peptide biomarkers as a way to determine meat authenticity ma - icomst ghent 2011.pdf · peptide...
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Peptide biomarkers as a way to determine meat authenticity
Miguel A. Sentandreu
Laboratory of Meat ScienceIATA (CSIC), SPAIN
Clear and reliable information about food is demanded nowadays by consumers
The problem of meat authentication
Honest and accurate food labeling is essential to assure food safety and choice
Robust and reliable methodologies are needed to assure that fraudulent or accidental mislabeling does not arise
Quantitative Ingredient Declaration (QUID)
Lifestyle affects individual’s choice on food consumption
Ballin, N.Z. (2010). Authentication of meat and meat products. Meat Science 86, 577
Meat origin
-Geographical origin-Feeding/production
systems- Breed- Sex- Meat cuts- Slaughter age
Processing
- Irradiation- Fresh/thawed- Preparation
Non-meat additions
- Additives- Water
Meat substitution
Meat
Fat
Protein
-Species-Tissue
- Vegetable- Animal
- Vegetable- Animal- Organic
Meat authenticity problem sources
Common strategies traditionally used to assess meat authenticity
1) Analysis of stable isotope ratios:
2H / 1H
13C/12C
18O/16O
15N/14N
Incorporation into animal tissues
Geographical origin
Feed intake
Common strategies traditionally used to asses meat authenticity
Fraudulent addition of water to meat Increase of size and weight
- Mass measurement before and after drying of meat
2) Determination of the water / protein ratio:
Easy to do
3) Methods based on magnetic resonance: NMR, MRI
- Allow the study of water distribution into meat
- Non-destructive
Addition of waterAddition of substances to increase
water holding properties
It can be masked by addition of exogenous proteins
4) Metabolomics
Common strategies traditionally used to asses meat authenticity
LC-MSLarge set of data analysisGC-FIDGC-MS
Identification and quantification of as many Low MolecularWeight Compounds as possible
- Identification of the presence of pasture in animal diets Sivadier et al. 2010
- Detection of Mechanically Recovered Meat Surowiec et al. 2011
- Authentication of typical Slavonian Salami Jerkovic et al. 2010
Examples:
“Non-targeted approach”
Differentiation of Mechanically Recovered pork Meat (MRM) by CG-MSmetabolite profiling
METABOLOMICS
Surowiec, Fraser, Pater, Halket & Bramley (2011). Food Chemistry, 125, 1468
MRM
Hand-deboned meatDesinewed meat
Clear discrimination of samples is not possible in all cases
Search for specific biomarkers
Common strategies traditionally used to asses meat authenticity
- High throughput
- Easy to use- High sensitivity
• Need for specific antibodies
• Cross-reactions• Processing of foods can affect
the immunoassay
False positives
A) Protein detection methods (ELISA):
Search for specific biomarkers
“Targeted approach”
Identification of meat species in foods
Giovannacci, I. et al. (2004). Int. J. Food Sci. Technol. 39, 863
B) Methods based on DNA analysis (PCR):
- High discrimination power (species-specific)
• Difficulties on DNA extraction
• DNA degradation: pH, heat, hydrolytic enzymes…
- High sensitivity
Limitations on processed foods:
Low reliability
Need to develop alternative analytical approaches for species identification
Identification of biomarker peptides
MASS SPECTROMETRY
Proteomics
Identification of Meat Species in Foods
Proteomic technologies in Meat Science
