tools for biomarker-detection also related to food quality or safety. The classical bottom-up proteomic approach, that integrated 2-DE for protein separation and mass spectrometry for protein identification, was initially applied by Pineiro et al. to discriminate five closely related hake species (Fig. 1). Protein spots responsible for differences in 2-DE maps obtained from the analysis of sarcoplasmic extracts from white muscle of Merluccius merluccius (European hake), Merluccius australis (Southern hake), Merluccius hubbsi (Argentinian hake), Merluccius gayi (Chilean hake), and Merluccius capensis (Cape hake) were subjected to in-gel tryptic digestion and the obtained peptide mixtures were 4
ACCEPTED MANUSCRIPT analyzed by MALDI-TOF-MS and MS/MS. Using mass spectrometric data, PRVBs and a nucleoside diphosphate kinase protein were identified. Peptide mass maps obtained from the nucleoside diphosphate kinase allowed the differential classification of the hake species into two groups: the East Atlantic coast group and the West Atlantic coast group. Moreover, the presence of a specific signal in the peptide mass
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maps of PRVBs, unambiguously discriminated Southern hake from the other species [42]. A similar
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approach was used to discriminate three European marine mussel species: Mytilus edulis, Mytilus
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galloprovincialis and Mytilus trossulus. MALDI-TOF peptide mass maps were generated from proteins contained in six randomly selected prominent spots present in the 2-DE gels obtained from foot protein extracts. Two peptides were selected as biomarkers of Mytilus edulis and Mytilus galloprovincialis, whereas another peptide was selected to discriminate Mytilus trossulus. MS/MS followed by database searching and
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de novo sequencing determined the primary structure of these peptides [43].
Analysis of sarcoplasmic extracts from raw white muscle by 2-DE and MALDI-TOF-MS allowed
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differentiating ten closely related species of commercial relevance. A method for fish authentication was drawn relying on differences of the PRVBs pattern in the 2-DE maps and on the presence or absence of species-specific signals in MALDI-TOF-MS spectra originated from the analysis of in-gel digested PRVBs of each analyzed species. This method led to discriminate nine hake species of the genus Merluccius
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(Merluccius merluccius, Merluccius capensis, Merluccius polli (Benguela hake), Merluccius paradoxus
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(Deep-water Cape hake), Merluccius hubbsi, Merluccius gayi, Merluccius australis australis and Merluccius australis polylepsis) and one grenadier species Macruronus novaezelandiae with two populations
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(novaezelandiae and magellanicus).[44]. Integrating this approach with de-novo sequencing of specific tryptic peptides of nucleoside-diphosphate kinase B and using these peptides to design ad hoc selective ion reaction monitoring (SIRM) experiments, it was possible to confirm the previous results and to include new species (Merluccius bilinearis, Merluccius senegalensis and Merluccius productus) in the differential
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classification of hakes [45] (Fig. 1). Two species-specific peptides originated from aldolase A were identified by a similar analytical strategy and used to discriminate Merluccius paradoxus and Merluccius capensis that could not be differentiated on the basis of MALDI-TOF-MS peptide maps [46]. The strength of this method was also ascertained analyzing sarcoplasmic protein extracts from seven different shrimp species. A selective differentiation between the Penaeoidea and Pandaloidea superfamilies and among the Penaeidae, Solenoceridae, and Pandalidae families was achieved. The unequivocal shrimp identification relied on the 2-DE separation of arginine kinase isoforms and on similarities and differences in the sequence of their tryptic peptides as deduced by MS/MS data [37]. A similar approach was applied to discriminate the closely related species S. seenghala and S. aor, two commercially important species of the tropical rivers, considering the presence of diagnostic signals in MALDI-TOF fingerprints obtained from in-gel digestion of triosephosphate isomerase isoforms [48]. These promising results established proteomics as a feasible tool for fish authentication. At the same time, these studies showed that the major limits of this strategy were the laboriousness of 2-DE analysis and the lack of fish protein sequences in databases that could hamper protein identification. These concerns 5
ACCEPTED MANUSCRIPT prompted the application of novel high-throughput proteomic strategies encompassing high-resolution mass spectrometry and MALDI-TOF MS molecular profiling strategies.
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3. High-throughput proteomics for fish authentication
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The identification of unique biomarkers for fish authentication, i.e. tryptic species-specific peptides
development of advanced targeted proteomic approaches.
