HPLC-MS/MS of species-specific fibrinogen peptide as a qualitative
method for fibrin based meat binder
FINAL REPORT
Report Number:
FD05/36
Authors:
P.Reece, H.Grundy
Date:
10th Feb 2007
Sponsor:
Food Standards Agency
Sponsor’s Project Number:
Q01093
CSL Project Number:
M6EC
CSL File Reference:
FLN 8039
Principal Workers:
H.Grundy, J.Harrold, M.Sykes, P.Reece
Contract manager:
P.Reece
Distribution:
1. Dr. M. Woolfe (FSA)
2. Miss R. Hodgson (FSA)
3. Prof. J. Gilbert (CSL)
4. Dr. J. Dennis (CSL)
5. Information Centre (CSL)
6. Authors and Principal Workers
Central Science Laboratory
Sand Hutton
York
YO41 1LZ
Page 1 of 41
CONTENTS
EXECUTIVE SUMMARY.
3
ANALYTICAL SUMMARY.
3
1. INTRODUCTION.
5
1.1 THE TECHNICAL PROBLEM BEING ADDRESSED.
5
1.2 BACKGROUND.
5
1.3 MILESTONES OF THE PROJECT.
6
2. EXPERIMENTAL.
7
2.1 MATERIALS.
7
2.2 METHODS.
8
3. RESULTS AND DISCUSSION.
11
3.1 OPTIMISATION OF HPLC SEPARATION OF PEPTIDES.
11
3.2 OPTIMISATION OF LC-MS/MS DETECTION OF PEPTIDES.
12
3.3 EVALUATION OF METHOD.
14
3.3.1 Spiking studies.
14
3.3.2 Effect of cooking.
15
3.3.3 Further sample cleanup.
15
3.3.4 Analysis of commercial samples spiked with Fibrimex®.
16
4. CONCLUSIONS.
16
5. APPENDIX.
5.1 ADDITIONAL STUDIES ON TUNA STEAK.
CONTAINING BOVINE FIBRIMEX.
17
5.1.1 The problem being addressed.
17
5.1.2 Materials and Methods.
17
5.1.3 Results.
17
5.1.4 Conclusions.
18
6. BIBLIOGRAPHY.
18
7. GLOSSARY.
18
8. TABLES.
19
9. FIGURES.
23
Page 2 of 41
EXECUTIVE SUMMARY.
1
The developed technique detected the presence of both bovine and porcine fibrinbased meat binding agents at the 5% spiking level in beef, pork, lamb and
chicken, based on the detection of one MS/MS transition for the bovine agent and
two for the porcine agent.
2
The method appeared less sensitive in chicken and the binding agent markers
could not be detected in white fish (cod) although they were detected in other fish
matrices (tuna).
3
The effect of storage of the samples prior to analysis was not studied. Prolonged
storage of the products after addition of the binding agents may have a significant
impact on the limit of detection of the markers, especially in chicken.
ANALYTICAL SUMMARY.
1
This report documents the findings of a study into the detection of porcine and
bovine blood fibrinopeptides A and B as a means of detecting the use of blood
based cold setting agents and the species from which the blood was derived.
2
The amino acid sequence of the A and B fibrinopeptides of pig and cow were
obtained from public databases (Table 1) and the peptides synthesised by a
commercial organisation (Severn Biotech Limited).
3
Commercial bovine blood based cold setting agent (Fibrimex®) was obtained
from Harimex UK Limited (the supplier) and a porcine fibrinogen extract was
prepared as an in-house equivalent of the commercial porcine product.
4
The methodology for the separation of the synthetic peptides was established
using C4 reversed phase HPLC.
5
MALDI TOF-MS studies confirmed that the terminal glutamine of the synthetic
bovine Fibrinopeptide B underwent a chemical transformation when the peptide
was exposed to the acidic conditions during the HPLC separation, resulting in
conversion of the residue to pyroglutamic acid.
6
Extraction and MALDI TOF-MS analysis of the gelled bovine Fibrimex® and the
porcine fibrinogen, gelled with commercial thrombin, showed that all four
fibrinopeptides were present. Bovine Fibrinopeptide B was present in the
pyroglutamic acid form.
7
LC-MS conditions were optimised for the separation and characterisation of the
four fibrinopeptides, including the pyroglutamic acid form of bovine
fibrinopeptide B (bovine Fibrinopeptide B pGlu).
8
MS/MS analysis of the peptides identified characteristic fragment ions for all five
peptides (including bovine Fibrinopeptide B pGlu) such that theoretically the
presence of bovine blood binding agent could be confirmed by the presence of up
to 3 MS/MS transitions, one for Fibrinopeptide A and two for the pyroglutamic
acid version of the Fibrinopeptide B. The presence of porcine blood binding agent
could be confirmed by the presence of 6 MS transitions, two for the
Fibrinopeptide A and four for the Fibrinopeptide B.
9
Analysis of beef and pork meat bound with commercial bovine Fibrimex®, or
porcine fibrinogen and commercial thrombin, showed that the fibrinopeptides
Page 3 of 41
could be detected in samples spiked at less than the 5% commercial level of
addition.
10 Analysis of samples of lamb and chicken meat bound with commercial bovine
Fibrimex®, or porcine fibrinogen plus commercial thrombin, showed that the
fibrinopeptides could be detected in samples spiked at the 5% level based on a
minimum of one MS/MS transition. MS/MS peak heights for the fragment ions in
chicken muscle suggested that a significant amount of both the bovine and porcine
fibrinopeptides were not detected.
11 Spiking and storage experiments of both raw chicken muscle and denatured
chicken extract showed that the peptides were lost during storage of native
chicken muscle, suggesting enzymic breakdown of the peptides by intramuscular
peptidases.
12 Analysis of samples of cod muscle spiked with 5 and 10% commercial bovine
Fibrimex®, or with porcine fibrinogen plus commercial thrombin, showed that the
fibrinopeptides were barely detectable. A possible hypothesis to explain this is the
presence of a very active intramuscular peptidase.
13 Analysis of cooked samples of beef, pork, lamb, chicken and fish, spiked with 5%
bovine Fibrimex® showed that approximately 50% of the peptide remained with
the solid meat after cooking, allowing detection of the added Fibrimex® in the
meat based on the presence of the MS/MS transition for the Fibrinopeptide A.
14 Analysis of fourteen commercial uncoated and eighteen commercial coated
products blended with 5% Bovine Fibrimex® revealed that the detection of the
bovine Fibrinogen A could clearly be detected in all of the samples suggesting
that typical commercial ingredients do not interfere with the detection of
Fibrimex®.
15 A follow on study, examining the presence of bovine fibrinopeptides in tuna
samples containing Fibrimex showed that the bovine Fibrinopeptide A could be
detected in the tuna samples at the level of 1% Fibrimex® addition, even after
cooking, suggesting the problem of loss of the fibrinopeptides was restricted to
white fish muscle.
Page 4 of 41
1. INTRODUCTION.
1.1 THE TECHNICAL PROBLEM BEING ADDRESSED.
In an attempt to maximise the use of lower value carcass meats and trimmings, the
food industry has developed binders to incorporate these cuts into reproducibly sized
and shaped consumer portions. These binders have traditionally been extracts of the
meat that gel on heating. The recent demand for cold-setting agents has prompted the
introduction of protein gels extracted from bovine and porcine blood. These gelling
agents are being recommended for use in a variety of meat and fish products since the
agent is both colourless and odourless.