The study of a genome expression products, with the objective to obtain an integrated and global vision of the cell processes
Genome
Proteome
Proteomics
Evolution of Proteomics
70’s: Cell proteins
2D-PAGE
Identification
ImmunodetectionN-terminal sequencing (Edman)
- Not suitable for great scale protein analysis
- Low sensitivity- Slow and complicated
Remarkable limitations
Evolution of Proteomics
90’s:
Development of “soft” ionization techniques coupled to mass spectrometry
ESI MALDI+
+
Massive genome sequencing + development of protein databases web-accessible
Development of high-throughput bioinformatic tools
MODERN PROTEOMICS“Great-scale Proteomics”
Postgenomic Revolution
Workflows in current Proteomics
1D, 2D-PAGE MS:+
Sample2D-PAGE
Digestion(“In-gel”)
216
1399.499 1723.567
1200.4761077.382
1530.482 1927.700
B
0
2
4
6
4x10
1000 1200 1400 1600 1800 2000m/z
MALDI-TOF MS Identity
Peptide fingerprint
MS/MS fragmentation
Peptide sequence
Identity
Ambiguity
A)
Workflows in current Proteomics
B) Gel-free Proteomics: Sample
Digestion (“In-solution”)
Fractionation
(LC)-LC-ESI-MS/MS
Protein 2
Protein 1Protein 3
Protein 4
Lametsch R. et al. (2003). J. Agric. Food Chem. 51, 6992
Immediately after slaughter 72 h postmortem
Changes in the pig muscle proteome during postmortem meat storage
Tenderness Actin & Myosin degradation
In-gel digestion + MALDI-TOF MS Protein identification
Proteomic contributions to Meat Science: Postmortem proteolysis
Mora, L. et al. (2010). Food Chem. 123, 691
Dry-cured ham
Proteomics: Characterisation of proteolysis products in dry‐cured ham
Extraction
Identification of peptide sequences
Deproteinisation
Size-exclusion chromatography
Reverse phase HPLC
LC-ESI-MS/MS MALDI-TOF/TOF
TAPKIPEGEKVDFDDIQKKRQNKDLAPKIPEGEKVDFDDIQKKRQNKDLKIPEGEKVDFDDIQKKRQNKDLIPEGEKVDFDDIQKKRQNKDL
TAPKIPEGEKVDFDDIQKKRQNKDAPKIPEGEKVDFDDIQKKRQNKDTAPKIPEGEKVDFDDIQKKRQNKAPKIPEGEKVDFDDIQKKRQNKKIPEGEKVDFDDIQKKRQNKIPEGEKVDFDDIQKKRQNK
TAPKIPEGEKVDFDDIQKKRQAPKIPEGEKVDFDDIQKKRQKIPEGEKVDFDDIQKKRQ
Identified sequence57-8158-8160-8161-8157-8058-8057-7958-7960-7961-7957-7758-7760-77
123456789
10111213
PositionPeptide
Fragments generated from Troponin T degradation
MS/MS
Peptide biomarkers as a reliable and accurate way to reveal meat composition
Development of a methodology capable to overcome the existing limitations on the identification of animal species that can be found in meat products
OBJECTIVE
Avoid fraudulent or accidental mislabelling of meat constituents
Proteomics
Identification of species-specific peptide biomarkers
Extraction of muscle proteins
SDS-PAGE
In-gel digestion with proteolytic enzymes
Identification of species-specific peptide biomarkers
Selection of target proteins
MALDI-TOF MS ESI-MS/MS
Workflow:
1 2 3 4 5 6 7 8 9
1 % chicken in pork
+
Detection of chicken in a mix with pork meat
Selection of target protein:
1 2 3 4 5 6 7 8 9 Std 1 2 3 4 5 6 7 8 9
MLC-3MLC-2
MLC-1
100 % pork 100 % chicken
MLC-1MLC-2
MLC-3
Trypsin digestion
Isoelectric focusing + SDS PAGE of myofibrillar proteins:
It is possible to detect 1% chicken in pork meat?
In-gel tryptic digestion of myosin light chain 3
Reflex III
1 % chicken in pork
100 % chicken
100 % pork 2163.005
1514.731
2289.081
1284.6861723.828
1399.773 2562.139 2760.2231993.951
2383.974
09-11-07offgel9\0_G2\1\1SRef
0
1
2
3
4
4x10
Inte
ns. [
a.u.
]
2331.795
2162.7841200.681
1512.5642803.937
1340.513 1723.626
1845.588 2051.700
09-11-07offgel10\0_H1\1\1SRef
0
2
4
64x10
Inte
ns. [
a.u.