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originated from sarcoplasmic proteins, led the scientific community to turn its attention towards the
The recent significant technological advancements in mass spectrometric instruments and the development of new bioinformatics tools made possible this important step forward. Moreover, in the last years, the
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genomes of several commercial species have been sequenced [49] thus increasing the number of fish protein sequences available in the databases and facilitating the discover of peptide biomarkers. Targeted proteomic approaches were firstly based on Selected Reaction Monitoring (SRM) experiments, a
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mass spectrometric technique that was specifically designed for the detection and quantification of selected biomolecules in complex mixtures. In a SRM experiment, molecular ions within a m/z range centered around the m/z of the targeted peptide are selected in a mass analyzer, fragmented by CID and one or more fragment
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ions are selectively monitored during the LC run, thus producing a chromatographic trace in which retention
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time and signal intensity are the coordinates. The pair precursor ion-fragment ion is termed SRM transition, and more transitions could be sequentially and repeatedly monitored at a periodicity that is fast compared to
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the peptide’s chromatographic elution, thus leading to a chromatographic trace for each transition. This allows performing the detection and quantification of different peptides in complex samples, and, by inference, of target proteins even present in very low concentration [50]. Interestingly, in modified SRM experiments, termed “Selected MS/MS ion monitoring” (SMIM) scanning mode, the MS analyzer is
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programmed to execute continuous MS/MS scans on the selected precursor ions along the chromatographic separation and to obtain virtual chromatogram traces for the different fragment ions. These experiments are particularly suitable for the analysis of very complex samples as the acquisition of MS/MS spectra of the monitored peptides is crucial to confirm peptide identities [51]. In 2010, Carrera and colleagues carried out the de-novo sequencing of twenty-five new PRVBs belonging to eleven different species from the Merlucciidae family, combining a classical bottom-up proteomics approach with accurate molecular weight determination of intact proteins by FTICR-MS and SMIM experiments of peptide mass gaps [52]. These results provided the basic knowledge for the development of a high sensitive, specific, and relatively fast method for authentication of Merlucciidae species that used SMIM experiments performed on an ESI-ion trap mass spectrometer to monitor eleven specific PRVBs peptide biomarkers (Fig. 1). This strategy included the selective purification of PRVBs from sarcoplasmic proteins by means of a short heat treatment, the tryptic digestion of PRVBs under an ultrasonic field provided by high-intensity focused ultrasound (HIFU), thus reducing hydrolysis time to two minutes, and the analysis of peptide mixtures by LC-MS/MS operating in SMIM scanning mode. The discrimination of eleven main commercial 6
ACCEPTED MANUSCRIPT Merluccius species and two subspecies belonging to Macruronus novaezelandiae was performed monitoring specific SMIM transitions that demonstrated the presence or the absence of each selected PRVBs peptide in the analyzed protein fraction following a systematic flow-chart. The entire procedure led to the unequivocal discrimination in less than two hours and was validated on protein extracts from ten commercial hake
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products including raw and precooked fillets and sticks [53]. The power and applicability of the described
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methodology was tested analyzing a wide set of several fish species of relevant commercial value such as
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Gadus morhua (cod), Merluccius species, Sparus aurata (gilthead seabream), Diplodus sargus (white seabream), Thunnus albacares (yellowfin tuna), Solea solea (common sole), Salmo salar, (salmon), Genypterus blacodes (pink cusk-eel) and Lophius piscatorius (angler). Furthermore, the method allowed also the authentication of commercial fish foodstuff such as dried salted cod fillets, frozen surimi and baby food.
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As PRVBs are the major fish allergens, this approach provided an alternative method for the detection of fish allergens in food matrices that represent a serious health risk [54].
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Similarly, the discrimination of seven shrimp species belonging to Penaeidae, Solenoceridae and Pandalidae families was achieved using transitions of diagnostic peptides originated from arginine kinases [55]. The studies performed by Carrera and colleagues defined a reliable proteomics pipeline to investigate
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different issues of the seafood quality and safety (Fig. 1). The first phase of this pipeline, the discovery phase, consists of a throughput proteomic analysis of sarcoplasmic proteins for biomarker identification. The
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second phase, the target-driven phase, includes ad hoc LC-MS/MS experiments used to monitor selected peptide biomarkers in fish based products. Of note, this strategy could be theoretically applied in each field
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of food authentication based on protein analysis [56,57]. Differently, Wulff et al. developed a shotgun proteomic method for fish authentication. Protein extracts of muscle fish samples from twenty-two species belonging to Salmoniformes, Siluriformes, Perciformes,
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Zeiformes, Pleuronectiformes and Gadiformes orders were digested with trypsin and the peptide mixtures were directly analyzed by LC-MS/MS to obtain a dataset of reference MS/MS spectra for each analyzed species. These datasets were used to build up a spectral library database and identification of unknown fish samples was achieved by querying the spectral library database with the MS/MS dataset produced analyzing protein extracts from unknown samples. The highest number of matching mass spectra unambiguously determined the reference dataset and consequently the fish species. To demonstrate the broad applicability of the method, heavily processed samples, i.e cooked, deep fried and smoked samples, were analyzed and successfully identified [58]. Noteworthy, this method did not require any information on the sequences of analyzed proteins or the selection of species-specific peptide biomarkers. Shotgun proteomics, including chemical labeling and label free methods for protein quantification has been recently applied to discriminate farmed and wild gilthead seabreams based on qualitative and quantitative changes in proteome profiles, thus demonstrating the ability of proteomics to assess differences induced by diverse life conditions on fish of commercial interest [59].
