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The samples were heated to 95°C for 30 mins, then cooled. Urea was added to a final
concentration of 0.5M. Trypsin solution (1mg/ mL) was added in a 1:30 enzyme to substrate
weight ratio. The samples were then vortexed and digested overnight at 37°C.
The digests were diluted 1:2 with water and desalted using a Strata-X 33μ reversed phase
cartridge (Phenomenex, Macclesfield, UK). The cartridge was washed then activated using 1
mL of methanol then equilibrated with 1 mL of 1% formic acid. The cartridge was loaded with
the diluted sample and washed with 1 mL 5% methanol/1% formic acid. Peptides were then
eluted by adding 1 mL acetonitrile/water (90:10; 0.1% formic acid). The samples were
evaporated to dryness using the centrifugal evaporator then dissolved in 250 μL
acetonitrile/water (3:97; 0.1% formic acid).
3.6. Predictive Tools
The likely peptides resulting from the tryptic digestion of yellowfin (Thunnus albacares),
albacore (Thunnus alalunga), skipjack (Katsuwonus pelamis) and bigeye (Thunnus obesus) Mb
were predicted using the PeptideCutter tool 70
set to the ‘simple model’ option. Potential
transitions produced by fragmentation during MRM-MS were then predicted using Open
Source Skyline.71
Skyline setting included the following: 0 missed cleavages, conventional
trypsin rules [KR|P], no modifications, exclude N-terminal, amino acids set to 1, No cysteine
modifications, amino acids set to 1 (to avoid initiator methionine) peptide length 6 – 50,
collision energy of ‘ABI 4000 Q Trap’.
3.7. LC-MS/MS Workflow
Skyline-predicted transitions were used to create one MS/MS method file for each tuna
species. The presence of these transitions was then determined. This was accomplished by
testing each species of purified Mb with its own method in unscheduled monitoring mode.
The peptides useful as markers were then selected based on signal quality and species
specificity. The retention times and four most intense transitions (based on peak height) for
all selected peptides were then recorded.These were used to create a single multiplex MS/MS](https://image.slidesharecdn.com/7a54994a-ee73-4867-8935-7b2e9b5ffa6c-160919133657/85/Masters-Research-Project-27-320.jpg)
Masters Research Project
- 1. 1 NAT-MN0Y 2015-16 Natural SciencesProject Multiple Reaction Monitoring Mass Spectrometry of Myoglobin Peptides to Determine Tuna Authenticity Author: 6157106 Supervisor: Dr Andrew Mayes
- 2. 2 TABLE OF CONTENTS 1.Abstract.................................................................................................................................... 4 2. Introduction.......................................................................................................................... 4 2.1. The Tuna Industry........................................................................................................... 6 2.2. Tuna Authentication Protocols................................................................................. 9 2.2.1. DNA-based Analysis.............................................................................................. 9 2.2.1.1. PCR-RFLP..................................................................................................... 10 2.2.1.2. Immunological Analysis............................................................................ 10 2.2.2. Proteomics............................................................................................................. 11 2.2.3. Mass Spectrometry.............................................................................................. 13 2.2.3.1. Separation Techniques.............................................................................. 13 2.2.3.2. Ion Sources.................................................................................................. 15 2.2.3.3. Mass Analysers.......................................................................................... 16 2.2.3.4. Tandem Mass Spectrometry.................................................................... 17 2.3. The Present Work.......................................................................................................... 20 2.3.1. Objectives & Hypotheses............................................................................... 24 3. Methodology...................................................................................................................... 25 3.1. Materials........................................................................................................................... 25 3.2. Purified Myoglobin Preparation............................................................................. 25 3.3. Raw Sample Preparation........................................................................................... 26 3.4. Processed Sample Preparation............................................................................... 26 3.5. Proteolysis........................................................................................................................ 26 3.6. Predictive Tools.............................................................................................................. 27 3.7. LC-MS/MS Workflow................................................................................................... 27 3.8.LC-MS/MS Analyses...................................................................................................... 28
- 3. 3 4. Results..................................................................................................................................... 29 4.1.Peptide Marker Selection.......................................................................................... 29 4.2. Multiplex Myoglobin Analyses................................................................................ 32 4.3. Multiplex Raw Tuna Analyses.................................................................................. 34 4.4. Multiplex Processed Tuna Analyses...................................................................... 35 5. Discussion............................................................................................................................. 36 5.1. Marker Reliability......................................................................................................... 36 5.1.1. Purified Myoglobin.............................................................................................. 37 5.1.2. Raw Samples......................................................................................................... 39 5.1.3. Processed Samples.............................................................................................. 40 5.2. Marker Specificity......................................................................................................... 41 5.2.1. Carryover................................................................................................................ 42 6. Conclusion............................................................................................................................ 45 7. Acknowledgments.......................................................................................................... 47 8. References............................................................................................................................ 47 9. Appendix............................................................................................................................... 56 9.1. Research Proposal........................................................................................................ 56
- 4. 4 1. ABSTRACT The improperdescription of tuna products is of concern, due to the frequent, fraudulent substitution of species within these commodities. This creates a demand for novel authentication protocols to accurately identify species within tuna products. The application of a recently developed approach to four species of tuna: yellowfin, albacore, skipjack and bigeye is presented here. This method, known as the CPCP approach (corresponding proteins corresponding peptides), was originally developed to rapidly distinguish between species in red meat products. Peptides selected as markers for species identification using predictive tools, are detected via multiple reaction monitoring mass spectrometry (MRM MS). These are derived solely from the digestion of myoglobin (Mb) from the species of interest. In the present work, Mb peptide markers able to distinguish between the four species of tuna are identified. These were visible in both purified myoglobin and in raw crude extracts in all four species. These could not be used to distinguish between species however, due to the occurrence of carryover. Further development of the technique should focus on the elimination of carryover within the system to restore marker specificity. The application of this technique to heavily processed tuna samples, as found in canned tuna, should also be studied further. Both were attempted here, though unsuccessfully. KEYWORDS: authentication, food, fish, tandem mass spectrometry, peptide biomarker 2. INTRODUCTION It is the right of consumers that the origins and contents of any food product are described clearly and accurately. This offers patrons the ability to make informed choices about these commodities that directly impact their diet, health and lifestyle. Proper description is also important to honest practitioners in the food industry, as the fraudulent labelling of food can provide an economic advantage to neglectful or deceitful competitors.1 Food authentication is the verification that a food corresponds to its label description. The scale and complexity of the industry provides many avenues of exploitation for fraudulent processors, which have long been a documented issue warranting a global response (Box 1).2 One such avenue involves the partial, or complete, substitution of an ingredient for a similar
- 5. 5 alternative. This canbe accidental or intentional, motivated by a difference in value between the original and its substitute, the adulterant.2 When intentional, the adulterant is cheaper or easier to come by than the original, to provide commercial gain from substitution. This practice was demonstrated in Europe, when an array of beef products were shown to contain or consist entirely of undeclared horse meat.3 The scale of this incident, known as the European horsemeat scandal of 2013, increased consumer and government awareness of issues in food fraud regulation and deterrence.4 At5 the6 time7 of the horsemeat scandal, EU legislation concerning food regulation was largely concerned with issues of food safety, not fraud.4, 8 Procedure has since been established to facilitate increased cooperation between member states, as well as the implementation of union wide controls in the supply chain. New agencies have also been developed, and existing agencies bolstered, to directly combat food fraud.4 An example of this is the development of the National Food Crime Unit (NFCU) within the Food Standards Agency (FSA). The FSA is the government body responsible for ensuring food is of the standard expected by consumers in the UK.9 Tuna production is another area in the food industry at risk of fraudulent practice.10 This fact is recognised by the European Union (EU), whose regulations state the appropriate contents COMMERCIAL TREATMENT The misrepresentation of how a food is produced or treated. Certain treatments are desirable to consumers which indicate a product of good quality (e.g. pasteurized), whilst others are treated with suspicion (e.g. frozen). Investigators found that seven leading brands of olive oil were falsely labelled as extra virgin. Extra virgin olive oils are products of high value due to the use of more time consuming and arduous processes to yield oil of better quality.5 PRODUCT EXTENSION The adulteration of a product with a base ingredient, such as water, to extend foodstuffs increasing the product yield at minimal cost. This has been observed in a wide array of both solid and liquid food products. An investigation into the authenticity of wine vinegar revealed 60% of 92 tested vinegars imported from the Italian market were shown to have been produced by diluting from a concentrate with water, making them fraudulent.6 BRAND COUNTERFEITING The replication of a food using the brand name of the original. Similarly to geographical location and commercial treatments, brand names are often associated with product quality. This provides economic benefit to the perpetrator by selling inferior foodstuffs at a higher price rate, based purely on the reputation of the imitated brand. It was observed in London 2002 that 3 out of 5 bottles of seized Johnny Walker Black Label whisky were not authentic causing both financial and health detriments as the false products contained dangerous levels of methanol.5 The authenticity of alcohol is particularly difficult to determine as most protocols involve the destruction of the product once bottled, making luxury brands at high risk of imitation.7 Box 1. Various authenticity issues. The multitude of methods and cases of fraudulent food representation illustrates that continued vigilance is required. This should involve the development of dependable and efficient food authentication protocols worldwide.