The binding agent is a fibrinogen-enriched plasma, which is mixed with thrombin
enzyme to coagulate and bind the ground meat together. The process does provide
opportunity for unscrupulous producers to fraudulently increase the declared meat
content of products, and the process also raises the issue of products of one animal
being added as undeclared processing aids in the manufacture of other meat species
and fish products. This raises issues of accurate labelling of these foods for religious
and ethical purposes. Therefore there was a need for a method to be developed to
detect the use of these agents, to speciate the blood used, for use by enforcement
officers in their investigations.
1.2 BACKGROUND.
Whole blood addition to meat products is relatively easy to detect because of the
presence of haemoglobin. Red meat contains an average of 0.5% blood (results of the
FSA survey, personal communication) and therefore levels significantly above this
usually indicate deliberate addition. The detection of plasma addition to meat is more
difficult, requiring the quantitative detection of specific blood proteins. At high levels
of addition, blood specific proteins, e.g. serum albumins, can be detected by
electrophoretic techniques while antibody techniques are required for the lower levels
of addition and addition of purified blood products. The detection of the plasma
proteins could be used to detect the addition of blood based gelling agents however it
would not discriminate between the simple addition of plasma to meat products and
the addition of blood products as a processing aid.
In the case of a blood-based binding agent, the concentration of the binding protein is
relatively high so electrophoretic techniques could be used to quantitatively detect the
presence of the binding gel protein if this could be consistently solubilised. However
fibrin, the blood based binding protein, is cross-linked by transglutaminase in blood
shortly after it is formed, making it resistant to common solubilising agents such as
SDS and requiring peptide bond cleavage to solubilise a ‘hard’ blood clot.
Transglutaminase is also present in the commercial blood based gelling agent
Fibrimex (personal communication).
Fibrinogen, the precursor protein of the blood clotting protein fibrin, is composed of
three pairs of non-identical peptide chains, α, β, and γ. The insoluble protein fibrin is
formed by the protease thrombin, which cleaves sequentially Fibrinopeptide A from
the N-terminus of the α chain, then Fibrinopeptide B from the β chain of fibrinogen
[1]. The resulting fibrin monomers [(αβγ) 2] then polymerise, forming rod-like fibrils
which form cross links due to the activities of the calcium-activated transglutaminase
to form a ‘hard’ blood clot (Figure 1). The amino acid sequence of a number of
Page 5 of 41
fibrinopeptides is known to differ between a number of species making them ideal
candidates for markers to identify the species of clotted blood (NCBI Genbank).
The peptide sequences of bovine and porcine fibrinopeptides A and B are shown in
Table 1.
A RP-HPLC procedure has been reported for the differentiation of eight mammalian
fibrinogen peptides [2] and this provides the potential means to identify the
fibrinogen/thrombin added to meat products as a cold set binder.
HPLC separation of the peptides does not however provide a definitive test for the
peptides, especially if the peptides are recovered from a complex food sample.
Improved selectivity and characterisation of the peptides, in the form of MS/MS
detection after HPLC separation, was anticipated to be required to resolve the
fibrinogen peptides from a potential multitude of peptides from complex food
matrices.
1.3 MILESTONES OF THE PROJECT.
Project Milestones.
Number
Milestone title
Achieved
01/01
Optimisation of HPLC separation of peptides
Yes
02/01
Optimisation of MS/MS detection of peptides
yes
03/01
Evaluation of the HPLC-MS/MS combined method
using in-house food products
Yes
Page 6 of 41
2. EXPERIMENTAL.
2.1 MATERIALS.
2.1.1 Food matrix samples.
Meat samples were selected to represent the range of species and muscle types likely
to be used commercially with cold setting agents. Chicken breast, pork loin,
beefsteak, lamb and cod fillet were purchased unfrozen from the local supermarket.
Samples were stored at –20°C in the laboratory until use. All samples were used
within 2 months of purchase.
Commercial samples were also selected for investigation as sample matrices (sample
types reported in Tables 4 and 5). These were selected on the basis of the complexity
of their ingredients, to determine whether commercial food ingredients interfered
with the analysis.
2.1.2 Preparation of synthetic peptides.
10 mg of each of the four fibrinopeptides were synthesised by Severn Biotech
Limited at an 85% purity level from the known peptide sequences (NCBI Genbank).
Bovine Fibrinopeptide B was later re-synthesised with the N-terminal glutamine
converted to pyroglutamic acid.
.
2.1.3 Fibrimex®.
Bovine Fibrimex® was supplied by Harimex UK Limited as a fibrinogen suspension
and a thrombin solution which were stored at -20ºC. To form the gelling agent,
fibrinogen and thrombin were mixed in a 10:1 ratio and allowed to set for 24 hours at
4ºC according to the manufacturer’s instructions.
2.1.4 In-house preparation of porcine fibrinogen based binding agent.
Porcine fibrinogen based binding agent was prepared in house by mixing fibrinogen
extracted from blood with commercial bovine thrombin (Sigma-Aldrich T9549).
To extract fibrinogen from fresh blood, sodium citrate was added to a 3.8% (w/v)
concentration. Red blood cells were removed by centrifugation at 1500 x g for 20
minutes. 1000 mL of the remaining serum was centrifuged at 5000 x g for 15 minutes
at 4ºC. The plasma supernatant was precipitated for 1 hour at 4ºC with 100%
saturated ammonium sulphate, pH 7.4 (3 volumes supernatant +1 volume saturated
ammoniun sulphate) followed by centrifugation at 5000 x g for 15 minutes at 4ºC.
The protein pellet was washed with 25% saturated ammonium sulphate and was taken
up in 50 mM potassium phosphate, 10 mM EDTA, pH 6.6. The proteins were
precipitated by mixing with 100% saturated ammonium sulphate (3 volumes
resuspended pellet: 1 volume ammonium sulphate) for 1 hour at 4ºC. The protein was
recovered by centrifuging at 5000 x g for 15 minutes at 4ºC. The protein was dialysed
against 50 mM Tris-HCl, pH 7.4, 150 mM NaCl prior to ethanol precipitation with
100% ethanol. A further dialysis was then carried out against 50 mM Tris-HCl, pH
7.4, 150 mM NaCl and the fibrinogen solution was recovered by centrifugation at
20000 x g for 15 minutes at 4ºC.
Page 7 of 41
The final concentration of fibrinogen in the extract was estimated at 2.9 mg/mL by
spectrophotometry. Meats were spiked with this material by adding 10 and 5% (v/w)
aliquots of the extracted fibrinogen solution. Commercial thrombin (Sigma-Aldrich
T9549) was then prepared as a solution of 50 μg/mL in water, adding 5 μg per 2.9 mg
fibrinogen protein.