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ACCEPTED MANUSCRIPT 4. Molecular profiling strategies based on MALDI-TOF MS Molecular profiling strategies based on the analysis of protein extracts from different food matrices using MALDI-TOF-MS asserted themselves as powerful tools to determine biomarkers potentially useful as
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indicators of food authenticity [60-64]. In 2008, Mazzeo et al. proposed this analytical approach as a
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straightforward and fast method for fish authentication. Proteins from white fish muscle were extracted in aqueous solution and the supernatants were directly analyzed by MALDI-TOF-MS. Mass spectra showed
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characteristic signal patterns constituted by few highly intense signals in the m/z range 11-12 kDa; further MS/MS analyses demonstrated that these signals were originated from PRVBs. Due to interspecific variability of PRVBs, the mass signal patterns were unique for the species under analysis. Reference mass spectra obtained from the analysis of fresh samples of few authenticated species are reported in Fig. 2. This
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method allowed to easily and unambiguously discriminate forty different fish species, including the most relevant commercial European species mainly belonging to Gadiformes, Perciformes and Pleuronectiformes
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orders. Its specificity was validated identifying closely related species such as Diplodus sargus sargus (white seabream), Diplodus vulgaris (common two-banded seabream), Diplodus puntazzo (sharpsnout seabream) and Diplodus annularis (annular seabream) and the two species Pagellus acarne (auxilary seabream) and
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Pagellus erythrinus (common pandora). The authentication of fish samples subjected to harsh heat treatment
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and commercial products (cod or sole fillets and fishsticks) demonstrated the robustness of the method. In fact, mass spectra obtained from the analysis of processed products showed the same biomarker patterns of the fresh products (reference mass spectra). More importantly, this method allowed, for instance, the
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unambiguous discrimination of Dentex dentex (common dentex) fillets from Pagrus pagrus (red porgy) ones or Pangasius pangasius (pangas catfish) and Tilapiine cichids (tilapias) fillets from cod ones. These evidences assessed the potential of this strategy in the rapid detection of fraudulent and dangerous practices
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such as the substitution of high value species with inferior ones [36,38]. Up to now, these data represent one of the most comprehensive repertoires of species included in fish authentication proteomic studies. MALDITOF mass spectra and biomarker patterns of forty species belonging to seven order and twenty families are now available on the web (http://bioinformatica.isa.cnr.it/MALDImare/). A similar approach has been recently used to discriminate among three commercial freshwater fish species very common in large Italian lakes (the shad Alosa agone, the whitefish Coregonus macrophthalmus and the roach Rutilus rutilus). In the mass spectra of protein extracts from muscle and liver tissues, m/z markers typical of each species were detected and enabled an unambiguous identification [65]. Profiling strategies are exploiting the improved performance of novel MALDI-TOF instruments and specific software developed to build up mass spectral databases and to perform database querying with mass spectral data obtained from the analysis of unknown samples. This new technological platform was used to construct a mass spectra reference library of six commercially important shrimp species (Litopenaeus setiferus, Farfantepenaeus aztecus, Sicyonia brevirostris, Pleoticus robustus, Pandalopsis dispar and Pandalus platyceros). Protein extracts from five individuals for each species were analyzed and twenty mass spectra were collected to generate the average profile for each species. In addition, mass spectra obtained from 8
ACCEPTED MANUSCRIPT seventy-four unknown shrimp samples were data base-searched and identification at the species level was achieved with 97 % accuracy, thus assessing the robustness of the methods [37]. It should be underlined that molecular profiling strategies do not demand preliminary information on the sample under investigation (origin, putative species) and the identity of proteins generating the biomarker
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pattern. They require a short time for sample preparation, data acquisition and processing could be
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completely automated, and mass spectra of novel fish species could be easily added to reference database, so
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that these approaches could help to solve emerging problems of fish market.
5. Conclusions
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The assessment of food quality and safety is a major challenge to guarantee consumers both from health risk and illegal economic practices. In the last decades, the introduction of proteomic platforms has significantly contributed to explore several aspects of food science and provided new highly accurate and sensitive tools
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to verify food authenticity, also in the field of fishery products.
In particular, MALDI-TOF molecular profiling strategies, based on direct and straightforward protocols, can lead to fish identification within minutes. On the other hand, targeted mass spectrometric approaches make
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feasible not only fish authentication but, more interestingly, the rapid detection of the major fish allergen, β-
tests for other food matrices.
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PRVBs, in any foodstuff. Of note, these methods could be easily adapted for setting up specific authenticity
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Therefore, the successfully application of mass-spectrometric based methods clearly demonstrates that proteomics holds the potential to become a reliable an operative first-line testing resource in food authentication, offering all the desirable features (flexibility, reliability, straightness, short time of analysis)
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of official protocols for screening tests.
Acknowledgments
MFM and RAS were partly supported by a dedicated grant from the Italian Ministr of conom and inance to
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for the roject “Inno a ione e S il ppo del Me ogiorno - onoscen e Integrate
per Sosteni ilit ed Inno a ione del Made in Ital
groalimentare ( ISI )” Legge n.191/2009.