- 6. 6 and labelling oftuna products.11 The current work concerns the development of a novel method of product analysis usable in the tuna supply chain, to enable these regulations to be properly enforced. The approach utilizes multiple reaction monitoring mass spectrometry to discriminate between the myoglobin of different tuna species. To provide context, it is first necessary to discuss issues in the tuna industry, as well as reviewing technologies currently relevant to tuna authentication. The use of chromatography and mass spectrometry will also be detailed, before the current work is expanded upon. 2.1. THE TUNA INDUSTRY The production of raw and processed tuna is of great economic importance. Between 1952 and 2010, the annual production of tuna has increased from 0.4 million tonnes to a peak of over 6 million tonnes.10,12 Tuna and tuna-like species used in food products are caught globally, primarily via longline, purse seine or pole and line fishing.13 Longliners cast nets at the greatest depths, and purse seine and pole and line fisherman harvest fish close to the surface with nets or poles respectively. The size of both catch and individual fish tends to increase with the depth of catch.13 This means the fish caught by pole and line or purse seine vessels are smaller. These are most suitable for canning, as larger fish yield more intact meat. For this reason, the larger fish caught via longline tend to be sold whole or as steaks. (Figure 1). 0° 40° E 80° E 120° E 160° E160° W 120° W 80° W 40° W 0° 30° S 60° S 90° N 60° N 30° N LONGLINE POLE & LINE, PURSE SEINE, LONGLINE PURSE SEINE, LONGLINE PACIFIC OCEAN ATLANTIC OCEAN INDIAN OCEAN PACIFIC OCEAN ARCTIC OCEAN 1 2 3 4 5 6 6 7 7 Figure 1 Tuna stocks, catch methods and distribution. A stock in this context is a subpopulation of a particular species of fish.13 The numbers bounded by the dotted lines indicate the locations of the main tuna stocks and their boundaries. Yellowfin (Thunnus albacares), albacore (Thunnus alalunga), skipjack (Katsuwonus pelamis) and bigeye (Thunnus obesus) are caught from all stocks. Atlantic (Thunnus thynnus), and Pacific (Thunnus orientalis) bluefin are caught from all stocks excluding stock 5. Southern bluefin (Thunnus maccoyii) are only caught in stocks 2 and 4.10 The main locations of tuna canneries are indicated by blue circles.14
- 7. 7 Once14 caught,10 tuna13are frozen prior to transportation for processing, packaging and then sale. Dependent on the operation, a single product can be transported multiple times between or within any stage of production.13 It is important that operations are monitored at each stage of production, as there are more opportunities for fraudulent practice to occur in supply chains of increased complexity. Authentication becomes more difficult as the number of locations and practitioners involved in production increases. This is because it becomes less clear where and how fraud occurs in the supply chain. Authenticity issues arise due to the deviation in value of species used as raw material for tuna products. Of the 61 tuna and tuna-like species within the Scombridae family, seven are attributed with two-thirds of the total catch.15 These species, termed the principal market tunas by the FAO,10 are yellowfin (Thunnus albacares), albacore (Thunnus alalunga), skipjack (Katsuwonus pelamis), bigeye (Thunnus obesus), Atlantic bluefin (Thunnus thynnus), Pacific bluefin (Thunnus orientalis) and southern bluefin (Thunnus maccoyii). Within these species of tuna there is even further deviation in commercial significance, based on the availability of each species (Figure 2). This is reflected in the price of tuna products.10 Skipjack, the most abundantly caught species, yields the cheapest products. Bluefin species are in danger of extinction due to overfishing, as recognised by the IUCN, the leading authority in conservation ecology.16-18 As a result the capture of all three species is restricted, making their products both rare and expensive. This price differential creates financial motive for species substitution. Effective authentication via product speciation (i.e. species identification) protocols is required to counter this. Figure 2 The global catch of principal market tuna. The total catch in 2010 weighed approximately 4 million tonnes in total.12 Yellowfin, bigeye and bluefin species are used in sashimi, a delicacy in Japanese cuisine. In this market, the three bluefin species are the most valuable (US$30 per kg minimum) followed by bigeye. Yellowfin fetch much lower prices on this market.10 The canned tuna market primarily uses skipjack, albacore and yellowfin. Canned market value are inverse to species catch proportion, with skipjack products being the cheapest, followed by canned yellowfin then albacore products being the highest value. Bigeye species have been used for canning though these products are rare. Bluefin species are not used for canning.10 Skipjack 58% Atlantic, Pacific & Southern Bluefin 1% Yellowfin 27% Bigeye 8% Albacore 6%
- 8. 8 The threat ofoverfishing further highlights the need for effective speciation in tuna products, to facilitate the recovery of bluefin populations. Certain catch methods, known as Fish Aggregation Devices (FADs), encourage fish to congregate, allowing larger numbers to be caught.19 This can be unsustainable when endangered fish outside the target species are attracted, leading to their capture amongst the desired fish. This is known as bycatch, and is reported to contribute to the observed overfishing in bluefin populations, as well as damaging other ecosystems.20 This can also lead to accidental species substitution when substitutes go unnoticed during tuna operations. By developing new methods of tuna product authentication, tuna operations can be better regulated, compelling organisations to fish sustainably.20 The risk of fraudulent substitution is compounded when fish products are made. Tunas can be sold whole, but can also be skinned, filleted and portioned to make products consisting of only a section of the flesh. As a result, these products bear little resemblance to their species of origin, due to a loss of most morphological characteristics during production. Some characteristics are maintained, such as the pink flesh coloration of tuna. The exception to this is albacore, which has white muscle tissue. This feature is translated to albacore products. However, visual discrimination is still difficult for consumers, as the flesh of most tuna species is similar in appearance. Species substitution occurs frequently as a result, using substitutes both within the Scombridae family and outside of it. This is exemplified in an investigation by Warner et al.21 The study carried out across the US between 2010 and 2012 revealed that 59% of 114 tuna samples purchased from retailers were mislabelled. In some cases, escolar (Lepidocybium flavobrunneum) was often used in place of albacore due to their similar morphologies (white flesh). Escolar is a species that is not digestible by humans without negative health effects.21 It becomes impossible for consumers to verify tuna species when products are canned, as the flesh cannot be seen before purchase. As the raw material loses even more identifying features during the canning process, it is also harder to regulate the fraudulent substitution of species in these products.22 In particular many authentication techniques are challenged by two heating stages that occur during processing, once to cook the flesh and once to
- 9. 9 sterilise the cannedproduct. The sterilisation step is particularly severe, with cans being heated to a minimum of 115.6 °C in a retort for at least 40 mins.23 This leads to further changes in the morphology of tuna flesh, and can sometimes result in damage to, or loss of analytes used by authentication techniques. Additionally, tuna can be cut into smaller pieces (i.e. slices, chunks or flakes) prior to canning. This increases the risk of partial substitution within these products, compared to those consisting of whole portions of flesh (i.e. steaks), as individual pieces within a product could be sourced from different species. This translates to the frequent occurrence of canned tuna fraud. Investigation into popular brands of tinned tuna in twelve countries (including Australia, the UK and the US) illustrates this issue. It was revealed that 30.3% of the 165 products tested contained, or consisted of, species not declared on the label.24 2.2. TUNA AUTHENTICATION PROTOCOLS The cases of fraudulent practice, and the risk of its occurrence in the future, accents the need for novel methods of tuna classification to ensure adulteration detection. Techniques most widely used rely on DNA based analysis, though a variety of immunological, chromatographic and spectroscopic tools are also relevant. 2.2.1. DNA-based Analysis The analysis of species-specific DNA generally involves methods derived from the polymerase chain reaction (PCR). This replicates specific genes of interest to create an amplicon (i.e. amplified material) for further testing.1 The use of species-specific primers, base sequence comparison, fragment length comparison, or other approaches can then reveal further information about a sample.25 The level of detail obtainable from non-coding regions of DNA makes these techniques useful.26 They have been successfully used for the commercial identification of species and geographical origin as well in other areas of authentication for a range of commodities.27 When determining an analyte, MT-CYB, a region of mitochondrial DNA (mtDNA) is often selected for the differentiation of fish species.22 This gene exhibits considerable inter-species variation due, in part, to the rapid evolution of mtDNA. Additionally, the reported
- 10. 10 thermostability of DNArelative to most proteins, combined with the abundance of mitochondria in a cell, makes MT-CYB suitable for canned tuna analyses. This is because the thermal treatment during canning is claimed to result in the irreversible degradation of many analytes within a sample.22 2.2.1.1. PCR-RFLP In terms of tuna, restriction fragment length polymorphism (RFLP) preceded by PCR amplification is widely used for fish species identification.27 PCR-RFLP is an inexpensive and simple technique carried out by the digestion of an amplicon, using a restriction enzyme (endonuclease). The fragments (termed alleles) can then be separated by length using gel electrophoresis. Fragment length polymorphism is observed when there is variation of corresponding allele lengths between individuals. By comparing the presence of certain alleles with the allelic frequencies observed in a given species, the origin of a sample can be established. An investigation carried out by Lin & Hwang 28 demonstrates the ability of the technique to differentiate between tuna. The study utilized PCR-RFLP comparison of MT-CYB to correctly identifying eight species amongst 18 commercially canned tuna products.28 Despite the strengths of PCR-RFLP, there are shortcomings intrinsic to the technique. Firstly, the use of PCR-RFLP is dependent on the allelic database used for species determination. As DNA is also variable intra-specifically, substantial polymorphisms are observed between the restriction fragment lengths of conspecific individuals. Substantial sample sizes from each species of tuna are therefore required to account for these variations.22, 27 Another issue is that the restriction patterns of a species are not necessarily unique. This could lead to the occurrence of false positives when carrying out analyses on unidentified samples.27 To avoid difficulties associated with these and other limitations, standard practice specifies that PCR- RFLP cannot be relied upon solely for the purposes of tuna authentication, due to the risk of unknown adulterants in tuna products.27 2.2.1.2. Immunological Analysis Enzyme-linked immunosorbent assays (ELISA) have many applications in authentication including allergen,29 irradiation30 and genetically modified food detection.31 They are also
- 11. 11 used for thespecies assignment of milk and meat products. This involves the detection of a species-specific analyte using a complementary antibody whose presence can be revealed via the use of a flourogenic or chromogenic substrate.32 A variety of kits for numerous species have been developed for commercial use, containing microtiter plates or immunosticks coated with the relevant molecules (antigens/antibodies) for simple and rapid analysis. It is for this reason that some of these kits are used by regulatory agencies to detect meat adulteration on site, in order to enforce legislation.31 The analyte is usually an antibody or an antigen though other macromolecules have been used, including DNA sequences. ELISA targeting DNA has recently been adapted to hierarchically distinguish between tuna species.33 This version of the method is preceded by PCR, focusing on regions of the MT-CYB gene as analytes. The method utilized five probes bound to biotin (biotinylation) specific to T.albacares (yellowfin), T. alalunga (albacore), T. orientalis (Pacific bluefin), T thynnus (bigeye) and K. pelamis (skipjack). A hierarchical ELISA system was developed consisting of two stages. The first established the presence of Thunnus in the sample and the second established the presence of one of the five aforementioned principal market tunas in the sample. After method validation, 11 commercial samples were analysed, revealing that 2 were mislabelled.33 With further development, this technique could be beneficial for the field screening of tuna in industry, as it is in meat production. In its current form however, analysis is still quite time consuming as kits are not commercially available for the plethora of species involved in the tuna industry. The reliability of ELISA based speciation has also been criticised, as extensive purification is required to eliminate cross-reactivity, causing fluctuations in the speed and efficacy of analysis between species.32 2.2.2. Proteomics The analysis of proteins for tuna authenticity is controversial, due to their reported lack of heat stability.22 This implies they are likely to be denatured (i.e. the loss of a protein’s natural conformation within an organism) and degraded (i.e. the breaking of covalent bonds in the protein’s amino acid sequence) when heat is applied. This would make them unsuitable
- 12. 12 biomarkers for cannedtuna which is sterilized by prolonged exposure to high temperatures.23 However, this claim is often disputed in the literature.34 Investigation into the physiochemical mechanisms behind protein thermostability has established that this property varies greatly between proteins. This deviation can be attributed to singular changes in the amino acid sequence of a protein, particularly if the number of hydrogen bonds or ion pairs are affected.35, 36 Additionally, thermal denaturation and degradation, is tolerable to a certain extent, when the desired biomarker is one of the protein’s constituent peptides. Analyses using peptides are only affected when the primary structure (the amino acid sequence) of that peptide is degraded. As the amino acid sequences of a peptide are shorter than those of a protein, and shorter still than many DNA sequences, the risk of critical peptide degradation is greatly reduced.37, 38 This implies that the selection of appropriate proteins (and peptides) for species identification, can bypass this issue of thermostability. Analytes can be selected using mass spectrometry via top-down, bottom up or shotgun strategies (Figure 3).39 The40 shotgun41 approach is effective for this application, as exemplified by von Bargen et al 42 . The study successfully identified 12 peptide sequences that were characteristic of beef, horse and pork which were later successfully detected below the 1% level in mixed samples (horse/ pork in beef) via tandem mass spectrometry. This study highlights the relevance of proteomic study in authentication. It also demonstrates that the adaptation of successful techniques used on related commodities should be considered for Figure 3. Workflows for proteomic study. Three approaches to mass spectrometry: top-down (blue), bottom-up (orange) and shotgun (green). Top-down proteomics is the analysis of intact proteins whilst bottom-up proteomics involves the analysis of peptides generated by protein digestion. Protein and peptide mixtures tend to be separated, or certain molecules isolated, prior to digestion in order to reduce system throughput and simplify data analysis. Separation can be achieved via chromatography (e.g. HPLC) or gel electrophoresis (GE) A variant of the bottom-up approach, known as shotgun proteomics, removes this separation step to provide information on all the constituents of a given sample, though this reduces the sensitivity and selectivity of analysis.40, 41 Sample Protein Mixture Peptide Mixture Separated Peptides LC/MS or LC-MS/MS Separated Proteins ENZYMATIC DIGESTION EXTRACTION HPLC/ GE HPLC/ GE
- 13. 13 novel tuna authenticationprotocols. These commodities include other fish, seafood or even meat products, as just described.42 2.2.3.Mass Spectrometry The mass and isotopic signature of a sample and it’s constituents can be determined by mass spectrometry (MS). Mass spectrometers utilize three different components to carry out each process within an instrument. The ion source ionises the sample, followed by ion separation in the mass analyser, based on their mass to charge ratio (m/z). Detectors then record when the sorted ions pass by (or hit) its surface producing an ion signal as a function of m/z. Ions will behave differently depending on their weight and charge, allowing their characterization.43 For high throughput analyses (i.e. the analysis of samples containing many proteins or peptides), MS can be improved with an appropriate separation technique in order to produce results of better clarity and higher specificity. As the current work centres on mass spectrometry, it is important to consider suitable MS instrument configurations for the analysis of tuna products. Additionally, as these products are complex mixtures (i.e. contain many proteins), it is also important to couple a suitable separation technique to enhance analysis. 2.2.3.1. Separation Techniques The present work uses chromatography, which will be the focus of this section. However, other methods of separation, such as electrophoresis44 or ion mobility spectrometry (IMS)45 are also used in food authentication. Chromatography separates a sample based on the differential interaction of it’s constituents with two phases, a stationary phase and a mobile phase. A sample is carried through a structure containing the stationary phase, by the mobile phase. Constituents with varying chemical structures have differing affinities for the stationary phase, causing them to be adsorbed onto it for different lengths of time, known as retention times (Rt). This causes them to be desorbed (termed eluted) separately. They are then injected into the mass spectrometer for detection in chromatography-MS coupled systems. The nature of the stationary phase, the mobile phase and the structure (or bed) housing the stationary phase are ways in which different chromatography techniques are classified.