2.2 METHODS.
2.2.1 MALDI TOF-MS.
MALDI TOF-MS experiments were performed on an Applied Biosystems VoyagerDE STR Biospectrometry Workstation. Peptide mass spectra were obtained in
positive ion reflector mode at an accelerating voltage of 20 kV by accumulating 150
laser shots. Monoisotopic peaks were automatically labelled by the Data Explorer
software supplied by the manufacturer. Calibration was performed with bullfrog
angiotensin I, des-Arg-Bradykinin, somatostatin and ACTH (1-17) and verified
against the commercially synthesised fibrinopeptides. The matrix was prepared with
10 mg/mL alpha-cyano-4-hydroxycinnamic acid, in acetonitrile-water (50:50 v/v)
containing 0.05%(v/v) trifluoroacetic acid. MALDI-TOF-MS Post Source Decay
(PSD) was performed using the same instrument using angiotensin as the standard. A
PSD spectrum was produced from 7-8 spectral segments which were amalgamated in
Data Explorer.
Extracted samples (as described in 2.2.2 and 2.2.3) were resuspended in 20 µL of
60% acetonitrile. 0.5 µL of this sample, or of the calibration standards, were mixed
with 0.5 µL of matrix prior to loading on the MALDI target.
2.2.2 Extraction of peptides from meats and cooking juices.
2.0 g of meat or cooking juice matrix was homogenised with an Ultra Turrax
homogeniser (Janke & Kunkel) on ice with 6.0 mL of 6.2% (w/v) trichloroacetic acid
(chilled) and precipitated on ice for 1 hour. Following centrifugation at 4000 x g for
10 minutes at 4ºC the supernatant was taken and mixed 1:1 with diethyl ether
(chilled). The solvent (upper) phase was discarded following centrifugation at 4000 x
g for 10 minutes at 4ºC. This diethyl ether step was repeated on the remaining peptide
solution and the solvent fraction discarded. Butanol (chilled) was then added to the
peptide solution (1:1), mixed and centrifuged at 4000 x g for 10 minutes at 4ºC. The
solvent (upper) fraction was discarded and 2 mL of hexane (chilled) mixed with the
peptide solution. The solvent (upper) phase was discarded following centrifugation at
4000 x g for 10 minutes at 4ºC. The peptide solution was then freeze dried for 18
hours.
One quarter (mass) of the resulting freeze dried material was taken up in 1 mL of
phosphate buffer (9 volumes phosphate buffered saline (Sigma-Aldrich P-4417) to 1
volume 1 M potassium dihydrogen orthophosphate pH 7.2). The mixture was
vortexed and agitated at room temperature for 1 hour. Undissolved solids were
removed by centrifugation at 14000 rpm for 2 minutes at room temperature. The
sample was applied to an Oasis® 30 mg HLB cartridge (Waters Corporation)
previously equilibrated in 1 mL of methanol and 1 mL of phosphate buffered saline.
Peptides bound to the cartridge were washed with 1 mL of 10%(v/v) aqueous
methanol and then eluted from the cartridge in 1 mL of 40% (v/v) aqueous methanol,
Page 8 of 41
containing 2% (v/v) ammonium hydroxide. This peptide sample was then transferred
to a low binding micorcentrifuge tube (Axygen) and dried in a centrifugal evaporator.
2.2.3 Additional cleanup step.
Additonal cleanup of samples was investigated by applying the peptide solution
eluted from the HLB cartridge onto an Oasis® MAX (anion exchange) cartridge
previously washed in 100% methanol and equilibrated in deionised water. The
cartridge was washed with 1 mL of 40% (v/v) aqueous methanol containing 0.2%
(v/v) acetic acid pH 5.2 prior to peptide elution in 1 mL 40% (v/v) aqueous methanol
containing 0.2% (v/v) formic acid, pH 3.2. The eluant was dried in a centrifugal
evaporator in preparation for LC-MS/MS analysis. Analysis of the commercially
coated and uncoated samples and the tuna samples was carried out using this
additional cleanup step.
2.2.4 HPLC separation of peptides.
Peptides were separated on a Phenomenex Jupiter 10 µM C4 300 Å reversed phase
250 x 4.6 mm HPLC column with an increasing acetonitrile gradient containing 0.1%
(v/v) TFA and flow rate of 1 mL/min. (See 2.2.6 for details of gradient). Preliminary
studies were carried out with UV monitoring of the eluant at 210nm.
2.2.5 Glutamine derivatisation step.
Peptide solution was incubated in 0.1 M triethylamine for 18 hours at room
temperature. The reaction was halted by neutralising the solution.
2.2.6 LC-MS/MS conditions.
The instrumentation used was a Waters (MicroMass) Quattro Ultima triple
quadrupole mass spectrometer with Waters Alliance 2695 HPLC system. The mass
spectrometer used the electrospray source in the positive ionisation mode. The
capillary voltage was 3.0 kV, the source temperature 120C, the desolvation
temperature 400C with cone gas flow at 100 L/hr and desolvation gas flow at 700
L/hr. Cone voltages and collision energies were set for individual transitions. The
same column and gradient were used for LC-MS/MS separation as were used for the
HPLC method with the exception that 0.1% (v/v) TFA was replaced with formic acid
pH 2.2.
HPLC solvent gradient:
Time min
A%
B%
0.00
95
5
3.00
70
30
10.00
70
30
11.00
95
5
26.00
95
5
A = Aqueous ormic acid pH 2.2.
B = Acetonitrile
Page 9 of 41
Dried samples were taken up in 0.1 mL of mobile phase and injection volumes were
10 µL. The prescribed HPLC flow rate of 1 mL/min was too high for the ion source
of the mass spectrometer so this was split post-column at a ratio of 4:1 so that only
200 µL/min of eluent entered the ion source. A switching valve was used to divert
unretained material during the first three minutes of chromatography to avoid
contaminating the ion source.
2.2.7 Preparation of meat samples spiked with binding agent.
2.2.7.1 Addition of bovine Fibrimex®.
Meat was first homogenised in a household blender before addition of the appropriate
bovine fibrinogen suspension (for a 10% (v/w) addition, 1 mL of Harimex UK
Ltd.fibrinogen suspension was added per 10 g of meat). The mixture was then
homogenised prior to addition of the thrombin (Harimex) (100 µL of thrombin per 10
g of meat). The matrix was then further homogenised and allowed to cold set for 24
hours at 4ºC.
2.2.7.2 Addition of in-house porcine gelling agent.
1 g meat samples were minced with a scalpel blade then homogenised with a PTFE
rod in a polypropylene tube. Fibrinogen solution was added to the required
concentration (v/w) and the meat remixed with the PTFE rod. Finally commercial
thrombin (Sigma-Aldrich) was added (0.1 mL of a 5 μg/mL solution of thrombin per
1 mL of fibrinogen solution added) and the mince re-homogenised before storing at
4ºC for 24 hr.
2.2.8 Cooking procedure.
Samples of meat matrix spiked with 5% (v/w) bovine Fibrimex® were heated to 80ºC
for 15 min in sealed containers in a water-bath. After cooling the solid cooked meat
was removed from the container and the remaining cooking liquor centrifuged to
remove suspended solids. Both the cooked solid and the clarified cooking liquor were
collected and analysed for the bovine fibrinopeptides according to Method 2.2.3
Page 10 of 41
3 RESULTS AND DISCUSSION.
3.1 MILESTONE 01/01 – OPTIMISATION OF HPLC SEPARATION OF PEPTIDES.
It was anticipated that the HPLC separation of the peptides would be straightforward
and synthetic peptides would take some time to produce, so the original proposal was
to recover the fibrinopeptides from blood while the peptide standards were being
synthesised. HPLC separation was however not straight forward (see section 3.2) and
therefore synthetic peptides were used throughout the project as reference standards.
