Peptide biomarkers as a way to determine meat authenticity

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1 Peptide biomarkers as a way to determine meat authenticity Miguel A. Sentandreu Laboratory of Meat Science IATA (CSIC), SPAIN

2 The problem of meat authentication Clear and reliable information about food is demanded nowadays by consumers Lifestyle affects individual s choice on food consumption Honest and accurate food labeling is essential to assure food safety and choice Quantitative Ingredient Declaration (QUID) Robust and reliable methodologies are needed to assure that fraudulent or accidental mislabeling does not arise

3 Meat authenticity problem sources Meat origin -Geographical origin -Feeding/production systems - Breed -Sex -Meat cuts -Slaughter age Meat substitution Meat Fat Protein -Species -Tissue - Vegetable -Animal - Vegetable -Animal -Organic Processing -Irradiation - Fresh/thawed -Preparation Non-meat additions -Additives -Water Ballin, N.Z. (2010). Authentication of meat and meat products. Meat Science 86, 577

4 Common strategies traditionally used to assess meat authenticity 1) Analysis of stable isotope ratios: Geographical origin Feed intake 2 H / 1 H 13 C/ 12 C 18 O/ 16 O 15 N/ 14 N Incorporation into animal tissues

5 Common strategies traditionally used to asses meat authenticity Fraudulent addition of water to meat Increase of size and weight 2) Determination of the water / protein ratio: - Mass measurement before and after drying of meat Easy to do It can be masked by addition of exogenous proteins 3) Methods based on magnetic resonance: NMR, MRI - Allow the study of water distribution into meat Addition of water Addition of substances to increase water holding properties - Non-destructive

6 Common strategies traditionally used to asses meat authenticity 4) Metabolomics Identification and quantification of as many Low Molecular Weight Compounds as possible GC-FID GC-MS Large set of data analysis Examples: LC-MS - Identification of the presence of pasture in animal diets Sivadier et al Detection of Mechanically Recovered Meat Surowiec et al Authentication of typical Slavonian Salami Jerkovic et al Non-targeted approach

7 METABOLOMICS Differentiation of Mechanically Recovered pork Meat (MRM) by CG-MS metabolite profiling Surowiec, Fraser, Pater, Halket & Bramley (2011). Food Chemistry, 125, 1468 Desinewed meat Hand-deboned meat MRM Clear discrimination of samples is not possible in all cases Search for specific biomarkers

8 Common strategies traditionally used to asses meat authenticity Search for specific biomarkers Targeted approach A) Protein detection methods (ELISA): -Easy to use - High sensitivity - High throughput Need for specific antibodies Cross-reactions False positives Processing of foods can affect the immunoassay Identification of meat species in foods Giovannacci, I. et al. (2004). Int. J. Food Sci. Technol. 39, 863

9 Identification of Meat Species in Foods B) Methods based on DNA analysis (PCR): - High discrimination power (species-specific) - High sensitivity Limitations on processed foods: Difficulties on DNA extraction DNA degradation: ph, heat, hydrolytic enzymes Low reliability Need to develop alternative analytical approaches for species identification Identification of biomarker peptides Proteomics MASS SPECTROMETRY

10 Proteomic technologies in Meat Science

11 Genome Proteome Proteomics The study of a genome expression products, with the objective to obtain an integrated and global vision of the cell processes

12 Evolution of Proteomics 70 s: Cell proteins Identification Immunodetection N-terminal sequencing (Edman) 2D-PAGE Remarkable limitations - Not suitable for great scale protein analysis - Slow and complicated - Low sensitivity

13 Evolution of Proteomics Postgenomic Revolution 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

14 Workflows in current Proteomics A) 1D, 2D-PAGE + MS: x B Peptide fingerprint Sample 2D-PAGE Digestion ( In-gel ) m/z MALDI-TOF MS Identity Ambiguity Peptide sequence MS/MS fragmentation Identity

15 Workflows in current Proteomics B) Gel-free Proteomics: Sample Fractionation Digestion ( In-solution ) (LC)-LC-ESI-MS/MS Protein 1 Protein 3 Protein 2 Protein 4

16 Proteomic contributions to Meat Science: Postmortem proteolysis Lametsch R. et al. (2003). J. Agric. Food Chem. 51, 6992 Immediately after slaughter 72 h postmortem In-gel digestion + MALDI-TOF MS Protein identification Changes in the pig muscle proteome during postmortem meat storage Tenderness Actin & Myosin degradation