- 14. 14 In gas chromatography(GC), the mobile phase is gaseous and in liquid chromatography (LC) the mobile phase is liquid. In the current context, the former is not relevant, as liquid chromatography is required for the analysis of biological material. This is because it is more suitable for the analysis of non-volatile and thermally fragile molecules.46 In liquid chromatography the stationary phase can be contained within a column or present on a plane. Column chromatography is more efficient than planar chromatography, as the mobile phase can be pressurised in a technique known as high-performance liquid chromatography (HPLC). HPLC further improves upon other low pressure chromatography, by utilising smaller adsorbent particles in the stationary phase (known as packing particles), providing a larger surface area. This increases the resolution of separation, the degree to which two similar compounds can be separated. 47 For the analysis of organic analytes (those containing carbon) such as proteins, hydrophobic stationary phases are preferred. In these systems, analytes are usually dissolved in a polar mobile phase and molecules within the sample are retained in the column via hydrophobic interactions. The most hydrophobic molecules are late-eluting whilst the more hydrophilic ones elute first.48 This is known as reversed-phased chromatography (RPC). A commonly used stationary phase in (RPC) is an octadecyl carbon chain (18 carbon atoms) bound to silica. These are found within C18 columns, and provide a large surface area and pore volume to increase the retention of hydrophobic analytes.48 This increases the selectivity and efficiency of separations.47 When analysing samples that consist of molecules that exhibit a range of hydrophobic behaviour, the most hydrophobic molecules can be retained for long periods of time, increasing overall run times.49 To overcome this gradient elution is used. This is where the composition of the mobile phase is altered during separation to become more hydrophobic as separation progresses. This decreases the retention times of the most hydrophobic constituents, whilst still maintaining effective separation.49
- 15. 15 2.2.3.2. Ion Sources Animportant component that can dictate the selection of entire mass spectrometry configurations, is the selection of a sample-appropriate ion source.43 For the analysis of tuna proteins, or other biological molecules, soft ionisation sources are required. These sources result in minimal sample fragmentation, by reducing the amount of residual energy exerted on a sample.43 In macromolecular analysis this is important to ensure that the molecular (heaviest) ion is always observed, making these techniques valuable for protein identification via top-down workflows (Figure 3). Modern spectrometry typically uses two techniques for the soft ionisation of proteins; matrix-assisted laser desorption/ionisation (MALDI) and electrospray ionisation (ESI). In a MALDI system, the sample is first mixed with a UV absorbing crystalline matrix to serve as a proton source for ionisation, as well as to protect the sample from unwanted fragmentation. The mixture is then placed on a metal plate and vaporised before being ionised by the gases in the chamber.50 MALDI-MS has been used to successfully identify protein biomarkers able to identify 25 commercially sold species of fish, outside of the Scombridae family.51 This demonstrates the technique’s ability to discriminate between closely related fish species, though MALDI is limited to analysing larger molecules.50 This is because of the interference caused by the matrix in MALDI analyses, limiting the sensitivity and specificity of these systems. Additionally, the limited charged species produced by MALDI-MS (predominately 1+ ions) are unsuitable for fragmentation, a difficulty overcome by the variety produced by electrospray (2+, 3+, 4+ and other ions). Electrospray ionisation (ESI) employs an electric field to disperse a liquid analyte into a fine aerosol. Prior to the application of current, the sample is mixed with a conductivity increasing solvent which acts as a proton source to facilitate ionisation.52 The range of charge species produced by ESI allows fragmentation. As a result, electrospray ionisation greatly enables tandem mass spectrometry (MS/MS), more so than MALDI. This is in part, the reason why MALDI-MS/MS is not routinely used in proteomic study. Additionally, MALDI cannotbe paired with HPLC, removing the added specificity provided by chromatographic separation.
- 16. 16 The technique’s aptitudefor speciation has been demonstrated on meats.53 ESI-MS was used to examine purified haemoglobin and myoglobin sourced from beef, lamb, pork and horse. Analysis revealed that enough variation was present interspecifically in their amino acid sequences to make these proteins suitable biomarkers for species identification. This was observed in the study by the deviation in the weights of myoglobin and haemoglobin between species. The analytes were also deemed diagnostically viable even when subjected to high pressure steam at 121°C (autoclaved) for 1 hour. This indicates that ESI-MS on myoglobin and haemoglobin could be used to characterise cooked and processed products.53 2.2.3.3. Mass Analysers The second component of a mass spectrometer is the mass analyser. This is where ions are sorted and separated. Fourier transform ion cyclotron resonance mass spectrometry (FTICR- MS) is a type of mass spectrometry useful for the high throughput analysis of proteins. The technique utilizes a Penning trap mass analyser to deduce the mass to charge ratio of ions based on their movement in a fixed magnetic field (termed ion cyclotron resonance). 54 Due to the increased stability provided by the superconducting magnet in the mass analyser, FTICR-MS is able to achieve levels of resolution higher than any other MS instruments, making it ideal for detailed compositional and structural analyses of large molecules.55 However, these instruments are among the most expensive tools used for proteomic analysis. In addition, slow scan speeds are associated with the use of magnetic fields for mass discrimintation.56 For the purposes of developing a rapid, widely accessible technique, quadrupole based mass spectrometers are found more suitable. These instruments do not rely on magnetic fields, are relatively inexpensive and are found in most academic and industrial laboratories.56 Within quadrupole mass analysers, ions travel between four parallel metal rods. These rods have a voltage applied to them, producing an oscillating electric field. This electric field alters the trajectory of the ions. Ions with differing m/z values have varied trajectories: some within a narrow m/z region are able to pass through the quadrupole and get detected, while others are forced to collide with the quadrupole.56 Electron multipliers are often used as detectors in conjunction with quadrupole mass analysers.57 These consist of many dynodes (electrodes
- 17. 17 in a vacuum)that create secondary electrons upon impingement to increase signal intensity. This facilitates the detection of ions at low levels, providing increased sensitivity in MS analyses.57 Altering the voltage applied to quadrupoles allows the detected mass region to be altered, allowing selectivity.56 Quadrupoles can also be used to fragment molecular ions via collision induced dissociation. It is for these reasons that they are utilised in triple quadrupole instruments to provide additional structural information about molecules in tandem mass spectrometry.58 2.2.3.4. Tandem Mass Spectrometry The sensitivity and selectivity of single-stage mass spectrometry is restricted to relatively large peptide sequences. They therefore remain vulnerable to adulteration via false negatives. The presence of undeclared substances above 1% is considered actionable gross negligence or adulteration.37 With the aim of complete authentication in mind, the limit of detection (LOD) of analytes in a homogeneous sample must be below 1%. The ability of tandem mass spectrometry (MS/MS) to meet this requirement rapidly in comparison to other authentication protocols is where its value lies.42 It is largely for this reason that the present work utilises this technology. Tandem mass spectrometry overcome the issues associated with MS by the addition of a fragmentation step. In MS/MS systems, the initial ionisation and separation by m/z in the first mass analyser, is followed by the selection of ions (termed precursor ions) for fragmentation. This is carried out by collision induced dissociation or other Figure 4. An expanded MS/MS peak. The shown signals were derived from skipjack myoglobin. The transitions are indicated by each individual signal. The weights of each peptide fragment (product ion) are shown by the figure legend in daltons (Da). These were all fragmented, and therefore represent, the same peptide (precursor ion) and elute at the same retention time. The peptide weight is 565.3 Da. Intensity(cpsx10 6 ) Retention Time (minutes)
- 18. 18 processes, to formproduct ions. The product ions are then separated by a second mass analyser, followed by detection.59 A precursor ion and product ion pair is known as a transition. Each transition produces a unique signal upon its detection, which is representative of a peptide fragment (Figure 4). This causes MS/MS to yield very high levels of specificty, as the likelihood of multiple peptides having the same mass and fragments of the same mass is very small.41 This surpasses single-stage MS analyses which relies solely on peptide masses for information. A study by Wulff et al60 demonstrates the value of triple quadrupole tandem mass spectrometry in authentication. The study uses a bottom-up proteomics approach to build a database of spectra derived from the MS/MS analysis of whole proteomes. This database consisted of 22 fish species inculding yellowfin, albacore and skipjack tunas. The comparison of spectra produced from unknown samples to the database allowed the correct identification of raw, cooked and processed samples.60 This demonstrates the successful use of quadrupole instruments in tuna identification. The study also shows that tandem mass spectrometry remains reliable when testing various product forms, making it robust. However, the use of whole proteomes instead of selected proteins or peptides, makes this technique vulnerable to partial substitutions. Spectra produced from a mixture of species would not correspond to a database consisting of single-species samples. For the proper authentication of tuna products, this method would not be reliable unless mixed spectra were produced and included in the database. This would be arduous considering the number of potential adulterants. The use of whole proteomes also limits the sensitivity, and therefore the limits of detecion, of analyses.41 By selecting specific markers, partial substitution can be detected by the presence of markers from more than one species. Limits of detection and quantitation are improved by the use of selected reaction monitoring mode (SRM) (Figure 5).39, 41 This43 MS mode determines the presence of peptides based on a select number of transitions. This reduces interference caused by irrelevant ion signals, which selected for detection, increasing spectra clarity, run efficiency and detection capabilities.61 It is important that the reliabilty of transitions is established before disregarding other signals, to ensure accurate results are obtained. In the context of species identification, the signals
- 19. 19 deemed relevant wouldfunction as species markers when analysing samples of unknown origin. The application of SRM to multiple product ions orignating from the same precursor ion can further increase the sensitivity of analyses. This variation is referred to as multiple reaction monitoring (MRM) in the current work.59, 62 It is important to note that the use of this term varies in the literature, as it is not recognised by IUPAC as a standard term. The strengths associatied with the use of MRM MS for the analysis of peptides were demonstrated by a recently developed protocol for meat authentication.63 The adaptation of this study is central to the work at hand. MASS ANALYSER (Q1) Sample Selection Selection Selection Selection ScanningScanning Scanning Scanning Ionisation COLLISION CELL (Q2) MASS ANALYSER (Q3) Figure 5. Tandem-in-space mass spectrometry modes. When tandem-in-space, two coupled mass analysers are separated by spatially distinct controlled fractionation. In triple quadrupole instruments these processes take place in three quadrupoles. The pathways above indicates the four experiments primarily conducted with tandem-in-space MS. In product ion scans (red), the precursor ion is specified in the first mass analyser (Q1) and the resultant product ions from fragmentation (in Q2) are scanned then detected after the second mass analyser (Q3). Precursor ion scans (blue) involve the selection of the product ion in Q3 and the detection of precursor ion masses in Q1. In selected reaction monitoring (yellow), both Q1 and Q3 are set to a specific mass. In neutral loss scanning (green), mass analysers are set to scan all ions at an offset to account for the observed neutral loss typically observed in the analyte.43 ION SOURCE
- 20. 20 2.3. THE PRESENTWORK A method developed by Watson et al. 63 could be effectively applied to tuna speciation if modified. The study successfully used an intelligence-led method to identify horse, pork, beef and lamb via MRM MS. The approach differs from previous research by using the peptides derived from one protein to determine species identity, as opposed to the sets of proteins used in conventional shotgun strategies. Myoglobin (Mb) was exploited due to the presence of inter-specific variation in Mb amino acid sequences.63 Enzymatic digestion of the Mb enabled the production of characteristic peptide sets detectable by LC-MRM MS. This use of corresponding proteins for speciation via the analysis of representative peptide sets is referred to as CPCP for brevity. Corresponding proteins are nominally similar proteins with different species of origin. 63 This method is an attractive candidate for application to tuna products for a number of reasons. The original study demonstrated that species identification via this approach is more rapid than currently-used PCR-based approaches. An issue common to DNA-based methods is the time required for analysis, as extraction to final testing can take a number of days at best.42 This can be surpassed by MS analyses, particularly when analysing nominally similar proteins as markers. The use corresponding proteins for characterisation simplifies preparation, run times and data handling. The efficiency of analysis is further enhanced by the multiplex capabilities of MRM MS. A multiplex method is one that is able to detect multiple analytes in one test. This attribute allows the presence of many species in a sample to be determined rapidly, which is especially useful when considering the complexity of the tuna industry. The anaylsis of peptide fragments and the peptides they are derived from is also important to tuna anaylsis. For this tandem mass spectrometry is required. As many of the principal market tunas primarily belong to the same genus, the differences between the structures of their proteins are likely to be few. This will result in a small number of peptides suitable for species identification upon digestion. In order to reliably identify substitutions of these closely related species, the detection of multiple transitions (as in MRM MS) is a necessity to provide high sensitivity and specificity.