The sequence of the bovine and porcine fibrinopeptides (Table 1) was obtained from
NCBI Genbank. and 10 mg of each synthesised by Severn Biotech Limited as the free
peptides (i.e. terminal amino and acid group left unblocked). MALDI-TOF-MS
analysis at CSL confirmed the theoretical molecular masses and that the peptide
preparations were essentially free of significant other peptide contamination,
degradation products and synthetic precursors (Figures 2 and 3).
Preliminary experiments to develop an HPLC separation of the four peptides were
based on the published method [2] using far UV detection. A lower pH mobile phase
than that published was desirable to aid ionisation for the planned subsequent LCMS/MS detection. The studies homed in on a C4 reverse phase column with TFA as
ion pair reagent in an acetonitrile gradient (Method 2.2.4.)
The optimised HPLC procedure resolved the two porcine fibrinopeptides and the two
bovine fibrinopeptides but could not resolve all four together satisfactorily.
Furthermore the bovine Fibrinopeptide B appeared to produce two peptide peaks, in
spite of apparent purity, as observed by MALDI-TOF-MS (Figure 4).
MALDI-TOF-MS analysis of both forms of bovine Fibrinopeptide B, using post
source decay, revealed that the original form of bovine Fibrinopeptide B co-eluted
with porcine Fibrinopeptide B while the second form of the peptide produced a
discrete peak on the chromatogram which was a derivative of the peptide formed by
conversion of the N-terminal glutamine residue of the peptide under acidic conditions
to form pyroglutamic acid (Figure 5).
Consideration was given to carrying out a derivatisation step to force the conversion
of the bovine Fibrinopeptide B to the pyroglutamic acid form, (Method 2.2.5)
however in subsequent studies it was discovered that the reaction proceedd to
completion in the acidic extraction buffer used to recover the peptides from complex
food matrices and so a derivatisation step was not necessary.
In conclusion, the HPLC separation of the peptides was therefore achieved by
resolving the bovine Fibrinopeptide B as the pyroglutamic acid derivative.
Bovine Fibrinopeptide B was synthesised by Severn Biotech Limited as the
pyroglutamic acid derivative for use as a reference standard.
Having optimised the separation of the peptides, attention was then turned to
optimisation of the detection and characterisation of the peptide standards by MS/MS.
As a prelude to optimising MS/MS analysis of the synthetic peptides, attempts were
made to determine whether MALDI-TOF-MS could detect the fibrinopeptides in the
binding agents alone, without HPLC separation of the peptides. MALDI-TOF-MS is
a more direct approach to identifying the peptides since it provides information on the
unfragmented molecular mass of the peptide. This would prove a valuable tool in
Page 11 of 41
confirming the presence of the peptides in subsequent experimental studies on
complex matrices (Figure 6). Electrospray MS/MS, on the other hand, fragments the
peptides and provides information on the masses of the fragments from the peptides.
This provides much more information on the sequence of the peptides and therefore is
a more robust approach to confirming the identity of the peptides in unknown
samples. Electrospray MS/MS is also usually coupled to HPLC separation of the
peptides (LC-MS/MS) which further aids in the characterisation of the peptides.
The results of the MALDI-TOF-MS study showed that both the A and B peptide from
bovine Fibrimex® and in-house porcine binding agent, could be detected by MALDITOF-MS. There was significantly more Fibrinopeptide A than Fibrinopeptide B from
both sources. While MALDI-TOF-MS is not a quantitative method this difference
could be attributed to the differing ionisation potentials of the peptides on the MALDI
target under the conditions used (method 2.2.1), these results are also in keeping with
those of Blombäck et al [1] who showed that the Fibrinopeptide A was produced
before the Fibrinopeptide B during blood coagulation and therefore could be expected
to be at a higher concentration if the proteolysis reaction had not gone to completion.
3.2 MILESTONE 02/01 OPTIMISATION OF LC-MS/MS DETECTION OF
PEPTIDES.
Unlike MALDI-TOF-MS, electrospray MS/MS produces multiply charged ions for
any given analyte, usually the larger the analyte the more charge it carries. Since the
mass spectrometer defines the mass in terms of m/z, the apparent size of the analyte is
reduced by a factor proportional to the charge. The outcome of this is that each
peptide produces a series of charged species during MS/MS, each significantly lower
than the mass of the peptide as observed by MALDI-TOF-MS. In addition the various
salts of the peptide (e.g sodium and ammonium salts) add to the complexity of the
profile from the first MS dimension and require some interpretation to arrive at the
precise molecular mass of the peptide (Figure 7).
Individual peptide standards were infused at a rate of 10 µL/min into the electrospray
source of the mass spectrometer. Mass spectra of each peptide were acquired in the
positive mode. Electrospray mass spectra of peptides are known to be characterised
by multiply-charged ions which bring the observed m/z of the ions into the range of
the quadrupole mass analyser. Ions were sought which were representative of the
peptide’s 2+, 3+ or 4+ charge state with ions formed by protonation, ammoniation or
sodiation or a combination of these. Having established these precursor ions,
fragmentation was achieved by collisionally-activated dissociation with argon gas.
Fragment ions were chosen based on their selectivity, response and mass
spectrometer parameters, (principally cone voltage and collision energy). Selected
transitions are given in Figure 8.
One analysis of the initial mass spectra revealed that many of the peptides
preferentially formed sodiated ions rather than protonated ions. Sodiation is
undesirable because the level of sodium in the system is uncontrolled, so to counter
this, formic acid was added to promote protonation.
The LC part of the method used the same column, flow rate and initial gradient as the
conventional HPLC method. The mobile phase differed in that TFA replaced with
formic acid to achieve an optimal pH of 2.2 (TFA is not a desirable additive to use
with mass spectrometry due to potential ion suppression problems.) The
chromatography produced retention times of the four major peptides as follows :
Page 12 of 41
Bovine Fibrinopeptide A
6.86 min
Bovine Fibrinopeptide B (pGlu form)
6.79 min
Porcine Fibrinopeptide A
6.48 min
Porcine Fibrinopeptide B
5.83 min
Ion suppression was found to be a particular issue for the ionisation of peptides.
Before using the instrument, the ion source was carefully cleaned and the HPLC
system thoroughly flushed through with the mobile phase. This procedure was strictly
adhered to after initial ionisation problems and the method was subsequently very
robust.
Samples were made up in the aqueous mobile phase. After some initial concerns over
stability of samples in solution, the final dried-down samples were only reconstituted
immediately prior to analysis with the autosampler temperature set to 5C.
Having optimised the MS/MS analysis of the synthetic peptides a linearity of
response for each of the transitions was confirmed for concentrations of peptide
between 0.1 and 10 μg/mL. This was to assist in confirming recovery data for the
peptides from different food matrices.