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ACCEPTED MANUSCRIPT [41] M. Pazos, L. Méndez, M. Vázquez, S.P. Aubourg, Proteomics analysis in frozen horse mackerel previously high-pressure processed, Food Chem. 185 (2015) 495-502. [42] C. Piñeiro, J. Vázquez, A.I. Marina, J. Barros-Velázquez, J.M. Gallardo, Characterization and partial sequencing of species-specific sarcoplasmic polypeptides from commercial hake species by mass
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[48] S.K. Barik, S. Banerjee, S. Bhattacharjee, S.K. Das Gupta, S. Mohanty, B.P. Mohanty, Proteomic analysis of sarcoplasmic peptides of two related fish species for food authentication, Appl. Biochem. Biotechnol. 171 (2013) 1011-1021. [49] B. Louro, D.M. Power, A.V. Canario, Advances in European sea bass genomics and future
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[62] L.F. Ciarmiello, M.F. Mazzeo, P. Minasi, A. Peluso, A. De Luca, P. Piccirillo et al., Analysis of different European hazelnut (Corylus avellana L.) cultivars: authentication, phenotypic features, and phenolic profiles, J. Agric. Food Chem. 62 (2014) 6236-6246. [63] M. Sassi, S. Arena, A. Scaloni, MALDI-TOF-MS platform for integrated proteomic and peptidomic
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[64] D. Resetar, M. Marchetti-Deschmann, G. Allmaier, J. Peter Katalinic, S. Kraljevic Pavelic, Matrix assisted laser desorption ionization mass spectrometry linear time-of-flight method for white wine fingerprinting and classification, Food Control 64 (2016) 157e164. [65] P. Volta, N. Riccardi, R. Lauceri, M. Tonolla, Discrimination of freshwater fish species by MatrixAssisted Laser Desorption/Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF MS): a pilot study, J. Limnol. 71 (2012) 164-169.
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ACCEPTED MANUSCRIPT Figure Captions
Fig. 1- Proteomics pipeline used for fish authentication. In the discovery phase, 2-DE and mass
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spectrometric analyses of sarcoplasmic proteins are applied to identify and characterize species-specific
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peptide biomarkers. In the target-driven phase, SMIM experiments are designed for monitoring speciesspecific peptide biomarkers. (Reproduced from Carrera et al. Food Res. Int. 2013 with permission from
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Elsevier, License Number: 3805210903494).
Fig. 2- Fish authentication by MALDI-TOF molecular profiling strategy. A specific biomarker pattern for each fish species is obtained from the direct MALDI-TOF-MS analysis of sarcoplasmic protein extracts. Biomarker patterns obtained from the analysis of unknown raw and processed products samples are compared with the reference ones to achieve species authentication.
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Highlights
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Fish authentication and frauds detection are main issues in quality control process MALDI-TOF mass spectra of sarcoplasmic proteins allow authentication of fish species Targeted proteomics has been applied to assess fish authenticity and detect allergens
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Proteomics offers tools potentially suitable as routine test in food authentication
Graphical Abstract
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Significance The assessment of fishery products authenticity is a main issue in the control quality process as deceptive
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practices could imply severe health risks. Proteomics based methods could significantly contribute to detect
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falsification and frauds, thus becoming a reliable operative first-line testing resource in food authentication.
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S1874-3919(16)30064-1 doi: 10.1016/j.jprot.2016.03.007 JPROT 2446
To appear in:
Journal of Proteomics
Received date: Revised date: Accepted date:
17 December 2015 15 February 2016 1 March 2016
Please cite this article as: Mazzeo Maria Fiorella, Siciliano Rosa Anna, Proteomics for the authentication of fish species, Journal of Proteomics (2016), doi: 10.1016/j.jprot.2016.03.007
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Proteomics for the authentication of fish species
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Maria Fiorella Mazzeo, Rosa Anna Siciliano*
Centro di Spettrometria di Massa Proteomica e Biomolecolare, Istituto di Scienze dell'Alimentazione, CNR,
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E-mail: [email protected]; [email protected]
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via Roma 64, 83100 Avellino, Italy. Tel +39 0825 299363, Fax +39 0825 781585.
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*corresponding author
Keywords: fish authentication; proteomics; mass spectrometry; molecular profiling; fish frauds
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ACCEPTED MANUSCRIPT ABSTRACT
Assessment of seafood authenticity and origin, mainly in the case of processed products (fillets, sticks, baby
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food) represents the crucial point to prevent fraudulent deceptions thus guaranteeing market transparency and
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consumers health. The most dangerous practice that jeopardies fish safety is intentional or unintentional
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mislabeling, originating from the substitution of valuable fish species with inferior ones. Conventional analytical methods for fish authentication are becoming inadequate to comply with the strict regulations issued by European Union and with the increase of mislabeling due to the introduction on the
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market of new fish species and market globalization.
This evidence prompts the development of high-throughput approaches suitable to identify unambiguous
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biomarkers of authenticity and screen a large number of samples with minimal time consumption. Proteomics provides suitable and powerful tools to investigate main aspects of food quality and safety and has given an important contribution in the field of biomarkers discovery applied to food authentication. .
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This report describes the most relevant methods developed to assess fish identity and offers a perspective on their potential in the evaluation of fish quality and safety thus depicting the key role of proteomics in the
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authentication of fish species and processed products.