- 21. 21 These qualities arealso important to ensure that low level adulterations can be detected. Due to time constraints, quantitation will not form part of the present study on tuna. However, given the wider context of food authentication, it is beneficial to consider a technique likely to be able to distinguish between tuna species in a homogenous mixture, for the puposes of future research. It is also important to be able to determine the amount of the adulterant present in these mixtures relative to the total product. This is referred to as relative quantitation. Preliminary investigation of the CPCP approach indicates that this is within its capabilities. When applied to red meats, levels of relative quantitation as low as 1% in various horse, beef, pork and lamb mixtures were achieved.63 This supports the proposition that the CPCP approach is able to effectively identify the species present in a mixture of tuna species, using the peptides derived from corresponding proteins. This is contingent only on the presence of interspecific variation in the amino acid sequence of said proteins, which can be determined using an online database (such as UniProt). The likely transitions to arise from the digestion of proteins of interest can also be predicted by software (such as Open Source Skyline). This removes the labour intensive process of identifying sets of species-specific proteins, as required by high sensitivity identifications using whole proteomes (as in shotgun proteomics).42 An ideal technique for tuna species identification should also be versatile to reflect the numerous forms in which tuna is commercially sold. To reflect this the technique would remain robust when testing raw, cooked or heavily processed products, such as canned tuna. Again, initial testing of the CPCP approach on these product forms is encouraging as peptide markers for the identification of red meats were successfully recorded.63 Prior to analysis, it is first important to select an appropriate protein as a source of peptide marker sets. As stated previously, it is necessary to select a relatively heat stable protein for tuna authentication, to avoid the excessive thermal degradation of the analyte during processing. Cytochrome b, a mitochondrial protein used in the electron transport chain in aerobic respiration, initially seems like an intuitive candidate. This is due to the freuquent use of its coding gene, MT-CYB, in the DNA-based identification of fish species. However, initial comparison amongst principal market tunas indicates that corresponding cytochrome b
- 22. 22 proteins offer nodiscriminatory value, unlike its highly variable coding gene. This is due to a lack of interspecific variation in their amino acid sequences, revealed by using the BLAST tool on the UniProt database.64 The use of myoglobin could be beneficial, as it was in the original study by Watson et al.63 This protein facilitates the movement of oxygen within muscle tissue, and is also responsible for the pink colourationg of certain meats. The amino acid sequence of the most easily sourced tunas, yellowfin, albacore, skipjack and bigeye vary between these species, allowing their characterisation via via CPCP (Figure 6). 65 Study of myoglobin concentrations in both light (0.37-1.28mg/g) and dark (5.3-24.4mg/g) yellowfin muscle tissue indicates that Mb abundance will be adequate for analyses in species regardless of their flesh colouring.66 This is an important observation in the case of albacore, the white tuna. Additionally the pigmentation, location and water solubility of Mb, make them an attractive candidate for use in rapid analysis, as all of these properties simplify preparatory procedure.63,167 The thermostability of various Scrombidae Mb has also been explored. Investigation revealed that fish Mb, though more prone to degradation than horse Mb, are still relatively thermostable, suggesting that the peptides of interest would survive the retort stages of canned tuna production.68 There is also added value in using the same protein as the previous study of the CPCP approach, to explore the versatility of Mb whilst keeping already optimised preparation protocols universal between products. It is for these reasons that myoglobin was selected as the protein of interest in this investigation. P02205|ADFDAVLK*CWGPVEADYTTMGGLVLTR*LFK*EHPETQK*LFPK*FAGIAQADIAGNAAISAHGATVLK*K*LGELLK*A Q9DGJ2|ADFDAVLK*CWGPVEADYTTIGGLVLTR*LFK*EHPDTQK*LFPK*FAGIAQADLAGNAAISAHGATVLK*K*LGELLK*A Q9DGI8|ADLDAVLK*CWGAVEADFNTVGGLVLAR*LFK*DHPETQK*LFPK*FAGIT-GDIAGNAAVAAHGATVLK*K*LGELLK*A Q76G09|ADFDAVLK*CWGPVEADYTTIGGLVLTR*LFK*EHPETQK*LFPK*FAGIAQADIAGNAAVSAHGATVLK*K*LGELLK*A P02205|K*GSHAAILK*PLANSHATK*HK*IPINNFK*LISEVLVK*VMHEK*AGLDAGGQTALR*NVMGIIIADLEANYK*ELGFSG Q9DGJ2|K*GSHASILK*PMANSHATK*HK*IPINNFK*LISEVLVK*VMQEK*AGLDAGGQTALR*NVMGIIIADLEANYK*ELGFTG Q9DGI8|K*GNHAAIIK*PLANSHAK*QHK*IPINNFK*LITEALAHVLHEK**AGLDAAGQTALR*NVMGIVIADLEANYK*ELGFTG Q76G09|K*GSHAAILK*PLANSHATK*HK*IPINNFK*LISEVLVK*VMHEK*AGLDAGGQTALR*NVMGIIIADLEANYK*ELGFSG Figure 6. Corresponding Tuna Mb Amino Acid Sequences. The amino acid sequences of (from top to bottom) yellowfin Mb (P02205), albacore Mb (Q9DGJ2), skipjack Mb (Q9DGI8) and bigeye Mb (Q76G09). Cleavage sites arising from tryptic digestion are marked by “*”. The resultant peptides unique to one species are indicated by blue (yellowfin), green (albacore), red (skipjack), or purple (bigeye) text. Deviations from the ancestral amino acid sequence are marked in yellow. Amino acid sequences were obtained and aligned using UniProt database.65 Methionine from position 1 has been removed to reflect its cleavage from most mature proteins.63
- 23. 23 With this inmind, the protocols used for sample preparation were largely in accordance with those used in Watson et al.63 Two key preparatory stages are an extraction process and an additional purification process used to produce purified myoglobin. Extraction is a necessary preparatory step in proteomic analysis, to separate the protein and non-protein parts of a sample. This ensures that other cellular components, known as particulate matter, do not contaminate the results. In addition, some particulate matter is inhibits the function of the instrumentation and must therefore be removed prior to LC-MS/MS.69 As peptides and transitions in this approach are determined theoretically as opposed to experimentally, it is important to establish their visibilty prior to high throughput analyses on complex samples. For this reason, prior to enzymatic digestion (termed proteolysis), an additional purification step was employed to isolate myoglobin from the other proteins in certain samples. This reduces the intereference caused by other peptides within an unpurified mixture. Useful peptides were then selected as markers in these purified samples, based on the visibility of their transitions and their discrimnatory power. A peptide that is present in only one species offers the most discriminatory power as a species marker, whilst one that appears in many offers little. A multiplex method capable of scanning for the presence of all species at once can then be created by compiling these markers. As this purification is labourious and time consuming, once the presence of markers has been established in the purified Mb, this purification step is removed. Marker presence can then be tested in more complex extracts from raw tunas, and commercially canned tunas. This aligns with the intended purpose of this technique: to rapidly identify the species within a tuna product. Again, for the purposes of establishing peptide presence, purified samples were initally analysed using unscheduled monitoring. This is a mode in MRM MS where ion signals are scanned for at all retention times. Once the retention times of the selected peptides have been determined, samples can be tested using scheduled monitoring. This MRM MS mode only scans for ion signals at specified retention times, further increasing the clarity of results by removing background at other retention times.
- 24. 24 2.3.1.Objectives & Hypotheses Tobetter understand the versatility of the CPCP method, and assess the ability of the approach to combat tuna fraud, the aims and objectives of this investigation were as follows: 1. To establish whether the CPCP approach can be used to distinguish between various species of tuna myoglobin using MRM MS: H1 – An unscheduled MRM method searching for predicted transitions in purified myoglobin yields signals that do not differ sufficiently between tuna species. The results of this analysis could not be used to select markers for a multiplex method searching for signals at scheduled retention times. The CPCP approach cannot be used to distinguish between various species of tuna via MRM MS. 2. To establish whether the CPCP approach can be used to rapidly distinguish between various species of tuna myoglobin using multiplex MRM MS: H2 – A multiplex MRM method performed on purified myoglobin produces signals that do not differ sufficiently between tuna species at schedules retention times (Rt). The CPCP approach cannot be used to rapidly distinguish between various species of tuna via MRM MS. 3. To establish whether the CPCP approach can be used to rapidly distinguish between various species of tuna myoglobin in raw tuna samples using MRM MS: H3 – A multiplex MRM method performed on raw tuna extracts produces signals that do not differ sufficiently between tuna species at scheduled retention times (Rt). The CPCP apprach cannot be used to rapidly distinguish between various species of tuna in readily available tuna samples via MRM MS.
- 25. 25 4. To establishthe whether the CPCP approach can be used to rapidly distinguish between various species of tuna myoglobin in extensively processed samples using MRM MS: H4 – A multiplex MRM method performed on canned tuna extracts produces signals that do not differ sufficiently between tuna species at scheduled retention times (Rt). The CPCP apprach cannot be used to rapidly distinguish between various species of tuna in extensively processsed tuna samples via MRM MS. 3. METHODOLOGY The detailed workflow and protocols are based upon previous work by Watson et al.63 3.1. Materials Methanol and acetonitrile were purchased from Fisher Scientific (Loughborough, UK). Urea, trifluoroethanol and trypsin (from bovine pancreas, TYPCK treated) were purchased from Sigma-Aldrich (Gillingham, UK) and formic acid from BDH Chemicals (Poole, UK). Both raw and processed yellowfin (Thunnus albacares), albacore (Thunnus alalunga) and skipjack (Katsuwonus pelamis) were sourced from local supermarkets or an online fishmonger (King Catch, Reading, UK). Due to sourcing difficulty, only raw bigeye (Thunnus obesus) was obtained for study. Prior to preparation, raw tuna was stored at -40 °C. Processed tuna remained within cans at room temperature. Prior to the production of purified reference myoglobin and raw sample preparation raw tuna meat was diced then frozen in liquid nitrogen. Samples were then ground using a Waring blender and stored at -40 °C. 3.2. Purified Myoglobin Preparation Ground tuna samples (30 g) were extracted at 4 °C in 30 mM TRIS/HCl (pH 8.4). The extract then underwent gravity filtration and the filtrate was centrifuged (4 °C, 30 mins 14000 g). The supernatant was then filtered through syringe filters of decreasing size (0.8 μm, 0.45 μm and 0.2 μm). Sodium azide (0.02%) and potassium sorbate were added (1 mg/ml) and then the sample was syringe filtered (0.2 μm).
