1. 1
NAT-MN0Y
2015-16 Natural Sciences Project
Multiple Reaction Monitoring Mass Spectrometry of
Myoglobin Peptides to Determine Tuna Authenticity
Author:
6157106
Supervisor:
Dr Andrew Mayes
4. 4
1. ABSTRACT
The improper description 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 can be
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 of tuna 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 tuna13 are 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 of overfishing 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 canned product. 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 DNA relative 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 the species 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 canned tuna 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 authentication protocols. 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
An important 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 aptitude for 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 form product 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 would function 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 PRESENT WORK
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 are also 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 no discriminatory 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 in mind, 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
To better 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 establish the 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 mL aliquot 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 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
28. 28
methods file for the 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 this study 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 associated with 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.
32. 32
4.2. Multiplex Myoglobin Analyses
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 indicate that 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 Raw Tuna 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 Tuna Analyses
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. Marker Reliability
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 is often 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 missing transitions 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 the reduction 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 the present 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 organic and 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 acid residues 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 thorough inspection 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 impractical for 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, the occurrence 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