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ACCEPTED MANUSCRIPT 1. Introduction
Authentication of fishery products represents a central issue for the assessment of food quality and safety. In
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the fishery market, the most common falsification is the substitution of more valuable fish species with
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inferior ones. In particular the identification of processed products by visual inspection becomes quite
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challenging as anatomical and morphological characteristics (such as the head, fins, skin, or bones), essential for fish species authentication, are lost during processing. These frauds are constantly growing due to market globalization and are leading to an illegal economic gain but, more alarmingly to severe health risks for the consumers. Since 2000, EU issued directives and regulations regarding fishery and aquaculture products
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establishing what must be reported in label (fish species, geographical origin, and production method (wild or cultivated) (EC No 104/2000 and 2065/2001) [1,2]. []. Moreover, the European Food Safety Authority
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(EFSA) defined a detailed system of traceability for food (including fishery and aquaculture products) and feed businesses to control food safety at all stages, as reported in EC regulation no. 178/2002 [3] . More recently, EU introduced a more stringent regulation (EC No 1379/2013) [4] on different aspects of the common organization of the markets in fishery and aquaculture products (e.g. provisions on consumer
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information, recognition of producer organizations, extension of rules, production and marketing planning),
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and the Commission Implementing Regulation No 1420/2013 [5] to define in details the rules for the adoption of EC No 1379/2013 [6,7]. First analytical methods for fish authentication have been based on the
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on the analysis of protein extracts by electrophoretic, chromatographic and immunological methods.
In particular, IEF analysis of sarcoplasmic proteins has been applied to discriminate different fish and shrimp [8-12] and considered the validated method for species identification by the Association of Official
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Analytical Chemistry [13]. IEF profiles of sarcoplasmic proteins from different fish species have been collected by FDA US in the internet library Regulatory Fish Encyclopedia [14]. More recently, DNA-based approaches, centered on the amplification of specific DNA-fragments (cytochrome b gene [15-18], mtDNA control region [19] and 12S rRNA region [20,21]) using PCR have been developed. DNA-barcoding has been also proposed as a reliable method for fish authentication and DNA barcode data has been included in FDA's Regulatory Fish Encyclopedia 22,23]. These methods are sensitive and specific, even in the case of closely related species [24,25], however, processing of fishery products such as heat treatment and acidic conditions could damage DNA integrity, thus leading to non-specific identification. Moreover, the difficulties to standardize protocols for DNA analyses could cause inconsistencies in results from different laboratories that could lead to regulatory or legal implications. The high costs of analysis should also be taken into account [26,27]. Although the efforts to improve the legislation and the analytical methods for seafood identification have reduced the occurrence of fish mislabeling, in some European countries and, to a lesser extent, in US, this phenomenon still represents a severe risk for consumers health [28,29]. In fact, the possibility to get great 3
ACCEPTED MANUSCRIPT economic gains also throughout illegal, unreported and unregulated (IUU) fishing, exploiting the increasing worldwide fish consumption and the market globalization, continuously fed the fraudulent and dangerous practice of fish mislabeling [30,31] In this frame, it is mandatory the development of high-throughput approaches suitable to discover
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unambiguous biomarkers of authenticity and screen a large number of samples with minimal time
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consumption. In the recent years, among the omics approach, proteomics has significantly contributed to
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investigate the main aspects of food quality and safety, including traceability, authenticity, absence of contaminating and/or adulterating agents and impact of the processing/storage conditions [32]. In the field of fish authentication, first results were obtained applying the classical proteomic strategy that integrates 2-DE and mass spectrometry. Afterwards, methods based on the analysis of peptides and intact proteins by high
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resolution and tandem mass spectrometry opened the way to a new phase in biomarkers discovery, aimed to assess identity of fish-based food [33-35]. On the other hand, molecular profiling strategies based on
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MALDI-TOF-MS emerged as an alternative tool to define biomarkers of fish authenticity [36-38]. The topic of this report is the role that proteomics held in the discovery of biomarkers suitable for fish authentication. A special emphasis is given to the ability of advanced proteomics-based approaches to
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on the protection of consumer health.
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provide reliable quality control methods to detect deceptive practices and frauds, thus fulfilling EU directives
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2. The first proteomic approach integrating 2-DE and MS
Electrophoretic methods have been widely applied to achieve fish authentication. Noteworthy, IEF analyses of the sarcoplasmic proteins extracted from fish muscle tissues led to define species-specific profiles suitable to discriminate several fish species. Differences in protein patterns were originated by a class of proteins, the
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parvalbumins (PRVBs) that showed a high variability in their primary structure [10]. These proteins are calcium-binding proteins with molecular weight in the 11-12 kDa range, abundant in muscle tissue and highly soluble in aqueous buffers. They are also the major allergy-eliciting proteins in fish. Moreover, due to their structural features, PRVBs showed a remarkable thermal stability [39,40]. More recently, the stability of PRVBs to high pressure processing has also been assessed [41]. Therefore, PRVBs hold all the required features to be used as biomarkers to evaluate the identity of raw and processed fish products. The advent of proteomics in the 1994 and its relative rapid and widespread application in each field of protein science provided innovative tools for biomarker-detection also related to food quality or safety. The classical bottom-up proteomic approach, that integrated 2-DE for protein separation and mass spectrometry for protein identification, was initially applied by Pineiro et al. to discriminate five closely related hake species (Fig. 1). Protein spots responsible for differences in 2-DE maps obtained from the analysis of sarcoplasmic extracts from white muscle of Merluccius merluccius (European hake), Merluccius australis (Southern hake), Merluccius hubbsi (Argentinian hake), Merluccius gayi (Chilean hake), and Merluccius capensis (Cape hake) were subjected to in-gel tryptic digestion and the obtained peptide mixtures were 4
ACCEPTED MANUSCRIPT analyzed by MALDI-TOF-MS and MS/MS. Using mass spectrometric data, PRVBs and a nucleoside diphosphate kinase protein were identified. Peptide mass maps obtained from the nucleoside diphosphate kinase allowed the differential classification of the hake species into two groups: the East Atlantic coast group and the West Atlantic coast group. Moreover, the presence of a specific signal in the peptide mass
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maps of PRVBs, unambiguously discriminated Southern hake from the other species [42]. A similar
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approach was used to discriminate three European marine mussel species: Mytilus edulis, Mytilus
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galloprovincialis and Mytilus trossulus. MALDI-TOF peptide mass maps were generated from proteins contained in six randomly selected prominent spots present in the 2-DE gels obtained from foot protein extracts. Two peptides were selected as biomarkers of Mytilus edulis and Mytilus galloprovincialis, whereas another peptide was selected to discriminate Mytilus trossulus. MS/MS followed by database searching and
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de novo sequencing determined the primary structure of these peptides [43].