]
2162.762
1399.499 1723.567
1200.4762288.875
1077.3821530.482 2483.9841927.700 2804.020
Band 1 bis (1% chicken)\0_C1\1\1SRef
0
2
4
6
4x10
Inte
ns. [
a.u.
]
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800m/z
+
MALDI-TOF
Zoom-in for m/z range 1490-1550
DQGTFEDFVEGLRDQGSYEDFVEGLR
Sequence
Gallus gallus81-931512,6965Sus scrofa81-931514,683
OriginPositionM+H+
1514.731
1493.772
09-11-07offgel9\0_G2\1\1SRef
0 0
0.5
1.0
1.5
2.0
4x10
Inte
ns. [
a.u.
]
1512.564
1538.577
09-11-07offgel10\0_H1\1\1SRef
0 0
0.5
1.0
1.5
2.0
2.54x10
Inte
ns. [
a.u.
]
1514.683
1530.755
1494.749
Band 2 bis (1% chicken)\0_C2\1\1SRef
0
1
2
3
4
4x10
Inte
ns. [
a.u.
]
1490 1500 1510 1520 1530 1540 1550m/z
100 % pork
100 % chicken
1 % chicken in pork
1512.6965
Zoom-in for m/z range 1380-1450
1434.772
09-11-07offgel9\0_G2\1\1SRef
0
1000
2000
3000
4000
Inte
ns. [
a.u.
]
1399.613
1411.6091391.608 1446.579
09-11-07offgel10\0_H1\1\1SRef
0
1000
2000
3000
4000
5000
6000
Inte
ns. [
a.u.
]
1399.499
1411.4851433.5001381.477
Band 1 bis (1% chicken)\0_C1\1\1SRef
0
1
2
3
4
4x10
Inte
ns. [
a.u.
]
1380 1390 1400 1410 1420 1430 1440 1450m/z
100 % pork
100 % chicken
1 % chicken in pork
ALGQNPTNAEINKSequence
AcetylationModification
37-49Position
Gallus gallus1411.6091369.70Origin
Modified massM+H+
288.20L1338.58
345.22G1225.50
175.12R
474.27E1168.48
573.34V1039.44
720.40F940.37
835.43D793.30
964.47E678.27
1111.54F549.23
1212.59T402.16
1269.61G301.11
1397.67Q244.09D116.03
y ionsb ions
Peptide fragmentation (MS/MS):
MS/MS of DQGTFEDFVEGLR (peptide 1)
757.01 (2+)1512,6965 (1+)Ion trapMALDI-TOF Sequence
261.16N1223.60
374.24I1109.56
147.11K
503.28E996.47
574.32A867.43
688.36N796.39
789.41T682.35
886.46P581.30
1000.51N484.25
1128.56Q370.21
1185.59G242.15
1298.67L185.13
A72.04
y ionsb ions
MS/MS for ALGQNPTNAEINK (peptide 2)
685.68 (2+)1411.609 (1+), AcetylIon trapMALDI-TOF
Sequence
Proteomics
Suitable for quantitation ?