- 26. 26 A 2 mLaliquot of crude extract was then loaded onto a Superdex 75 size-exclusion column (GE Healthcare, Chalfont, UK) attached to a BioCad Sprint HPLC system (Applied Biosystems) for FPLC separation. This was equilibrated with 100 mM TRIS/HCl (pH 8.4) at a flow rate 1 mL/min with a fixed absorbance at 280nm. Resultant fractions were then analysed by SDS- PAGE to identify those that contain myoglobin (Mb) by weight (circa 16kDa). Selected fractions were then pooled and stored at 4°C. The purified samples were then loaded onto a PD10 column (GE Healthcare) and eluted with 25mM ammonium bicarbonate prior to proteolysis. 3.3. Raw Sample Preparation The phosphate extraction buffer (4 mL of 0.3 M KCl, 0.3 M phosphate buffer at pH 6.5) was added to 300 mg of ground sample and then vortexed (30 s). The sample then underwent extraction at room temperature for 2 hrs on a Stuart SB3 tube rotator at 20 rpm. Aliquots of sample (2 mL) were then centrifuged (5 min, 4°C, 17000 g). 200 μL of the supernatant were then evaporated to dryness at 50°C, using a Jouan RC 1022 centrifugal evaporator. 3.4. Processed Sample Preparation Cans of processed sample were drained of liquid and 20 g of sample weighed into a plastic beaker. The phosphate extraction buffer (100 mL of 0.3 M KCl, 0.3 M phosphate buffer at pH 6.5) was added. The sample then underwent extraction by blending for 1 min using an Ultra Turrax blender. Aliquots of the extract (2mL) were then centrifuged (5 min, 4°C, 17000 g) and 200 μL of the supernatant evaporated to dryness at 50°C using a Jouan RC 1022 centrifugal evaporator. 3.5. Proteolysis The dried samples, either raw or processed, were dissolved in 25 mM ammonium bicarbonate prior to the rest of proteolysis. This step is the final difference in the treatments of the purified myoglobin, the raw samples and the processed samples. Therefore, for brevity, all shall be referred to as “the samples” or similar from this point forward.
- 27. 27 The samples wereheated to 95°C for 30 mins, then cooled. Urea was added to a final concentration of 0.5M. Trypsin solution (1mg/ mL) was added in a 1:30 enzyme to substrate weight ratio. The samples were then vortexed and digested overnight at 37°C. The digests were diluted 1:2 with water and desalted using a Strata-X 33μ reversed phase cartridge (Phenomenex, Macclesfield, UK). The cartridge was washed then activated using 1 mL of methanol then equilibrated with 1 mL of 1% formic acid. The cartridge was loaded with the diluted sample and washed with 1 mL 5% methanol/1% formic acid. Peptides were then eluted by adding 1 mL acetonitrile/water (90:10; 0.1% formic acid). The samples were evaporated to dryness using the centrifugal evaporator then dissolved in 250 μL acetonitrile/water (3:97; 0.1% formic acid). 3.6. Predictive Tools The likely peptides resulting from the tryptic digestion of yellowfin (Thunnus albacares), albacore (Thunnus alalunga), skipjack (Katsuwonus pelamis) and bigeye (Thunnus obesus) Mb were predicted using the PeptideCutter tool 70 set to the ‘simple model’ option. Potential transitions produced by fragmentation during MRM-MS were then predicted using Open Source Skyline.71 Skyline setting included the following: 0 missed cleavages, conventional trypsin rules [KR|P], no modifications, exclude N-terminal, amino acids set to 1, No cysteine modifications, amino acids set to 1 (to avoid initiator methionine) peptide length 6 – 50, collision energy of ‘ABI 4000 Q Trap’. 3.7. LC-MS/MS Workflow Skyline-predicted transitions were used to create one MS/MS method file for each tuna species. The presence of these transitions was then determined. This was accomplished by testing each species of purified Mb with its own method in unscheduled monitoring mode. The peptides useful as markers were then selected based on signal quality and species specificity. The retention times and four most intense transitions (based on peak height) for all selected peptides were then recorded.These were used to create a single multiplex MS/MS
- 28. 28 methods file forthe detection of all species at once in scheduled monitoring mode (scan time: 2 s, nominal retention window: ±50 s). New purified Mb, raw samples and processed samples were then analysed with this multiplex method. 3.8. LC-MS/MS Analyses Analysis was carried out via high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). This was conducted on an Agilent 1200 Rapid Resolution LC system (Stockport, UK) connected to an AB Sciex 4000 QTrap triple quadrupole mass spectrometer (Warrington, UK). A Phenomenex XB C18 reversed-phase column (100 x 2.1mm, 2.6 μ particle size, Macclesfield, UK) using a 300 μl/min flow rate at 40°C was used for chromatographic separation. The binary gradient profile consisted of 97% A (water and 0.1% formic acid) and 3% to 28.4% B (acetonitrile and 0.1% formic acid) to 28.4%. Injection volume was 10 μl. Positive mode electrospray was used to detect eluted peptides using a TurboIonSpray probe (AB Sciex) ion source. Ion source settings were a curtain gas of 25 psi, desolvation gas (GSI) of 50 psi, and sheath gas (GS2) of 20 psi. Source temperature was 550°C. Data was acquired on Analyst 1.6.2 software (AB Sciex) as selected-reaction monitoring chromatograms. These were then converted into total ion current chromatograms, displaying the sum of all signals detected at any given retention time for illustrative purposes.
- 29. 29 4. RESULTS As thisstudy did not involve quantitation or limits of detection study, the relative intensities of ion signals were merely used as an indicator of peak visibility. Only the presence of a precursor and product ion pair (transition) can be determined using these chromatograms. No relationships can be accurately drawn between the area underneath each peak and the quantity of an analyte in this dataset. 4.1. Peptide Marker Selection The targeted search of the four myoglobin for the predicted transitions revealed a number of clearly defined signals (Figure 7). This established that the peptides and their fragments within the digests of the purified myoglobin were detectable by MRM mass spectrometry. 0 6 12 18 24 30 0 10 20 30 40 Intensity(cpsx10 6 ) 0 3 6 9 12 0 10 20 30 40 0 0.2 0.4 0.6 0.8 1 0 4 8 12 16 B C A Retention Time (minutes) D 858.5 565.3 450.8 336.2 423.2 832.4 1035.0 858.5 336.2 423.2 565.3 450.8 832.4 821.51127.1 825.4 737.4 572.3 336.2 423.2 565.3 450.8 336.2 423.2 832.4 875.5 Figure 7. Total ion current chromatograms resulting from the unscheduled screening of purified yellowfin (A), albacore (B), bigeye (C) and skipjack (D) myoglobin. Each ‘peak’ is the sum of all signals at any given retention time. The numbers indicate the m/z values of the peptides observed at that retention time. These can be common to more than one species (black) or unique to one species (green). It is important to note that not all observed peaks are visible in this format without magnification. Only the most visible or the most unique peptides are labelled here.
- 30. 30 21 peptides associatedwith these signals were deemed useful as markers for the differentiation of the tuna species examined, and were selected for use in a scheduled multiplex method for further analyses (Table 1). Among the selected peptides, at least one was detected in each sample that was unique to that species. These peptides offer the most discriminatory power, though the peptides unique to albacore and bigeye produced ion signals with very low intensities. As a result it was necessary to retain clearly visible peptides with less discriminatory power in future methods, to increase confidence in the species identification of a sample. It was therefore established that the signals produced by the unscheduled screening of purified myoglobin can be used to distinguish between the analysed species of tuna, as the differences in these ion signals allowed effective species discrimination.
- 31.
- 32. 32 4.2. Multiplex MyoglobinAnalyses During multiplex analysis, most of the selected peptides and their fragments produced ion signals at the scheduled retention times (Figure 8). An exception to this was the fragments of 875.5, the peptide specific to albacore. This implies that this peptide is not a reliable species marker. Additionally, some ion signals did not always occur in the expected species, as the transitions from previously injected samples were detected in the next samples (Table 1). This caused the species identity of a sample to be unclear, due to a loss of peptide specificity. It was established that this was not due to contamination prior to LC-MS/MS analysis via the by testing a sample known to contain no peptides, referred to as a blank sample (Figure 9). After analysis, peptides were observed in the blank, establishing that the contamination occurred within the LC-MS/MS system. 0 4 8 12 16 0 5 10 15 20 25 30 35 40 0 3 6 9 12 15 0 5 10 15 20 25 30 35 40 0 0.2 0.4 0.6 0.8 1 0 4 8 12 16 Intensity(cpsx10 6 ) B C A Retention Time (minutes) (minutes) D 858.5 565.3 450.8 336.2 423.2 832.4 1035.0 858.5 336.2 423.2 565.3 450.8 832.4 821.51127.1 825.4 737.4 572.3 336.2 423.2 565.3 450.8 336.2 423.2 832.4875.5 Figure 8. Total ion current chromatograms resulting from the multiplex analysis of purified yellowfin (A), albacore (B), bigeye (C) and skipjack (D) myoglobin. Each ‘peak’ is the sum of all signals at any given retention time. The numbers indicate the m/z values of the peptides observed at that retention time. These can be common to more than one species (black) or specific to one species (green). Certain peptides (grey) were not detected during analysis. It is important to note that not all observed peaks are visible in this format without magnification. Only the most visible or the most unique peptides are labelled here.
- 33. 33 These results indicatethat the CPCP approach could not be used to rapidly distinguish between the tested tuna species, due to the presence of within system contamination in this dataset. 0 0.005 0.01 0.015 0.02 0 5 10 15 20 25 30 35 40 YAB (450.8) YASB (336.7) YASB (423.2) S (565.3) YAB (832.4) S (989.5) Figure 9. Total ion current chromatograms resulting from the multiplex analysis of ammonium bicarbonate. This was run after multiplex myoglobin analyses. Each ‘peak’ is the sum of all signals at any given retention time. The species of origin of each peptide are coded as YF = yellowfin, AL = albacore, skipjack = SJ and bigeye = BE. The numbers indicate the m/z values of the peptides observed at that retention time. The presence of peptides in this sample indicates that peptides are retained within the LC-MS/MS system. It is important to note that not all observed peaks are visible in this format without magnification Only the most visible peptides are labelled here. Intensity(cpsx10 6 ) Retention Time (minutes)
- 34. 34 4.3. Multiplex RawTuna Analyses Again, the scheduled multiplex analysis of many of the selected peptides and their fragments were detected as clear ion signals at the specified retention times. More background and noise was observed in these chromatograms (Figure 10). Therefore, it can be established that the selected myoglobin derived peptides are detectable in raw tuna extracts. No fragments of the peptides 1035.0 or 989.5 were detected during the analysis of these samples (Table 1). This indicates that these peptides are not reliable species markers Some transitions from previously injected samples were again detected in the next samples (Table 1). This was confirmed to be caused within LC-MS/MS using a blank sample (Figure 11). The present work indicates that the CPCP approach cannot not be used to rapidly distinguish between the species tested in raw tuna, due to the lack of specificity detectable by MRM-MS analysis in this dataset. 0 1 2 3 4 5 0 5 10 15 20 25 30 35 40 0 1 2 3 4 5 6 0 5 10 15 20 25 30 35 40 0 1 2 3 4 5 0 0.2 0.4 0.6 0.8 1 Intensity(cpsx10 6 ) B C A Retention Time (minutes) D 858.5 565.3 450.8 336.2 423.2 832.4 1035.0 858.5 336.2 423.2 565. 3 450.8 832.4 821.51127.1 825.4 737.4 572.3 336.2 423.2 565.3 450.8 336.2 423.2 832.4 875.5 737.4 565.3 Figure 10. Total ion current chromatograms resulting from the multiplex analysis of raw yellowfin (A), albacore (B), bigeye (C) and skipjack (D) samples. Each ‘peak’ is the sum of all signals at any given retention time. The numbers indicate the m/z values of the peptides observed at that retention time. These can be common to more than one species (black) or specific to one species (green). Certain peptides (grey) were not detected during analysis, whilst some peptides were observed in the wrong species (red). It is important to note that not all observed peaks are visible in this format without magnification. Only the most visible or the most unique peptides are labelled here.