Analysis of sarcoplasmic extracts from raw white muscle by 2-DE and MALDI-TOF-MS allowed
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differentiating ten closely related species of commercial relevance. A method for fish authentication was drawn relying on differences of the PRVBs pattern in the 2-DE maps and on the presence or absence of species-specific signals in MALDI-TOF-MS spectra originated from the analysis of in-gel digested PRVBs of each analyzed species. This method led to discriminate nine hake species of the genus Merluccius
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(Merluccius merluccius, Merluccius capensis, Merluccius polli (Benguela hake), Merluccius paradoxus
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(Deep-water Cape hake), Merluccius hubbsi, Merluccius gayi, Merluccius australis australis and Merluccius australis polylepsis) and one grenadier species Macruronus novaezelandiae with two populations
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(novaezelandiae and magellanicus).[44]. Integrating this approach with de-novo sequencing of specific tryptic peptides of nucleoside-diphosphate kinase B and using these peptides to design ad hoc selective ion reaction monitoring (SIRM) experiments, it was possible to confirm the previous results and to include new species (Merluccius bilinearis, Merluccius senegalensis and Merluccius productus) in the differential
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classification of hakes [45] (Fig. 1). Two species-specific peptides originated from aldolase A were identified by a similar analytical strategy and used to discriminate Merluccius paradoxus and Merluccius capensis that could not be differentiated on the basis of MALDI-TOF-MS peptide maps [46]. The strength of this method was also ascertained analyzing sarcoplasmic protein extracts from seven different shrimp species. A selective differentiation between the Penaeoidea and Pandaloidea superfamilies and among the Penaeidae, Solenoceridae, and Pandalidae families was achieved. The unequivocal shrimp identification relied on the 2-DE separation of arginine kinase isoforms and on similarities and differences in the sequence of their tryptic peptides as deduced by MS/MS data [37]. A similar approach was applied to discriminate the closely related species S. seenghala and S. aor, two commercially important species of the tropical rivers, considering the presence of diagnostic signals in MALDI-TOF fingerprints obtained from in-gel digestion of triosephosphate isomerase isoforms [48]. These promising results established proteomics as a feasible tool for fish authentication. At the same time, these studies showed that the major limits of this strategy were the laboriousness of 2-DE analysis and the lack of fish protein sequences in databases that could hamper protein identification. These concerns 5
ACCEPTED MANUSCRIPT prompted the application of novel high-throughput proteomic strategies encompassing high-resolution mass spectrometry and MALDI-TOF MS molecular profiling strategies.
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3. High-throughput proteomics for fish authentication
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The identification of unique biomarkers for fish authentication, i.e. tryptic species-specific peptides
development of advanced targeted proteomic approaches.
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originated from sarcoplasmic proteins, led the scientific community to turn its attention towards the
The recent significant technological advancements in mass spectrometric instruments and the development of new bioinformatics tools made possible this important step forward. Moreover, in the last years, the
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genomes of several commercial species have been sequenced [49] thus increasing the number of fish protein sequences available in the databases and facilitating the discover of peptide biomarkers. Targeted proteomic approaches were firstly based on Selected Reaction Monitoring (SRM) experiments, a
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mass spectrometric technique that was specifically designed for the detection and quantification of selected biomolecules in complex mixtures. In a SRM experiment, molecular ions within a m/z range centered around the m/z of the targeted peptide are selected in a mass analyzer, fragmented by CID and one or more fragment
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ions are selectively monitored during the LC run, thus producing a chromatographic trace in which retention
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time and signal intensity are the coordinates. The pair precursor ion-fragment ion is termed SRM transition, and more transitions could be sequentially and repeatedly monitored at a periodicity that is fast compared to
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the peptide’s chromatographic elution, thus leading to a chromatographic trace for each transition. This allows performing the detection and quantification of different peptides in complex samples, and, by inference, of target proteins even present in very low concentration [50]. Interestingly, in modified SRM experiments, termed “Selected MS/MS ion monitoring” (SMIM) scanning mode, the MS analyzer is