Comparison of the relative responses for each of the fragment ions from the peptides
showed that the two most significant marker ions for bovine fibrinopeptides were
likely to be the transitions 783>171 and 783>296 both from the pyroglutamic acid
form of Fibrinopeptide B. The two most significant marker ions for porcine
fibrinogen binder were likely to be the transitions 588>201 and 551>157 of
Fibrinopeptides A and B respectively (Table 2).
A study on the development of a suitable peptide extraction procedure from complex
food samples was investigated in parallel to the LC-MS/MS studies. The optimised
extraction procedure (method 2.2.3) incorporated a TCA extraction step to precipitate
most of the high molecular mass proteins, followed by ether extraction to remove the
TCA and suspended lipid. A subsequent butanol washing step removed phospholipid
from solution, which interfered with the MALDI-TOF-MS analysis of the peptides.
Finally the peptides were purified by reverse phase separation using the Oasis® HLB
SPE cartridge.
Analysis of the peak areas of the transitions of the porcine peptides recovered from
145 μg porcine fibrinogen + thrombin incubated overnight at 4°C revealed that there
was approximately 5 times more Fibrinopeptide A than the Fibrinopeptide B (24.7 +/1.4 μg for the two transitions for Fibrinopeptide A and 5.2 +/- 1.2 μg for the four
transitions of Fibrinopeptide B). This result implied the most sensitive marker ions
would be the two transitions from Fibrinopeptide A for the porcine fibrinogen based
binder. This was not confirmed for the bovine fibrinogen but assumed to be similar
since the observation confirmed the reported sequential mechanism of thrombin first
cleaving off the Fibrinopeptide A then later in the reaction cleaving the fibrinogen to
release Fibrinopeptide B. The release of Fibrinopeptide A parallels the rate of
formation of fibrin, whereas the rate of release of Fibrinopeptide B becomes maximal
when formation of fibrin is near completion [1,3].
Page 13 of 41
3.3 MILESTONE 03/01 EVALUATION OF THE METHOD USING IN HOUSE
FOOD PRODUCTS.
3.3.1 Spiking studies.
Samples of beef, pork, lamb, chicken, and cod were spiked with both bovine
Fibrimex® and in-house porcine fibrinogen binding agent in order to determine the
specificity and limit of detection of the tests.
Analysis of the LC-MS/MS profiles of the unspiked tissues showed a number of
contaminating peaks in almost all of the transition profiles close to the retention times
for the fibrinopeptides. (Figures 9-12). However in most cases these peaks were just
above baseline noise (S/N <3). Two notable exceptions were; 1) the transitions for
bovine Fibrinopeptide B (783>171) where significant contaminating peaks were
present in lamb and chicken meat, and 2) the four transitions for porcine
Fibrinopeptide B (734>185; 734>136; 551>251, and 551>157) where significant
contaminating peaks were present again in lamb and chicken meat, particularly in
transition 551>157. These contaminating peaks would significantly reduce the
sensitivity of the method in these tissues.
3.3.1.1 Spiked beef studies.
All peptide transitions were detected from both the bovine and porcine binding agents
when spiked into minced beef at the 5% (v/w) level (Figures 13 & 18). The apparent
concentrations of porcine Fibrinopeptide A and B were within a factor of 2 of those
observed with the unspiked binding agent suggesting no significant loss of peptide in
the sample matrix. MS/MS profiles showed baseline resolved profiles for both
porcine Fibrinopeptide A transitions, and the single bovine Fibrinopeptide A. The
second clearest bovine transition (fibrinopeptide B 783>296) was visible above a
background of low peaks. This background became significant at the 2% (v/w)
spiking level, preventing clear identification of the Fibrinopeptide B transition (S/N
<3).
3.3.1.2. Spiked pork studies.
All peptide transitions were again detected from both bovine and porcine binding
agents spiked into pork muscle mince at the 5% (v/w) level. (Figures 13 & 18) LCMS/MS profiles showed baseline resolved profiles for the porcine Fibrinopeptide A
transition 588>201 and the Fibrinopeptide B transition 551>251. Confirmation of the
presence of the bovine binding agent was obtained from the baseline resolved bovine
Fibrinopeptide A transition 947>695 and bovine Fibrinopeptide B transition 783>171
which was clearly visible above a background of low peaks. Again, this background
became significant at the 2% (v/w) spiking level, preventing clear identification of the
Fibrinopeptide B transition (S/N <3).
3.3.1.3.Spiked lamb studies.
Only the one Fibrinopeptide A transition from bovine Fibrimex® was detected at the
5% (v/w) addition level to lamb meat (Figure 14). Similarly only the LC-MS/MS
transition 588>201 for porcine Fibrinopeptide A and 551>251 for Fibrinopeptide B
were detected when spiked with 5% (v/w) porcine binding agent (Figure 20). At the
10% (v/w) addition level the porcine Fibrinopeptide A transition 588>446 was also
detected.
Page 14 of 41
3.3.1.4. Spiked chicken studies.
The bovine Fibrinopeptide A transition was detected in the 5% (v/w) spiked chicken
meat samples and a porcine Fibrinopeptide A transition and one Fibrinopeptide B
transition were detected at the 5% (v/w) spiking level (Figures 16 & 21).
Examination of the transition peak areas suggested that significant loss of both bovine
and porcine peptide had occurred in the chicken spiked samples. To investigate this,
identical concentrations of the synthetic bovine and porcine peptides were added to
raw chicken and bovine muscle then incubated for 24 hr at 4°C prior to extraction.
The resulting LC-MS/MS analysis showed that the concentration of fibrinopeptides
had fallen, particularly for both the porcine and bovine Fibrinopeptide B. Spiking of
the TCA extracts of chicken muscle with the peptides confirmed that no loss of
peptide occurred after denaturation of the chicken muscle proteins suggesting
peptidase activity as the cause of the loss of the peptides .
3.3.1.5. Spiked cod studies.
None of the fibrinopeptide transitions were detected in the cod muscle spiked with
5% (v/w) of either the bovine or porcine binding agent. At the 10% (v/w) addition
level only bovine Fibrinopeptide A could be identified with transition 947>685 with
S/N>3 (Figures 17 & 22).
The absence of any detectable peaks for the porcine fibrinopeptides or bovine
Fibrinopeptide B suggested a more potent peptidase activity in cod muscle than in
chicken muscle.
In conclusion, the recovery of peptides from the different meats suggested that the
binding agents could be detected in beef, pork, lamb and chicken products, when
added at the 5% (v/w) level, albeit based on the detection of a single LC-MS/MS
transition. However, the addition of the binding agents to cod muscle could not be
confirmed because of significant loss of both bovine and porcine fibrinopeptides from
the muscle after gelling of the binding agent.
3.3.2 Effect of cooking.
Additional studies were carried out concurrently to investigate whether the
fibrinopeptides would be lost in drip liquor if products containing the binding agents
were cooked prior to analysis. Spiking the 5 meat samples with 5% (v/w) bovine
Fibrimex® then cooking after an overnight setting at 4ºC resulted in approximately
50% of the peptide being lost in the drip from the cooking (Table 3).