- 35. 35 Multiplex Processed TunaAnalyses None of the selected peptides were detected in any of the 9 samples of canned tuna in either scheduled or unscheduled monitoring mode (Figure 12). This indicates that the analysis of myoglobin- derived peptides using the CPCP approach cannot be used to distinguish between the species present in heavily processed tuna using MRM analysis. 0 0.004 0.008 0.012 0.016 0.02 0 10 20 30 40 0 0.003 0.006 0.009 0.012 0.015 0 10 20 30 40 0 0.005 0.01 0.015 0.02 0.025 0 10 20 30 40 A B C Intensity(cpsx10 6 ) Retention Time (minutes)Figure 12. Total ion current chromatograms resulting from the multiplex analysis of canned yellowfin (A), albacore (B) and skipjack (C) samples. Each ‘peak’ is the sum of all signals at any given retention time. It was not possible to associate signals to any peptides no signals were observed that were discernible from background. 0 0.03 0.06 0.09 0.12 0.15 0 5 10 15 20 25 30 35 40 Intensity(cpsx10 6 ) Retention Time (minutes) YAB (450.8) YASB (336.7, 423.2) S (565.3) YAB (832.4) S (989.5) Figure 11. Total ion current chromatograms resulting from the multiplex analysis of ammonium bicarbonate. This was run after multiplex raw tuna analyses. Each ‘peak’ is the sum of all signals at any given retention time. The species of origin of each peptide are coded as YF = yellowfin, AL = albacore, skipjack = SJ and bigeye = BE. The numbers indicate the m/z values of the peptides observed at that retention time. The presence of peptides in this sample indicates that peptides are retained within the LC-MS/MS system. It is important to note that not all observed peaks are visible in this format without magnification. Only the most visible or the most unique peptides are labelled here.
- 36. 36 5. DISCUSSION 5.1. MarkerReliability The majority of markers selected were detected in the scheduled analysis of both purified samples and raw tuna samples. This demonstrates that marker detection was largely reproducible and therefore reliable. The detection of peptides in the raw tuna samples also demonstrates the versatility of this approach. These are encouraging observations. The largest distinction of the CPCP approach from previous work, is the minimal experimentation required for marker identification. Laborious processes are usually used to select species- specific markers, particularly in crude extracts, where it is difficult to find visible markers. This is because peptides are not all equally detectable in proteomic analyses and additional proteins can mask the signals produced by peptides of interest. Additional challenges are that some peptides are only present with post-translational modifications (PTMs), or that cleavages can be missed during tryptic digestion. 72, 73 The latter occurs when trypsin fails to cleave (i.e. break apart) the amino acid sequence of a protein at an expected residue (i.e. an amino acid within a protein). This can lead to false negatives caused by a shift in the molecular mass of uncleaved peptides.73 To be thorough, missed cleavages were searched for initially in all purified myoglobin, though none were detected. This is likely due to the optimised proteolysis procedure involving heat denaturation and the addition of urea, both promotes complete digestion.63 PTMs are covalent changes made to the primary structure of a protein, often via the introduction of new functional groups to it’s amino acid residues. These can lead to a shift in the molecular mass of peptides, causing them to have unexpected m/z values. If unmodified m/z values were used to search for modified peptides, this would result in false negatives. This can be problematic in proteomic study, due to the large number of PTMs possible.72 The occurrence of certain modifications should be considered for future analyses on different samples. The presence of high levels of methionine in tuna is noteworthy.74 The sulfur atom of this residue is prone to oxidation, forming methionine sulfoxide.72 Methionine is found at the start of all amino acid sequences (at the N-terminal) to initiate protein synthesis (termed
- 37. 37 translation). It isoften cleaved from the mature protein after synthesis to increase stability and enable proper function.75 As a result only four methionine residues remain in the selected markers and do not appear to be a source of error in the present work. However, four markers were not consistently observed in all samples, implying thatthey were not reliable (Table 1). The 1026.0 peptide was not detected in albacore in any sample, though it was consistently observed in bigeye. This could be due to the presence of a cysteine residue in this peptide, as cysteine is prone to modification via the formation of disulfide bonds, bridging two peptides to form a dimer.74 The reactive SH in cysteine makes these disulfide bridges commonplace, though methionine cannot form disulfide bridges as its sulfur atom is in a more stable thioester (R-S-CH3) group. The consistent lack of detection of 1026.0 in albacore compared to its consistent detection in bigeye is of interest. It could imply that albacore Mb are more prone to this modification than bigeye Mb, however this could also be due to a difference in Mb concentrations between the species. The inconsistent detection of the other cysteine-containing markers (989.5 and 1035.0) supports the explanation that disulfide bridges interfered with analyses. To reduce the occurrence of disulfide bridges, an additional reduction step should be included prior to proteolysis using tris(2- carboxyethyl)phosphine (TCEP) or a similar reducing agent.76 This will facilitate the digestion of Mb via trypsin by reducing any disulfide bonds prior to proteolysis. Reduction should be followed up with an alkylation step with iodoacetamide to inhibit the formation of any disulfide bonds by modifying any free sulfur groups (SH).76 The MRM-MS method file would have to be adjusted accordingly to include these peptides containing modified cysteine residues. The presence of the remaining unreliable markers deviated between samples. The mechanisms causing their lack of detection are likely to be dependent on the form of the sample. They will therefore be discussed separately for clarity. 5.1.1.Purified Myoglobin As they were both carried out on purified Mb samples, it seems counterintuitive that transitions detected in unscheduled analysis were not detected during multiplex analysis.
- 38. 38 Specifically, these missingtransitions were all associated with the peptide uniquely found in albacore (875.5). It is possible that this occurred due to carryover effects, the likely cause of within system contamination. This is an issue of great complexity, and will therefore be expanded upon at the end of this discussion. If marker unreliability was caused by carryover, detection of these markers would be expected in subsequent samples. This was not observed, making it unlikely that carryover was a cause of marker absence. It is important that this peptide can be reliably detected, as it creates the specificity needed for albacore identification. This is made more difficult by the fact that, when present, these transitions produced ion signals with very low intensities. This implies they are only present in low amounts within albacore. It is therefore likely that this peptide was lost due to human error during the purification of crude extract. The isolation of myoglobin from a crude extract is a lengthy process, with a large number of steps involved. This creates many opportunities for a peptide to be lost as residue, particularly when the levels of that peptide are low. To accurately establish the reliability of this marker, a greater number of replicates need to be analysed on the same and different samples. Additionally, false negatives could have occurred via the uneven distribution of peptides within the sample. This would mean the aliquot injected did not contain all peptides present in the sample, leading to the observed lack of detection. The fact that the peptides derived from tuna myoglobin have a tendency to aggregate, supports this theory.77 This occurs due to the slight hydrophobic behaviour of tuna Mb in solution, caused by a low number of charged residues in their amino acid sequences.77 The disruption of these aggregations could therefore increase the reliability of these markers. One method of doing so would be to add a chaotropic agent to samples prior to their analysis. This is a molecule that interferes with hydrogen bonds and other non-covalent forces within an aqueous (polar) solution to increase entropy (disorder). This results in a reduction of the hydrophobic effect, decreasing the formation of aggregates.78-80 Examples of chaotropes are urea or guanidine hydrochloride. These have been used in previous investigations as a mobile phase modifier to improve separation efficiency in liquid chromatography. Unfortunately, these are not compatible with mass spectrometry, as they can cause the suppression of ion signals reducing the sensitivity of analyses.81-82 They would therefore not be suitable in a coupled LC-MS/MS system.
- 39. 39 Investigation into thereduction of hydrophobic interactions between peptides in solution could therefore be useful to further develop this approach. 5.1.2.Raw Samples Two peptides were not detected in the raw samples of crude extract, 1035.0 and 989.5. Though they are both unique peptides, the loss of 1035.0 is of the greatest concern, as this is the only marker unique to yellowfin. Without it, the approach cannot effectively distinguish yellowfin from the other three species tested. Both human error and peptide aggregation could be responsible for this outcome as they are in purified samples. An increased number of replications and the use of a denaturant post proteolysis are therefore still advised in future studies. However, it is less likely that experimental error is the cause of peptide loss in the raw samples. This is because the preparative process is far shorter for raw samples, and there are therefore fewer opportunities for peptides to be lost in residue. It is even less likely the cause behind the loss of 1035.0, as the fragments associated with this peptide produced ion signals of relatively high intensity. This indicates that this peptide is fairly abundant within yellowfin myoglobin and is therefore unlikely to be completely lost this way. The lack of purification is likely to increase the number of peptide-peptide interactions. This is due to the fact that there are peptides present from many proteins in crude extract, as opposed to just those derived from isolated myoglobin. This could cause an increase in the number of disulfide bridges and a greater amount of aggregation. Disulfide bridges are also more likely to occur in aggregates, due to the increased proximity of cysteine residues from separate peptides. This is the likely mechanism for the lack of detection of 1035.0 and 989.5, as these peptides both contain cysteine residues. As discussed above, the reduction of hydrophobic interaction and aggregation could increase the reliability of these markers.