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programmed to execute continuous MS/MS scans on the selected precursor ions along the chromatographic separation and to obtain virtual chromatogram traces for the different fragment ions. These experiments are particularly suitable for the analysis of very complex samples as the acquisition of MS/MS spectra of the monitored peptides is crucial to confirm peptide identities [51]. In 2010, Carrera and colleagues carried out the de-novo sequencing of twenty-five new PRVBs belonging to eleven different species from the Merlucciidae family, combining a classical bottom-up proteomics approach with accurate molecular weight determination of intact proteins by FTICR-MS and SMIM experiments of peptide mass gaps [52]. These results provided the basic knowledge for the development of a high sensitive, specific, and relatively fast method for authentication of Merlucciidae species that used SMIM experiments performed on an ESI-ion trap mass spectrometer to monitor eleven specific PRVBs peptide biomarkers (Fig. 1). This strategy included the selective purification of PRVBs from sarcoplasmic proteins by means of a short heat treatment, the tryptic digestion of PRVBs under an ultrasonic field provided by high-intensity focused ultrasound (HIFU), thus reducing hydrolysis time to two minutes, and the analysis of peptide mixtures by LC-MS/MS operating in SMIM scanning mode. The discrimination of eleven main commercial 6
ACCEPTED MANUSCRIPT Merluccius species and two subspecies belonging to Macruronus novaezelandiae was performed monitoring specific SMIM transitions that demonstrated the presence or the absence of each selected PRVBs peptide in the analyzed protein fraction following a systematic flow-chart. The entire procedure led to the unequivocal discrimination in less than two hours and was validated on protein extracts from ten commercial hake
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products including raw and precooked fillets and sticks [53]. The power and applicability of the described
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methodology was tested analyzing a wide set of several fish species of relevant commercial value such as
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Gadus morhua (cod), Merluccius species, Sparus aurata (gilthead seabream), Diplodus sargus (white seabream), Thunnus albacares (yellowfin tuna), Solea solea (common sole), Salmo salar, (salmon), Genypterus blacodes (pink cusk-eel) and Lophius piscatorius (angler). Furthermore, the method allowed also the authentication of commercial fish foodstuff such as dried salted cod fillets, frozen surimi and baby food.
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As PRVBs are the major fish allergens, this approach provided an alternative method for the detection of fish allergens in food matrices that represent a serious health risk [54].
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Similarly, the discrimination of seven shrimp species belonging to Penaeidae, Solenoceridae and Pandalidae families was achieved using transitions of diagnostic peptides originated from arginine kinases [55]. The studies performed by Carrera and colleagues defined a reliable proteomics pipeline to investigate
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different issues of the seafood quality and safety (Fig. 1). The first phase of this pipeline, the discovery phase, consists of a throughput proteomic analysis of sarcoplasmic proteins for biomarker identification. The
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second phase, the target-driven phase, includes ad hoc LC-MS/MS experiments used to monitor selected peptide biomarkers in fish based products. Of note, this strategy could be theoretically applied in each field
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of food authentication based on protein analysis [56,57]. Differently, Wulff et al. developed a shotgun proteomic method for fish authentication. Protein extracts of muscle fish samples from twenty-two species belonging to Salmoniformes, Siluriformes, Perciformes,
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Zeiformes, Pleuronectiformes and Gadiformes orders were digested with trypsin and the peptide mixtures were directly analyzed by LC-MS/MS to obtain a dataset of reference MS/MS spectra for each analyzed species. These datasets were used to build up a spectral library database and identification of unknown fish samples was achieved by querying the spectral library database with the MS/MS dataset produced analyzing protein extracts from unknown samples. The highest number of matching mass spectra unambiguously determined the reference dataset and consequently the fish species. To demonstrate the broad applicability of the method, heavily processed samples, i.e cooked, deep fried and smoked samples, were analyzed and successfully identified [58]. Noteworthy, this method did not require any information on the sequences of analyzed proteins or the selection of species-specific peptide biomarkers. Shotgun proteomics, including chemical labeling and label free methods for protein quantification has been recently applied to discriminate farmed and wild gilthead seabreams based on qualitative and quantitative changes in proteome profiles, thus demonstrating the ability of proteomics to assess differences induced by diverse life conditions on fish of commercial interest [59].