As a result of this it was not possible to confirm the presence of the Fibrinopeptide B
transitions in any of the samples although the Fibrinopeptide A was clearly detected
in all samples including the cod.
3.3.3 Further sample cleanup.
In order to resolve the problems over loss of sensitivity, particularly the bovine
Fibrinopeptide B transitions, an additional study was carried out on the use of anion
exchange SPE cartridges to further remove contaminating material (Method 2.2.4).
Extracts of lamb muscle spiked with 10% bovine Fibrimex® and also extracts spiked
with the synthetic porcine and bovine fibrinopeptides were extracted and purified
using the existing procedure and HLB SPE cartridge.
Page 15 of 41
LC-MS/MS profiles of the eluted peptides showed only a marginal reduction in
contaminating peaks and not specifically the transitions for bovine Fibrinopeptide B.
As a result the bovine Fibrinopeptide B transitions were still not clearly identified
after the further purification step of the samples containing 10% Fibrimex® (Figure
23).
3.3.4 Analysis of commercial samples spiked with Fibrimex®.
Eighteen commercial coated products (excluding cod products because of the likely
presence of a peptidase in fish) were spiked with 5% (v/w) bovine Fibrimex®,
allowed to bind then analysed for the presence of the bovine fibrinopeptides (Table
4). Fourteen of these samples also had the coating removed and samples similarly
analysed (Table 5). In all cases the bovine Fibrinopeptide A could be clearly detected
by it's single transition. Bovine Fibrinopeptide B could only be detected in five of the
uncoated samples and two of the coated samples (Table 4). This suggested bovine
Fibrinopeptide B pGlu transitions were not reliable markers for the presence of
bovine Fibrimex®. The porcine fibrinopeptides were also detected in one uncoated
sample suggesting either interstitial pork blood had been detected or porcine blood
clotting agents had been added to the product.
The detection of a single transition provided three points of confirmation of the
identity of a peptide,
1) the same HPLC retention time as the standard peptide,
2) the same parent ion mass as the standard peptide,
3) the same fragment ion mass as the standard peptide.
Conclusive proof usually relies on a 5-point confirmation of identity. Ideally a second
transition would need to be identified for the peptide, or a transition from the
complementary fibrinopeptide would need to be identified to provide irrefutable
proof of presence.
4. CONCLUSIONS.
An LC-MS/MS method has been developed which can detect the addition to meat of
5% (v/w) fibrin based meat binder extracted from bovine and porcine blood. The
bovine binding agent is characterised by a single LC-MS/MS transition of 947>695
from the bovine Fibrinopeptide A, while porcine blood is characterised by the
detection of 551>251 transition in lamb, pork and beef matrices and 588>201 in
chicken matrices. The method detected the major bovine Fibrinopeptide A transition
in meat samples after cooking, indicating the peptide is not lost in the drip liquor
during cooking and a range of commercial food ingredients were shown not to
interfere with the analysis.
However a single transition cannot provide irrefutable proof of the presence of the
specific blood-binding agent and a second transition of the same fibrinopeptide, or a
transition of the complementary fibrinopeptide would need to be detected. This could
possibly be achieved in commercial products since extended storage periods after
gelling are involved, during which time concentrations of Fibrinopeptide B can be
expected to increase.
Page 16 of 41
The method was unable to routinely detect the fibrinopeptide transitions in a cod
matrix and it is believed this was due to hydrolysis of the peptides by peptidases in
the muscle tissue. This was observed to some extent in chicken muscle.
The effects of different muscle tissues and storage of the samples prior to analysis
have not been studied as this was outside the remit of this project. Further work
should also be carried out to study the effect of the temperature and pH of the matrix
at the time of addition of binding agent. Analysis concerning these conditions would
be useful in order to check the robustness of the method.
Both bovine and porcine binding agents are produced commercially. It would seem
reasonable therefore that lamb, chicken and fish products containing these agents
would be the foods that could possibly cause offence. As a consequence the possible
presence of a peptidase activity in chicken and cod muscle deserves further
investigation. Work carried out later on tuna (Appendix 1) suggests peptidase activity
may be restricted to the white fish species.
The LC-MS/MS approach presented in this report could still be applicable for meats
containing peptidase activity since the fibrinopeptides may be cleaved by the
peptidases into shorter, but still distinct, peptides. These may require only a slightly
modified cleanup and MS/MS characterisation before they can be detected.
5. APPENDIX.
5.1 ADDITIONAL STUDIES ON TUNA STEAK CONTAINING BOVINE
FIBRIMEX®
5.1.1 The problem being addressed.
White fish such as cod are a relatively low value fish product and unlikely to involve
the use of Fibrimex® as a binding agent in the manufacture of products. The earlier
results showing a possible problem detecting Fibrimex® peptides in cod flesh may
therefore not be a serious problem in the application of the test to suspect commercial
samples. There is a need however to investigate higher value fish species such as tuna
to determine whether the loss of the fibrinopeptides in the flesh is a problem
associated with all fish species or only the white flesh fish species.
5.1.2 Materials and methods.
Samples of tuna steak were spiked with bovine Fibrimex® binding agent at the 1, 2, 5
and 10% (v/w) levels following the procedures outlined in the materials and methods
section above.
A tuna steak spiked to contain 5% (v/w) bovine Fibrimex® prior to cooking was also
prepared as above and all samples were extracted according to Method 2.2.3.
5.1.3 Results.
LC-MS/MS profiles of extracts from the spiked tuna samples showed baseline
resolved peaks for bovine Fibrinopeptide A (transition 947>695) at both 2% and 5%
(v/w) Fibrimex® addition (Figures 24 & 25). Neither the 783>296 nor the 783>171
transitions for bovine Fibrinopeptide B pGlu were detected in any of the spiked
samples.
Page 17 of 41
There was little difference in the intensity of the LC-MS/MS signal produced for the
raw tuna spiked with 5% (v/w) bovine Fibrimex® compared to that of the cooked
tuna containing 5% (v/w) bovine Fibrimex® (Figure 26). There was no cooking
liquor produced during the preparation of this lean fish confirming that any loss of
fibrinopeptides during the cooking of the other matrices was due to loss in the
cooking liquor.
5.1.4 Conclusions.
The results show that bovine Fibrimex® can be detected after addition to tuna and
bovine Fibrinopeptide A can be detected at the level of 1% Fibrimex® addition. The
problem of loss of the marker fibrinopeptides after addition to foods may therefore be
restricted to only white flesh fish.
6. BIBLIOGRAPHY
[1] Blombäck, B., Hessel, B., Hogg, D., and Therkildsen, L. (1978) A two-step
fibrinogen-fibrin transition in blood coagulation. Nature 275:501-505.
[2] Sellers, J.P. and Clark, H.G. (1981) High Pressure Liquid Chromatography of
fibrinopeptides derived from eight mammalian fibrinogens. Thrombosis research 23:9195.
[3] Blombäck, B. and Vestermark A. (1958) Ark. Kemi 12:173.
7. GLOSSARY
MALDI-TOF
Matrix Assisted Laser Desorption Ionisation Time of Flight
m/z
Mass to charge ratio
pglu
Pyroglutamic acid form of the bovine fibrinopeptide B
PSD
Post Source Decay (MALDI-TOF-MS)
S/N
Signal to noise ratio
SPE
Solid phase extraction
TFA
Trifluoroacetic acid
TCA
Trichloroacetic acid.