Identification of species-specific peptides
Capable to detect the presence of 1% chicken in pork meat
Quantitative proteomic approach
AQUA: “Absolute QUAntitation”
• Use of stable isotope peptides made from previously selected sequences
ALGQNPTNAEINK (Mr 1369.7)
AL*GQNPTNAEINK (Mr 1376.7)
L*(13C6, 15N) = +7 Da F*(13C9, 15N) = +10 Da
DQGTFEDFVEGLR (Mr 1512.7)
DQGTFEDF*VEGL*R (Mr 1522.7)
Quantification of biomarker peptides
Gerber, SA, Rush, J, Stemmann, O, Kirschner, MW, Gygi, SP (2003). PNAS. 100, 6940
Quantification of animal species
Quantitative proteomics
Meat mixes (0, 0.5, 1, 2, 5 and 10 % chicken in pork)
Protein extraction
Isoelectric focusing24
+ -2322212019181716151413121110987654321
MLC-3
Stable isotope labelled peptides (13C, 15N)
In-solution trypsin digestion
LC-ESI-MS/MS Quantification of peptide biomarkers
MLC-30,5 % chicken 95% pork Trypsin digestion
Stable isotope labelled peptides
LCQ Deca
ALGQNPTNAEINK (685.532+)
LC-MSClipeus C18 (150x0.5 mm)
y = 2,7523xR2 = 0,9921
0
5
10
15
20
25
30
0 1 2 3 4 5 6 7 8 9 10% chicken in pork meat
Pico
mol
ALG
QN
PTN
AEI
NK Suitable for
quantitation
Chicken Turkey Chicken Turkey Chicken Turkey
MSFSPDEINDFKEAFLLFDRTGDAKITLSQVGDIVRALGQNPTNAEINKI50 MSFSPDEINDFKEAFLLFDKTGDAKITLSQVGDIVRALGQNPTNAEMNKI LGNPSKEEMNAKKITFEEFLPMLQAAANNKDQGTFEDFVEGLRVFDKEGN100 LGNPSKEEMNAKKITFEEFLPMLQAAANNKDQGTYEDFVEGLRVFDKEGN GTVMGAELRHVLATLGEKMTEEEVEELMKGQEDSNGCINYEAFVKHIMSV150 GTVMGAELRHVLATLGEKMTEEEVEELMKGQEDSNGCINYEAFVKHIMSV
Chicken Turkey Chicken Turkey Chicken Turkey
MSFSPDEINDFKEAFLLFDRTGDAKITLSQVGDIVRALGQNPTNAEINKI50 MSFSPDEINDFKEAFLLFDKTGDAKITLSQVGDIVRALGQNPTNAEMNKI LGNPSKEEMNAKKITFEEFLPMLQAAANNKDQGTFEDFVEGLRVFDKEGN100 LGNPSKEEMNAKKITFEEFLPMLQAAANNKDQGTYEDFVEGLRVFDKEGN GTVMGAELRHVLATLGEKMTEEEVEELMKGQEDSNGCINYEAFVKHIMSV150 GTVMGAELRHVLATLGEKMTEEEVEELMKGQEDSNGCINYEAFVKHIMSV
High sequence homology between chicken and turkey muscle proteins
Discrimination between closely related species
Capable to differentiate between chicken and turkey meat?
Proteomic approach
?
Myosin Light Chain 3
Trypsin digestion + MALDI-TOF
Discrimination between closely related species
1010.561
996.615
09-11-07offgel10\0_H1\1\1SRef
0 0
0.5
1.0
1.5
2.0
2.5
4x10
Inte
ns. [
a.u.
]
982.332
1082.376
D1 (Fr 9)\0_D1\1\1SRef
0
200
400
600
800
Inte
ns. [
a.u.
]
940 960 980 1000 1020 1040 1060 1080 1100m/z
Chicken myosin light chain 3
Turkey myosin light chain 3
EAFLLFDKEAFLLFDRSequence
Meleagris gallopavo13-20982.332Gallus gallus13-201010.561
OriginPositionM+H+
OFFGEL separation of chicken and turkey proteins
MLC-3
Yes, we can !
CONCLUDING REMARKS
- The identification of species-specific peptide biomarkers using a proteomic approach constitutes and interesting and promising alternative to existing methodologies currently in use to assess meat authenticity
- High discriminating power
• More robustness with respect to actual major limitations of DNA analysis:
Differentiation of closely related species
- Standardized extraction procedures
Possibility to develop reliable quantitative determinations
Can be applied for both fresh and cooked meats- Food processing
- Possibility to use routine, user-friendly, mass spectrometry equipment
GOOD, GOOD…CHAPS!
Dr. Quique Sentandreu
Prof. Peter Bramley
Dr. KaisaKoistinen
Dr. Paul Fraser
Mr. Chris Gerrish
Thank you
Valencia, Spain