- 40. 40 5.1.3.Processed Samples In thepresent study, peptides were not detectable in the crude extracts derived from 9 cans of tuna. However, this is not in keeping with preliminary testing of the technique on the same system, which successfully detected peptides in other canned tunas. As well as this, the analysis of canned red meats via this technique also allowed successful product speciation.63 This indicates that the lack of detection in the present work was due to human error. This explanation is made more likely by the fact that the processed samples in this investigation were only analysed once, with one sample per can. These results therefore should not be considered a reflection of the capabilities of this technique, as the error should not reoccur during the analysis of new samples. As the largest difference between this work and the previous studies is the samples used, it is likely that the error occurred during sample preparation prior to LC-MS/MS analysis. Most errors in preparation are likely to cause incomplete extraction or partial proteolysis, if all the reagents used are genuine and functional. However, in these instances some peptides would be detected, as opposed to none at all. In order to cause the universal lack of detection in all processed samples, the error must be due to the complete failure of extraction or proteolysis. Unsuccessful proteolysis is most likely as Mb is known to be relatively resistant to digestion, but easy to extract.63, 83 This was countered by the heat denaturation of samples and the addition of urea prior to digestion. Despite this, proteolysis could have still failed due to the fragility of trypsin. To maintain proper function, trypsin must be stored in the correct conditions (i.e. below -20 °C). Failure to do so would result in unsuccessful proteolysis due to the inactivation of trypsin, which could explain the detection of no peptides in these samples.84 The functionality of the trypsin used should therefore be verified before using it in further study. The water solubility of myoglobin should also be considered in future processed analyses. Canned products consist of tuna portions stored in an organic or aqueous (water-based) solutions. In the current study, cans were drained of solution prior to extraction. For products stored in aqueous mediums, this could result in a loss of any myoglobin that has diffused into the solution. This is unlikely to be a large source of error in the current study, as processed
- 41. 41 samples within organicand aqueous solutions were analysed. However, aqueous solutions should be retained during the extraction of future samples, to maximise the yield of myoglobin for downstream analyses. This consideration could also be used to decrease the total time of analysis for this approach. If sufficient myoglobin has passively diffused, direct analysis of this liquid could be used to indicate the species composition of the product. The myoglobin content of various aqueous mediums in canned tunas should therefore be established to determine their utility for product authentication. 5.2. Marker Specificity The specificity of the selected peptides, was compromised during the multiplex analysis of consecutive samples. Despite this, the results indicate that these Mb peptides are still useful as markers. When determining the presence of predicted transitions, ion signals unique to yellowfin, albacore, skipjack and bigeye were produced. Discrimination between these species was therefore possible using these peptides. This implies that the current approach could allow the species composition of unidentified samples to be accurately determined with these markers. In this dataset, this is only true when samples are tested separately (with extensive LC-MS/MS cleans in between), to eliminate carryover, the cause of within system contamination. Once preventative measures for carryover are established, methods testing a number of samples in succession could, theoretically, be used for rapid species assignment with the selected markers. However, although the selected markers are able to discriminate between these four tuna Mb, the introduction of additional species may be problematic. Of particular concern is the fact that the amino acid sequences of the Mb in bigeye and Atlantic, Pacific and southern bluefin are identical. Therefore, these markers would not be able to discriminate between these species, creating a vulnerability to species substitution. In the sashimi market, there is a large economic incentive for the substitution of valuable bluefin products with cheaper bigeye. There is also the possibility of accidental bigeye substitution with bluefin, potentially via bycatch, which is concerning given the danger of bigeye extinction. 16-18, 56 To circumvent these risks, the use of alternative proteins with more interspecific variations in
- 42. 42 their amino acidresidues would be necessary, to distinguish between bluefin and bigeye species. 5.2.1.Carryover The main limiting factor in this investigation was the removal of peptide specificity by contamination. The fact that this was sourced within the LC-MS/MS system, confirms the occurrence of carryover, a cause of systematic error. This can be defined as the transfer of material from previously injected samples to the next, causing contamination.85 This is frequently observed in contemporary mass spectrometry, since the development of systems of greater sensitivity, with certain organic substances detectable at levels around 1 fg (1 x 1015 g).86 This means that even small amounts of carryover can produce ion signals via LC- MS/MS, causing false positives and negatives.87 In the current context, this could result in incorrect species assignment, when analysing samples of unknown origin. It is therefore necessary that the mechanisms that cause carryover be considered, before ways to reduce and eliminate it are determined. This would aid the development of this technique for tuna authentication, as well as enhance chromatography and tandem mass spectrometry as a whole. It is worth noting that carryover, though present, was not a critical issue when red meat samples were studied on the same LC-MS/MS system, with the same approach.63 This has two implications: first, that a certain amount of carryover is an issue universal to the system used; and second, that carryover is exacerbated by properties intrinsic to tuna myoglobin. The carryover specific to the system is most likely caused by the retention of residue from previous samples. This is commonly observed within the auto-sampler of LC-MS/MS systems. It can also be caused by analytes trapped in dead volume (i.e. empty space) elsewhere in the system. This can occur in poorly fitted connections between components, or due to damage within the components themselves. When a blank sample is run, carryover resulting from residue is reported to appear as mimicry of the previous chromatogram, which dilutes with subsequent runs.88 This is a fitting description of the carryover observed in the present study, indicating that residual sample is retained in the system. This can be avoided by running samples on an alternative system. The current system could also be deconstructed, followed
- 43. 43 by the thoroughinspection each of its constituents for damage and dead volume. In the present work, neither option was explored due to instrument availability and time constraints, though these avenues should be pursued in further research. With regards to the carryover specific to the sample, it is likely due to the adsorptive interaction of peptides with the surface of the LC-MS/MS system. This would lead to the retention of peptides within the system, until they are desorbed by subsequent injections.87 The amount of carryover observed in the same system would therefore vary between samples, depending on their chemical structures. This would explain the deviation in the amount of carryover observed in red meat Mbs and tuna Mbs. Studies have revealed that the number of charged residues in mammalian myoglobin is far higher than the number in tuna myoglobin, causing them to be relatively more hydrophobic in solution. 77 This could lead to an increase in the adsorption of tuna peptides to hydrophobic components within the LC- MS/MS system, such as any resinous materials (plastics). It could also extend their retention on the chromatography column.88 This would manifest as an increase in the levels of carryover in late eluting peptides. Late eluting peptides are the most hydrophobic, making them the most hydrophobic. They would therefore require the highest concentrations of organic solvent to elute, which occurs at the end of a gradient profile. In the present work, the late eluting tuna peptides were generally observed to cause the most intense and persistent carryover. This supports the theory that hydrophobic interactions were a cause of carryover in the system. Exclusion of parts of the LC-MS/MS configuration reinforced this, as carryover is present in both the auto-sampler, the column and elsewhere in the system. In previous works, Vespel, a durable polymer used in the rotor seals of sample injectors, has been criticized for its affinity to hydrophobic molecules. The replacement of these seals with ones made of PEEK (poly ether ether ketone) could bypass this issue, as these plastics are reported to be more resistant to hydrophobic compound adsorption.88 Additionally, measures were applied in this investigation to counter carryover by rinsing or flushing various parts of the LC-MS/MS system (Table 2).85 Though87 all89 reduced the levels of carryover observed, some even eliminating it, this was not without significant time penalty.
- 44. 44 This is impracticalfor use in rapid identification, as a key advantage of the CPCP approach is the speed of its analysis. It is therefore important that alternative solutions to peptide carryover be explored. Solution Rationale Method Efficacy Needle rinse Carryover can be caused by the transfer of analytes retained on the outside of the injection needle during each injection. Washing the outside of the needle can remove this residue. The outside of the injection needle is rinsed with in an organic wash (60% ACN, 30% i-PrOH, 10% water, 0.1% FA) in a flush port within the autosampler after each injection.87 Although the time penalty for this method was low (1 min per sample), only minor reductions in carryover were observed Blank injection The injection of a sample containing no peptides could flush any previous residue, removing carryover from the system. 10 μl of ACN/H2O (3:97; 0.1% FA) is injected between each sample on the same method. Using this method, the system is carryover safe after 4 injections. Unfortunately, this causes a significant time penalty increasing analysis time (52 mins per sample. TFE injection 2,2,2-trifluoroethanol (TFE) has a strong acidic character due to the electronegativity of its trifluoromethyl (CF3) group. This gives TFE a strong affinity for peptides, enhancing their solubility.87, 89 The injection of TFE could therefore disrupt peptides that have interacted with the LC- MS/MS system, eliminating carryover. 10 μl of TFE is injected between each sample on the same method. The system becomes carryover safe after 2 injections. Though this is less than injection with ACN/H2O (3:97; 0.1% FA) the time penalty is still substantial (76 mins per sample) Repeated gradient elution Targeting the occurrence of late eluting carryover, the fluctuation of the organic concentration of mobile phase can disrupt the hydrophobic interactions of the most organic signals by repeatedly creating a high organic solvent concentration that promotes their elution. After the initial gradient profile has run, the organic solvent (B) ramps to 100% and is held for 1 mins to allow pressure to subside. This was repeated 6 times. The system becomes carryover safe after 6 additional gradients. Time penalty is 110 mins per sample Table 2 Methods to reduce carryover in the LC-MS/MS analysis of tuna myoglobin. As quantitative analysis was not part of the current objectives, the efficacy of each method is commented on qualitatively, based on the observed reductions in the peak heights of carryover transitions. The system can be classed as carryover safe when the effect of carryover no longer interferes with the present analyses, it does not signify that all carryover has been eliminated.85 The most effective method currently seems to be a combination of a blank TFE injection with repeated gradient elution between samples.
- 45. 45 As described, theoccurrence of carryover interferes with effective sample speciation. However, in these circumstances it has allowed some positive conclusions to be made about the CPCP approach. Carryover, like all contamination, is essentially the unintentional creation of mixtures. Its presence in this study therefore indicates that some of the corresponding tuna myoglobin tested can be identified at very low levels in a homogeneous mixture. Though many carryover transitions did not occur in the expected species, they were still detected at the scheduled retention times. This allowed their species of origin to be identified. Based on this, the following is highly likely: skipjack is detectable in purified and raw yellowfin, albacore and bigeye; bigeye is detectable in raw yellowfin and albacore; yellowfin is detectable in purified albacore. These are powerful observations in the context of tuna authenticity, as the identification of species in a homogeneous mixture enables the detection of unlawful product adulteration. This is especially true for the detections listed concerning skipjack and yellowfin, as there is economic incentive for partial substitutions using these cheaper species. 6. CONCLUSION The MRM MS analysis of four species of tuna myoglobin yielded signals that differed significantly between each species of Mb. These results were then used to select 21 peptides suitable for use as markers in multiplex testing of samples for yellowfin, albacore, skipjack and bigeye. This allows the null hypothesis (H1) to be rejected. The CPCP approach can be used to distinguish between these four species of tuna. The multiplex analysis of purified myoglobin did not produce signals that differed significantly between tuna species. This was because of the occurrence of carryover within the LC-MS/MS system. Until a solution for carryover can be determined, samples cannot be analysed consecutively, without time consuming cleaning processes in between. For this reason, the null hypothesis (H2) that the CPCP approach cannot be used to rapidly distinguish between various tuna species via MRM MS, cannot be rejected. The multiplex analysis of raw tuna extracts did not produce signals that differed significantly between tuna species. This was again due to the occurrence of carryover within the system, removing the specificity of marker peptides. To allow rapid speciation, an efficient solution
- 46. 46 for carryover mustbe determined. Therefore, the null hypothesis (H3) cannot currently be rejected. The CPCP approach cannot be used to rapidly distinguish between various species of tuna in readily available samples. The multiplex analysis of extracts derived from 9 commercially canned tuna samples produced no signals at scheduled retention times. This is likely to have occurred due to failed proteolysis caused by human error. The null hypothesis (H4) cannot be rejected. The results support the conclusion that the CPCP approach cannot be used to rapidly distinguish between various species of tuna in extensively processed tuna samples. This support is not strong however, due to the high likelihood of error. To further develop this approach it is important to investigate ways to reduce hydrophobic interactions within tuna myoglobin. Based on these results and previous studies, the relative hydrophobicity of tuna Mb is likely to contribute to inconsistent marker detection (via peptide aggregation), as well as cause carryover with the LC-MS/MS system. Reducing these interactions could therefore allow discrimination between consecutively tested tuna samples, as well improve the reliability of the selected markers by disrupting aggregates. To further improve the latter, the addition of reduction and alkylation steps to sample preparation may be beneficial, to reduce the occurrence of disulfide bridges. It is challenging to find methods to reduce hydrophobicity that do not impede chromatographic separation, nor inhibit mass spectrometry. It is also important to identify a solution that does not cause a significant time penalty, to facilitate the intended purpose of this approach: to rapidly identify the constituent species in a tuna product. The myoglobin concentration in aqueous mediums should also be determined, to establish their utility for more rapid product authentication in relevant canned products. Despite the improvements to be made, the CPCP approach could still be a valuable contribution to tuna authenticity. Although accurate discrimination in tuna is not yet possible, the identification of markers visible in both purified Mb and raw tuna extracts indicates that this approach remains reliable in complex mixtures. Additionally, the fact that peptides were not detected in the processed extracts is not of great concern, as this is likely to have occurred
- 47. 47 due to humanerror. It is therefore not expected to reoccur in further testing, a theory supported by previous preliminary analysis of canned tuna with the same approach. The indication that low levels of tuna species will be identifiable in a homogeneous mixture, is also encouraging. This is based on the detection of carryover. For these reasons investigation into the application of the CPCP approach to tuna will continue at the Institute of Food Research. An aim of this will be to produce further publications, which the present work will be a part of. 7. ACKNOWLEDGEMENTS I acknowledge the financial support of the Institute of Food Research and the University of East Anglia. I thank Kate Kemsley, Neil Rigby, Mark Philo, Yvonne Gunning, Andrew Watson, Stephen Day, Andrew Mayes and Rebecca Lewis for their guidance and feedback throughout this project. 8. REFERENCES 1. Reid LM, O'Donnell CP, Downey G (2006); Recent technological advances for the determination of food authenticity, Trends in Food Science & Technology, 17(7): 344-353. 2. Dennis MJ (1998); Recent developments in food authentication, Analyst, 123(9): 151R- 156R. 3. Directorate General for Health and Consumers (2013); Horse Meat Issue, European Comission (Accessed: 15/05/2016) http://ec.europa.eu/food/food/horsemeat/tests_results_en.htm 4. Avery J (2014); Fighting food fraud, European Parliamentary Research Service (Accessed: 28/03/2016) http://www.europarl.europa.eu/RegData/bibliotheque/briefing/2014/130679/LDM_BRI(20 14)130679_REV1_EN.pdf 5. Squires N (2015); Italian olive oil scandal: seven top brands 'sold fake extra-virgin', The Telegraph (Accessed: 15/05/2016)
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- 56. 56 9. Appendix 9.1. ResearchProposal Analysis of Tuna via the CPCP method using Multiple Reaction Mode Mass Spectrometry Author: Joshua Kai Peazer Advisor: Dr Stephen Day BACKGROUND AND RATIONALE Events over the past few years have brought the issue of food contamination into the media spotlight. In 2013, it was discovered that an array of beef products in Europe contained or consisted entirely of undeclared horse meat. The implications of this were catastrophic for financial, health, ethical and religious reasons, accenting a flaws in current food authentication protocol (von Bargen et al 2013). The most widely used food authentication techniques currently rely on DNA-based testing. Unfortunately, these methods are slow, have a limited number of identifiable species, and have poor quantitative ability. This lead the Analytical Sciences Unit at the IFR (Institute of Food Research) to propose a new method of meat authentication using Multiple-Reaction- Mode Mass Spectrometry or MRM MS (Watson et al 2015). The approach determines the species composition of a sample, by detecting its constituent ratio of peptide markers. These peptides correspond to proteins characteristic of a single species, allowing identification. This process can be referred to as the CPCP method for concision. The technique has proven effective in identifying beef, pork, lamb and horse, both raw and cooked. The approach was also able to quantify adulterants in these meats at levels below 1%, surpassing traditional DNA-based authentication (Watson et al 2015). Similarly to the undeclared use of horsemeat, the fraudulent labelling of species as tuna is harmful both economically and medically. There are also ethical and environmental implications associated with tuna fraud. It occurs frequently, as it is unclear where the deception takes place in the supply chain. An investigation carried out by Warner et al. (2013) revealed that 59% of tuna was mislabelled from 2010 to 2012. Escolar (Lepidocybium flavobrunneum) was often used in its place, a species that is indigestible by humans without negative health effects (Warner et al 2013). As the CPCP method using MRM MS is a method still under development, its efficacy on many species has still not been tested. For this reason and the aforementioned issues associated with Tuna fraud, it would be both interesting and potentially profitable to test this novel method on tuna samples, both processed and raw. This would provide an interesting challenge for the method, to see if it is effective even on highly processed samples.