Page 18 of 41
8 TABLES.
Table 1: Peptide sequence of fibrinogen peptide A and B from bovine and porcine
blood (source: NCBI Genbank).
Peptide and species
Peptide sequence
Number of
amino acids
FibrinopeptideA_cow EDGSDPPSGDFLTEGGGVR
19
FibrinopeptideA_pig
AEVQDKGEFLAEGGGVR
17
FibrinopeptideB_pig
AIDYDEDEDGRPKVHVDAR
19
FibrinopeptideB_cow QFPTDYDEGQDDRPKVGLGAR
21
Table 2: Relative intensities of fragment ions from the four synthetic
fibrinopeptides
Fragment ion
Bovine
Fibrinopeptide
Relative Ion
Intensity
Porcine
Fibrinopeptide
Relative Ion
Intensity
A
Transition
947>695
1.0
B (pglu)
Transition
783>296
3.8
B (pglu)
Transition
180>171
5.3
A
Transition
588>201
1.0
A
Transition
588>446
0.3
B
Transition
734>185
0.06
Page 19 of 41
B
Transition
734>136
0.1
B
Transition
551>251
0.3
B
Transition
551>157
0.6
Table 3: Effect of cooking meat on the detection of 5% (v/w) added bovine
Fibrimex®.
Meat
Distribution of Fibrinopeptide A
after cooking (%)
Solid cooked
Relative concentration of total
Fibrinopeptide A detected in each
spiked meat sample
Cooking liquor
meat
Beef
60
40
100
Pork
56
44
120
Lamb
36
64
20
Chicken
85
15
10
Cod
50
50
5
Page 20 of 41
Table 4: Detection of Fibrimex® in spiked coated commercial products.
Product
Fibrinopeptide
Fibrinopeptide B
A detected
detected
Supermarket 1 Chicken breasts topped with cheese, leek
and ham
Y
Supermarket 2 Chicken breasts in red wine sauce
Y
Supermarket 1 Creamy garlic kievs
Y
Brand 1 BBQ mini chicken griddlers
Y
Brand 2 Southern Fried Crispy chicken
Y
Supermarket 1 Spicy Thai style chicken
Y
Supermarket 1 4 chicken burgers
Y
Supermarket 1 Pork and herb sausages with vegetables
and onion gravy
Supermarket 1 Gammon steaks in a creamy cheddar
cheese sauce
Y
Y
Y
Supermarket 1 ExtraSpecial Moussaka
Y
Supermarket 2 Lamb chops with mint glaze
Y
Supermarket 1 Beef stroganoff
Y
Supermarket 1 Pork with sauce and vegetables
Y
Supermarket 1 Peppered beef steaks and garlic butter
Y
Supermarket 1 Minced beef and onion crisp bakes
Y
Supermarket 2 Beef curry with rice (rice not analysed)
Y
Supermarket 1 Chinese takeaway beef in black bean
Y
Supermarket 1 Indian takeaway lamb rogan josh
Y
Supermarket 2 Cumberland sausage and mustard mash
(mash not analysed)
Y
Page 21 of 41
Y
Y
Table 5: Detection of Fibrimex® in spiked uncoated commercial products.
(coating removed)
Product
fibrinopeptide A
fibrinopeptide B
detected
detected
Supermarket 1 Chicken breasts topped with cheese, leek
and ham
Y
Supermarket 2 Chicken breasts in red wine sauce
Y
Brand 1 8 BBQ mini chicken griddlers
Y
Brand 2 Southern Fried Crispy chicken
Y
Brand 1 4 chicken burgers
Y
Supermarket 1 Easy Carve pork shoulder joint w/ a sage +
onion stuffing, coated with parsley
Y
Supermarket 1 Gammon steaks in a creamy cheddar cheese
sauce
Y
Supermarket 1 ExtraSpecial Moussaka
Y
Supermarket 2 Lamb chops with mint glaze
Y
Supermarket 1 Minced beef and onion crisp bakes
Y
Supermarket 2 Beef curry with rice (rice not analysed)
Y
Supermarket 1 Chinese takeaway beef in black bean
Y
Y
Supermarket 1 Indian takeaway lamb rogan josh
Y
Y
Supermarket 2 Cumberland sausage and mustard mash
(mash not analysed)
Y
Page 22 of 41
Y
Y
9. FIGURES.
Figure 1 Diagramatic presentation of the production of fibrinopeptides from
fibrinogen
Thrombin
Fibrin
monomers
(--)2
Fibrinogen
(A-B-)2
Fibrinopeptides A
Fibrin polymer
+B
Transglutaminase
‘Hard’ Clot
Figure 2. MALDI-TOF profile of synthetic bovine Fibrinopeptides A and B
Fibrinopeptide A
Fibrinopeptide B
1892.3
2365.6
Theoretical mass 1891.9Da
Theoretical mass 2364.1Da
Fibrinopeptide B after HPLC separation
Voyager Spec #1[BP = 2351.4, 8818]
2351.3669
100
90
8818.3
2351.4
80
% Int e ns it y
70
Theoretical mass of pyroglutamic
acid derivative of bovine
fibrinopeptide B 2347.1Da
60
50
40
30
2333.7853
20
10
954.4013
0
499.0
Page 23 of 41
1894.2184
1399.4
2310.4871
2317.1896
2299.8
3200.2
Mass (m/z)
4100.6
0
5001.0
Figure 3. MALDI –TOF profile of synthetic porcine Fibrinopeptides A and B
Fibrinopeptide A
Fibrinopeptide B
1762.5
2201.7
Theoretical mass 1762.9Da
Theoretical mass 2201.3Da
Figure 4 HPLC separation of the synthetic bovine and porcine Fibrinopeptides A
and B
Figure 5. Conversion mechanism of terminal glutamine residues in peptides
H2N
C CO NH~
H2NOC
C
2H+
H2O
C
NH
C CO NH ~
C
C
C
Glutamine
pyroglutamic acid
Page 24 of 41
+NH3
Figure 6. MALDI-TOF-MS profile of fibrinopeptides extracted from blood
products.
A) Bovine Fibrimex
1892
Fibrinopeptide B
Fibrinopeptide A
2351
B) Porcine plasma
1761.4
Fibrinopeptide A
Fibrinopeptide B
2199.6
Page 25 of 41
Figure 7. ES-MS of bovine Fibrinopeptide A.
+Q1: 0.114 to 0.739 min from Sample 2 (Job6979_002) of DataPR1.wiff (Turbo Spray), Smoo...
652.9
1.03e4
1.00e4
Max. 1.0e4 cps.
660.3
9500.00
667.5
9000.00
8500.00
8000.00
7500.00
645.7
In te n s ity , c p s
7000.00
6500.00
6000.00
665.7
5500.00
716.3
5000.00
4500.00
674.8
658.6
673.2
4000.00
705.4
3500.00
3000.00
638.1
650.9
682.0
2500.00
687.9
2000.00
643.6
1500.00
651.8
500.00
610
628.9
612.9
624.0
620
627.6
630
656.9
643.0
682.8
694.4
727.4
679.1
668.8
639.1
1000.00 608.6
671.2
669.8
676.6
686.9
689.4
693.1
699.7 710.6
649.7
640
650
706.4
660
670
680
m/z, amu
690
700
710
717.9 724.4
720
736.1
730
740
The molecular weight of the peptide as determined by calibrated MALDI-TOF MS, was 1891.2.