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ACCEPTED MANUSCRIPT 4. Molecular profiling strategies based on MALDI-TOF MS Molecular profiling strategies based on the analysis of protein extracts from different food matrices using MALDI-TOF-MS asserted themselves as powerful tools to determine biomarkers potentially useful as
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indicators of food authenticity [60-64]. In 2008, Mazzeo et al. proposed this analytical approach as a
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straightforward and fast method for fish authentication. Proteins from white fish muscle were extracted in aqueous solution and the supernatants were directly analyzed by MALDI-TOF-MS. Mass spectra showed
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characteristic signal patterns constituted by few highly intense signals in the m/z range 11-12 kDa; further MS/MS analyses demonstrated that these signals were originated from PRVBs. Due to interspecific variability of PRVBs, the mass signal patterns were unique for the species under analysis. Reference mass spectra obtained from the analysis of fresh samples of few authenticated species are reported in Fig. 2. This
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method allowed to easily and unambiguously discriminate forty different fish species, including the most relevant commercial European species mainly belonging to Gadiformes, Perciformes and Pleuronectiformes
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orders. Its specificity was validated identifying closely related species such as Diplodus sargus sargus (white seabream), Diplodus vulgaris (common two-banded seabream), Diplodus puntazzo (sharpsnout seabream) and Diplodus annularis (annular seabream) and the two species Pagellus acarne (auxilary seabream) and
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Pagellus erythrinus (common pandora). The authentication of fish samples subjected to harsh heat treatment
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and commercial products (cod or sole fillets and fishsticks) demonstrated the robustness of the method. In fact, mass spectra obtained from the analysis of processed products showed the same biomarker patterns of the fresh products (reference mass spectra). More importantly, this method allowed, for instance, the
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unambiguous discrimination of Dentex dentex (common dentex) fillets from Pagrus pagrus (red porgy) ones or Pangasius pangasius (pangas catfish) and Tilapiine cichids (tilapias) fillets from cod ones. These evidences assessed the potential of this strategy in the rapid detection of fraudulent and dangerous practices
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such as the substitution of high value species with inferior ones [36,38]. Up to now, these data represent one of the most comprehensive repertoires of species included in fish authentication proteomic studies. MALDITOF mass spectra and biomarker patterns of forty species belonging to seven order and twenty families are now available on the web (http://bioinformatica.isa.cnr.it/MALDImare/). A similar approach has been recently used to discriminate among three commercial freshwater fish species very common in large Italian lakes (the shad Alosa agone, the whitefish Coregonus macrophthalmus and the roach Rutilus rutilus). In the mass spectra of protein extracts from muscle and liver tissues, m/z markers typical of each species were detected and enabled an unambiguous identification [65]. Profiling strategies are exploiting the improved performance of novel MALDI-TOF instruments and specific software developed to build up mass spectral databases and to perform database querying with mass spectral data obtained from the analysis of unknown samples. This new technological platform was used to construct a mass spectra reference library of six commercially important shrimp species (Litopenaeus setiferus, Farfantepenaeus aztecus, Sicyonia brevirostris, Pleoticus robustus, Pandalopsis dispar and Pandalus platyceros). Protein extracts from five individuals for each species were analyzed and twenty mass spectra were collected to generate the average profile for each species. In addition, mass spectra obtained from 8
ACCEPTED MANUSCRIPT seventy-four unknown shrimp samples were data base-searched and identification at the species level was achieved with 97 % accuracy, thus assessing the robustness of the methods [37]. It should be underlined that molecular profiling strategies do not demand preliminary information on the sample under investigation (origin, putative species) and the identity of proteins generating the biomarker
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pattern. They require a short time for sample preparation, data acquisition and processing could be
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completely automated, and mass spectra of novel fish species could be easily added to reference database, so
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that these approaches could help to solve emerging problems of fish market.
5. Conclusions
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The assessment of food quality and safety is a major challenge to guarantee consumers both from health risk and illegal economic practices. In the last decades, the introduction of proteomic platforms has significantly contributed to explore several aspects of food science and provided new highly accurate and sensitive tools
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to verify food authenticity, also in the field of fishery products.
In particular, MALDI-TOF molecular profiling strategies, based on direct and straightforward protocols, can lead to fish identification within minutes. On the other hand, targeted mass spectrometric approaches make
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feasible not only fish authentication but, more interestingly, the rapid detection of the major fish allergen, β-
tests for other food matrices.
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PRVBs, in any foodstuff. Of note, these methods could be easily adapted for setting up specific authenticity
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Therefore, the successfully application of mass-spectrometric based methods clearly demonstrates that proteomics holds the potential to become a reliable an operative first-line testing resource in food authentication, offering all the desirable features (flexibility, reliability, straightness, short time of analysis)
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of official protocols for screening tests.
Acknowledgments
MFM and RAS were partly supported by a dedicated grant from the Italian Ministr of conom and inance to
and
for the roject “Inno a ione e S il ppo del Me ogiorno - onoscen e Integrate
per Sosteni ilit ed Inno a ione del Made in Ital
groalimentare ( ISI )” Legge n.191/2009.
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ACCEPTED MANUSCRIPT Figure Captions
Fig. 1- Proteomics pipeline used for fish authentication. In the discovery phase, 2-DE and mass
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spectrometric analyses of sarcoplasmic proteins are applied to identify and characterize species-specific
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peptide biomarkers. In the target-driven phase, SMIM experiments are designed for monitoring speciesspecific peptide biomarkers. (Reproduced from Carrera et al. Food Res. Int. 2013 with permission from
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Elsevier, License Number: 3805210903494).
Fig. 2- Fish authentication by MALDI-TOF molecular profiling strategy. A specific biomarker pattern for each fish species is obtained from the direct MALDI-TOF-MS analysis of sarcoplasmic protein extracts. Biomarker patterns obtained from the analysis of unknown raw and processed products samples are compared with the reference ones to achieve species authentication.
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Highlights
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Fish authentication and frauds detection are main issues in quality control process MALDI-TOF mass spectra of sarcoplasmic proteins allow authentication of fish species Targeted proteomics has been applied to assess fish authenticity and detect allergens
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Proteomics offers tools potentially suitable as routine test in food authentication
Graphical Abstract
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Significance The assessment of fishery products authenticity is a main issue in the control quality process as deceptive
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practices could imply severe health risks. Proteomics based methods could significantly contribute to detect
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falsification and frauds, thus becoming a reliable operative first-line testing resource in food authentication.
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