- 57. 57 OBJECTIVES & HYPOTHESES Tobetter understand the versatility of the CPCP method coupled with MRM MS, the aims and objectives of the proposed investigation are as follows: 1. To establish the whether the CPCP method coupled with MRM MS can distinguish between different species of tuna: H0 - MRM MS performed on purified myoglobin produces no signals that do not differ significantly between tuna species. CPCP coupled with MRM MS cannot be used to distinguish between different the species of tuna. H1 - MRM MS performed on purified myoglobin produces signals that differ significantly between tuna species. CPCP coupled with MRM MS can be used to distinguish between different the species of tuna. 2. To establish the whether the CPCP method coupled with MRM MS can be used to detect different species in readily available tuna samples: H0 - MRM MS performed on tuna steaks produces no signals corresponding to reference peaks. CPCP coupled with MRM MS cannot be used to detect species composition of readily available tuna. H1 - MRM MS performed on tuna steaks produces signals corresponding to reference peaks. CPCP coupled with MRM MS can be used to be used to detect species composition of readily available tuna. 3. To establish the whether the CPCP method coupled with MRM MS can be used to detect different species in tuna samples that have undergone extensive processing: H0 - MRM MS performed on tuna steaks produces no signals corresponding to reference peaks. CPCP coupled with MRM MS cannot be used to detect species composition of processed tuna. H1 - MRM MS performed on tuna steaks produces signals corresponding to reference peaks. CPCP coupled with MRM MS can be used to be used to detect species composition of processed tuna. METHODOLOGY It is important to note that all processes and will be repeated as many times as possible on as many species of tuna as time and sourcing allows. This will provide increased reliability of results. Predictive Tools for Comparison To predict the peptides produced from digestion, PeptideCutter will be used. Though the tool has received criticism for its limited dataset in respect to certain enzymes, its tryptic digest dataset has proven accurate and convenient (Wang et al. 2013; Watson et al. 2015; Afzal et al 2011).
- 58. 58 The Open SourceSkyline software will be used to predict MRM transition candidates for the ABI 4000 QTrap. This has proven to an analytical tool of great utility for MRM assay design on specific instruments (Liebler & Zimmerman 2013). Materials As in Watson et al (2015), methanol and acetonitrile will be purchased from Fisher Scientific (Loughborough, UK). Urea and trypsin (from bovine pancreas) will be purchased from Sigma-Aldrich (Gillingham, UK) and formic acid from BDH Chemicals (Poole, UK). Tuna products of various species, namely yellowfin (Thunnus albacares), albacore (Thunnus alalunga), skipjack (Katsuwonus pelamis) and bigeye (Thunnus obesus), will preferably be purchased from local supermarkets and fishmongers to avoid waste. If this is not feasible due to product availability, wholesalers and specialised fish suppliers will be utilized and tuna bought in bulk. Purified Myoglobin Preparation To test the first hypothesis, purified myoglobin will be prepared prior to proteolysis and LCMS analysis. This will also serve as a screening process, to identify biomarkers that can be used in further experimentation and to test hypotheses 2 and 3. Tuna samples (30g) will be extracted at 4°C for using 100mM TRIS buffer adjusted to pH 8.4 with HCl. The temperature and pH must be carefully controlled to reduce the risk of denaturing or otherwise affecting the purity of the desired myoglobin (Watson et al. 2015; Yee & Peyton 1995; Klis et al. 2007). The sample will then be filtered and centrifuged at 4°C for 30mins and the supernatant separated and filtered once more. Sodium azide and potassium sorbate act as biocides for anaerobic and aerobic respirators respectively. They will therefore be added for their utility as preservatives (Watson et al. 2015; Chefetz et al. 2006; Quintavalla & Vicini 2002). This crude extract will then be separated via FPLC, using a Superdex 75 size exclusion column (GE Healthcare, Chalfont, UK) attached to a BioCad Sprint HPLC system (Applied Biosystems). This was equilibrated with 100mM TRIS/HCl (pH 8.4) at a flow rate 1 mL/min. The use of FPLC over HPLC in protein identification and quantitation has proven favourable in past studies and slightly more cost effective (Tangvarasittichai et al 2007). A SuperloopTM will also be used as opposed to a manual sample loop to enable the purification process to become automated, increasing efficiency (Eisele et al. 2012). To monitor the eluent for protein, the absorbance will be fixed to 280 nm and the flow rate maintained at a low speed to avoid decreases in spectra resolution. Resultant fractions will then be analysed by SDS- PAGE to establish which contain the purified myoglobin. These will then be pooled and stored at 4°C. Tuna Sample Preparation Samples prepared in this section will be analysed via LCMS to test hypothesis 2. This is contingent on the success of hypothesis 1. If no species specific biomarkers are identified in the screening process, then H0 must be accepted for all hypotheses.
- 59. 59 Samples of groundtuna will be prepared and extracted using a phosphate extraction buffer (0.3 M KCl, 0.3 M phosphate at pH 6.5) again to prevent damage to the desired myoglobin by controlling pH. Phosphate buffers in previous study, have been shown to produce more extractable protein from meat samples (Nahar et al 2013). A 2 mL aliquot of the sample will then be centrifuged briefly at high speed and 200 μL of the supernatant evaporated to dryness at 50°C. This will be carried out using a Jouan RC 1022 centrifugal evaporator (Watson et al. 2015). Preparation of Processed Sample In order to test hypotheses 3 a canning process will be mimicked using known species of tuna. This will be kept as close to conditions in industrial canning as possible. Tuna will be steamed at 100°C for 60 min or until its temperature reaches between 60-85°C. The fish will then be water cooled and placed in a sealed container in a brine medium, then vacuum sealed to replicate the conditions of a can. The container will then be heated once more for 2 hours then preferably pressure cooled (Warne 1988). Sample Proteolysis The tuna samples either raw or processed will be dissolved in 25 mM ammonium bicarbonate prior to the rest of the proteolysis process. This step is the final difference in the treatments of the purified myoglobin, the raw samples and the processed tuna samples. Therefore for brevity, both shall be referred to as “the samples” or similar from this point forward. The samples will be heated to 95°C for 30 mins, then cooled. Urea will then be added to a final concentration of 0.5M (Watson et al. 2015). Low concentrations of urea has been shown to promote the unfolding which is beneficial in preparation for proteolysis (Schnarr & Maurizot 1981). The solution will then be digested by trypsin in a 1:30 enzyme to substrate weight ratio. The samples will undergo digestion overnight at 37°C as is standard protocol (Watson et al. 2015). Accelerated digestion times could result in an improper digest due to the heterogeneity of the samples (López-Ferrer D et al 2006). High concentrations of salt and urea are incompatible with analysis by mass spectroscopy (Gundry et al 2009). The tryptic digests will therefore undergo dilution with water and desalting using a Strata-X 33μ reversed phase cartridge (Phenomenex, Macclesfield, UK), as per standard operating procedure (Watson et al 2015). The desalted sample will then be evaporated to dryness via centrifugal evaporation. Finally, the samples will then be made ready for LC-MS/MS analysis by dissolving then in acetonitrile/ H2O (Watson et al 2015). LC-MS/MS Analysis Analysis will be carried out via high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). This will be conducted on an Agilent 1200 Rapid Resolution LC system (Stockport UK) connected to an AB Sciex 400 QTrap triple quadrupole mass spectrometer (Warrington UK). The capillary column used for chromatographic separations will be a Phenomenex XB C18 reversed-phase column using a 300 μl/min at 40°C (Watson et al 2015). Temperature must be controlled at relatively high levels during chromatographic separation to ensure a higher quality output (Blacker et al 2008). The
- 60. 60 binary gradient usedwill consist of 97% A (water and 0.1% formic acid) and 3% B (acetonitrile and 0.1% formic acid) to 24%. This will be increased for a period to 100% B at the end to ensure the extraction of all compounds of interest (Skoog & West 1980). Using the predictive tools, a MS/MS methods file will be created encompassing Myoglobin from all species of Tuna. Output from MS will be viewed on Analyst 1.6.2 software (AB Sciex). Positive electrospray in dynamic monitoring mode will be used to detect eluted peptides with the four MRM transitions of the highest intensity used to determine peak height (Watson et al 2015). The electrospray ionisation mass spectrometry (ESI-MS) can provide less structural information than other MS methods, the use of tandem MS negates this limitation (Ho et al 2003). REFERENCES Blacker AR, Speers AE, Wu CC (2008); Chromatographic benefits of elevated temperature for the proteomic analysis of membrane proteins, Proteomics, 8(19): 3956-64 Chefetz B, Stimler K, Shechter M, Drori Y (2006); Interactions of sodium azide with triazine herbicides: Effect on sorption to soils, Chemosphere, 65(2): 352-57 Eisele T, Stressler T, Kranz B, Fischer L (2012); Automated multi-step purification protocol for Angiotensin-I-Converting-Enzyme (ACE), Journal of Chromatography B, 911: 64-70 Gundry RL, White MY, Murray CI, Kane LA, Fu Q, Stanley BA, Van Eyk JE (2009); Preparation of Proteins and Peptides for Mass Spectrometry Analysis in a Bottom-Up Proteomics Workflow, Current Protocols in Molecular Biology, 88: 10.25 Ho CS, Chan MHM, Cheung RCK, Law LK, Lit LCW, Ng KF, Suen MWM, Tai HL (2003); Electrospray Ionisation Mass Spectrometry: Principles and Clinical Applications, Clinical Biochemist Review, 24(1): 3–12 Klis FM, de Jong M, Brul S, Groot PW (2007); Extraction of cell surface-associated proteins from living yeast cells, Yeast, 24(4): 253-8 Liebler DC, Zimmerman LJ (2013); Targeted Quantitation of Proteins by Mass Spectrometry, Biochemistry, 52(22): 3793-806 López-Ferrer D, Cañas B, Vázquez J, Lodeiro C, Rial-Otero R, Moura I, Capelo JL (2006); Sample treatment for protein identification by mass spectrometry-based techniques, Trends in Analytical Chemistry, 25(10): 996- 1005 Nahar MK, Zakaria Z, Hashim U (2013); Effect of Buffer on the Protein Extraction for Chicken Meat, Advanced Materials Research, 795: 206-10 Quintavalla S, Vicini L (2002); Antimicrobial food packaging in meat industry, Meat Science, 62(3): 373-80 Schnarr M, Maurizot JC (1981); Unfolding of lac repressor and its proteolytic fragment by urea: headpieces stabilize the core within lac repressor, Biochemistry, 20(21): 6164-9
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