Using this molecular weight, some of the ions were interpreted as follows:
638 = [M+Na+H2]3+
646 = [M+Na2+H]3+
653 = [M+Na3]3+ and/or unresolved [(M-H+Na)+Na2+H]3+
660 = [(M-H+Na)+Na3]3+ and/or unresolved [(M-H2+Na2)+Na2+H]3+
Page 26 of 41
Figure 8. LC- MS/MS transitions for the four Fibrinopeptides.
783>296
Prepared JAH 10/11/05 - PR1 added, divert adjusted, inlet method corrected20.0µg/ml
Job8304_033
MRM of 14 Channels ES+
783 > 296
4.28e5
6.79
100
Pglu
%
Bovine Fibpeptide B(pGlu)
0
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
MRM of 14 Channels ES+
783 > 171
6.88e5
Job8304_033
6.82
100
783>171 Bovine Fibpeptide B(pGlu)
%
Pglu
0
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
MRM of 14 Channels ES+
588 > 446
8.68e4
Job8304_033
6.43
100
588>446 Porcine Fibpeptide A
%
PR2
0
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
MRM of 14 Channels ES+
588 > 201
4.14e5
Job8304_033
6.43
100
PR2
%
588>201
Porcine Fibpeptide A
0
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
MRM of 14 Channels ES+
947 > 695
4.00e4
Job8304_033
6.86
100
PR1
%
947>695
0Prepared JAH 10/11/05 - PR1 added, divert adjusted, inlet method corrected20.0µg/ml
1.00
2.00
3.00
4.00
5.00
6.00
Job8304_033
Time
7.00
8.00
9.00
10.00
5.83
100
Bovine Fibpeptide A
11.00
MRM of 14 Channels ES+
551 > 251
5.07e5
551>251
PR3
%
Porcine Fibpeptide B
0
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
Job8304_033
11.00
MRM of 14 Channels ES+
551 > 157
1.33e6
551>157
5.83
100
PR3
%
Porcine Fibpeptide B
0
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
Job8304_033
11.00
MRM of 14 Channels ES+
734 > 185
5.79e4
734>185
5.79
100
%
PR3
Porcine Fibpeptide B
0
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
Job8304_033
5.83
100
11.00
MRM of 14 Channels ES+
734 > 136
9.57e4
734>136
%
PR3
Porcine Fibpeptide B
0
Time
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
Page 27 of 41
11.00
Figure 9. LC-MS/MS profiles of unspiked beef muscle.
Bovine Fibrinopeptide specific transitions.
Transition A947>695
Transition B783>296
Transition B783>171
Retention time of transition
Porcine Fibrinopeptide specific transitions.
Transition A588>201
Transition B551>251
Transition A588>446
Transition B551>A157
Transition B734>185
Transition B734>136
Page 28 of 41
Figure 10. LC-MS/MS profiles of unspiked lamb muscle.
Bovine Fibrinopeptide specific transitions.
Transition A947>695
Transition B783>296
Transition B783>171
Porcine Fibrinopeptide specific transitions.
Transition A588>201
Transition B551>251
Transition A588>446
Transition B551>A157
Transition B734>185
Transition B734>136
Page 29 of 41
Figure 11. LC-MS/MS profiles of unspiked chicken muscle.
Bovine Fibrinopeptide specific transitions.
Transition A947>695
Transition B783>296
Transition B783>171
Porcine Fibrinopeptide specific transitions.
Transition A588>201
Transition A588>446
Transition B551>251
Transition B551>A157
Transition B734>185
Transition B734>136
Page 30 of 41
Figure 12 LC- MS/MS profiles of unspiked cod muscle.
Bovine Fibrinopeptide specific transitions.
Transition A947>695
Transition B783>296
Transition B783>171
Porcine fibrinopeptide specific transitions.
Transition A588>201
Transition B551>251
Transition A588>446
Transition B551>A157
Transition B734>185
Transition B734>136
Page 31 of 41
Figure 13. Detection of bovine Fibrinopeptides in beef containing 5% (v/w)
Fibrimex®.
Transition A947>685
Transition B783>296
Transition B783>171
Figure 14. Detection of bovine Fibrinopeptides in Pork containing 5% (v/w)
Fibrimex®.
Transition A947>685
Transition B783>296
Transition B783>171
Page 32 of 41
Figure 15. Detection of bovine Fibrinopeptides in Lamb containing 10% (v/w)
Fibrimex®.
Transition A947>685
Transition B783>296
Transition B783>171
Figure 16. Detection of bovine Fibrinopeptides in chicken containing 5% (v/w)
Fibrimex®.
Transition A947>685
Transition B783>296
Transition B783>171
Page 33 of 41
Figure 17. Detection of bovine Fibrinopeptides in cod containing 10% (v/w)
Fibrimex®.
Transition A947>685
Transition B783>296
Transition B783>171
Page 34 of 41
Figure 18 Detection of porcine Fibrinopeptides in beef containing 5% (v/w)
porcine fibrinogen binding agent.
Transition B551>251
Transition A588>201
Transition B551>A157
Transition A588>446
Transition B734>185
Transition B734>136
Page 35 of 41
Figure 19. Detection of porcine Fibrinopeptides in pork containing 5% (v/w)
porcine fibrinogen binding agent.
Transition B551>251
Transition B551>157
Transition A588>201
Transition B734>185
Transition A588>446
Transition B734>136
Page 36 of 41
Figure 20. Detection of porcine Fibrinopeptides in lamb containing 5% (v/w)
porcine fibrinogen binding agent.
Transition A588>201
Transition B551>251
Transition A588>446
Transition B551>157
Transition B734>185
Transition B734>136
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Figure 21. Detection of porcine Fibrinopeptides in chicken containing 5% (v/w)
porcine fibrinogen binding agent
Transition A588>201
Transition B551>251
Transition B551>157
Transition A588>201
Transition B734>185
Transition B734>136
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Figure 22. Detection of porcine Fibrinopeptides in cod containing 10% (v/w)
porcine fibrinogen binding agent.
Transition B551>251
Transition A588 >201
Transition B551>157
Transition A588> 201
Transition B734>185
Transition B734>136
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Figure 23. Treatment of Fibrimex® spiked pork sample with anion exchange SPE.
Sample after treatment.
Transition A947>685
Transition B783>296
Transition B783>171
Sample before treatment.
Transition A947>685
Transition B783>296
Transition B780>171
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Figure 24. Detection of bovine Fibrinopeptide A in raw tuna spiked with 2%
(v/w) bovine Fibrimex®.
Figure 25. Detection of Fibrinopeptide A profile for raw tuna spiked with 5%
(v/w) bovine Fibrimex®.
Figure 26. Detection of bovine Fibrinopeptide A in cooked tuna spiked with 5%
(v/w) bovine Fibrimex®.
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