17 Proteomics: Characterisation of proteolysis products in dry cured ham Dry-cured ham Extraction Deproteinisation Size-exclusion chromatography MS/MS Fragments generated from Troponin T degradation Reverse phase HPLC LC-ESI-MS/MS MALDI-TOF/TOF Identification of peptide sequences Mora, L. et al. (2010). Food Chem. 123, 691 Peptide Identified sequence TAPKIPEGEKVDFDDIQKKRQNKDL APKIPEGEKVDFDDIQKKRQNKDL KIPEGEKVDFDDIQKKRQNKDL IPEGEKVDFDDIQKKRQNKDL TAPKIPEGEKVDFDDIQKKRQNKD APKIPEGEKVDFDDIQKKRQNKD TAPKIPEGEKVDFDDIQKKRQNK APKIPEGEKVDFDDIQKKRQNK KIPEGEKVDFDDIQKKRQNK IPEGEKVDFDDIQKKRQNK TAPKIPEGEKVDFDDIQKKRQ APKIPEGEKVDFDDIQKKRQ KIPEGEKVDFDDIQKKRQ Position

18 Proteomics Peptide biomarkers as a reliable and accurate way to reveal meat composition OBJECTIVE Development of a methodology capable to overcome the existing limitations on the identification of animal species that can be found in meat products Avoid fraudulent or accidental mislabelling of meat constituents

19 Identification of species-specific peptide biomarkers Workflow: Extraction of muscle proteins SDS-PAGE Selection of target proteins In-gel digestion with proteolytic enzymes MALDI-TOF MS ESI-MS/MS Identification of species-specific peptide biomarkers

20 Selection of target protein: Detection of chicken in a mix with pork meat Isoelectric focusing + SDS PAGE of myofibrillar proteins: Std MLC-1 MLC-2 MLC-3 MLC-1 MLC-2 MLC % pork 100 % chicken Trypsin digestion 1 % chicken in pork + It is possible to detect 1% chicken in pork meat?

21 In-gel tryptic digestion of myosin light chain 3 Intens. [a.u.] Intens. [a.u.] x x % pork % chicken offgel9\0_G2\1\1SRef offgel10\0_H1\1\1SRef Reflex III MALDI-TOF Intens. [a.u.] 0 x % chicken in pork Band 1 bis (1% chicken)\0_c1\1\1sref m/z

22 Zoom-in for m/z range M+H + Position Sequence Origin 1514, DQGSYEDFVEGLR Sus scrofa 1512, DQGTFEDFVEGLR Gallus gallus Intens. [a.u.] x offgel9\0_G2\1\1SRef 100 % pork Intens. [a.u.] 00 x offgel10\0_H1\1\1SRef 100 % chicken Intens. [a.u.] 00 x Band 2 bis (1% chicken)\0_c2\1\1sref % chicken in pork m/z

23 Zoom-in for m/z range M+H + Position Modification Modified mass Sequence Origin Acetylation ALGQNPTNAEINK Gallus gallus Intens. [a.u.] offgel9\0_G2\1\1SRef % pork Intens. [a.u.] offgel10\0_H1\1\1SRef 100 % chicken Intens. [a.u.] 0 x Band 1 bis (1% chicken)\0_c1\1\1sref 1 % chicken in pork m/z

24 MS/MS of DQGTFEDFVEGLR (peptide 1) MALDI-TOF Ion trap 1512,6965 (1+) (2+) Peptide fragmentation (MS/MS): b ions Sequence D Q G T F E D F V E G L R y ions

25 MS/MS for ALGQNPTNAEINK (peptide 2) MALDI-TOF (1+), Acetyl Ion trap (2+) b ions Sequence A y ions L G Q N P T N A E I N K

26 Proteomics Identification of speciesspecific peptides Capable to detect the presence of 1% chicken in pork meat Suitable for quantitation?

27 Quantitative proteomic approach AQUA: Absolute QUAntitation Use of stable isotope peptides made from previously selected sequences L*( 13 C6, 15 N) = +7 Da ALGQNPTNAEINK (Mr ) AL*GQNPTNAEINK (Mr ) F*( 13 C9, 15 N) = +10 Da DQGTFEDFVEGLR ( Mr ) DQGTFEDF*VEGL*R ( Mr ) Quantification of biomarker peptides Quantification of animal species Gerber, SA, Rush, J, Stemmann, O, Kirschner, MW, Gygi, SP (2003). PNAS. 100, 6940

28 Quantitative proteomics Meat mixes (0, 0.5, 1, 2, 5 and 10 % chicken in pork) Protein extraction Isoelectric focusing MLC-3 Stable isotope labelled peptides ( 13 C, 15 N) In-solution trypsin digestion LC-ESI-MS/MS Quantification of peptide biomarkers

29 0,5 % chicken 95% pork MLC-3 Stable isotope labelled peptides Trypsin digestion LCQ Deca LC-MS Clipeus C18 (150x0.5 mm) ALGQNPTNAEINK ( ) Picomol ALGQNPTNAEINK Suitable for quantitation y = 2,7523x R 2 = 0, % chicken in pork meat

30 Discrimination between closely related species Proteomic approach Capable to differentiate between chicken and turkey meat?? Chicken Turkey Chicken Turkey Chicken Turkey MSFSPDEINDFKEAFLLFDRTGDAKITLSQVGDIVRALGQNPTNAEINKI 50 MSFSPDEINDFKEAFLLFDKTGDAKITLSQVGDIVRALGQNPTNAEMNKI LGNPSKEEMNAKKITFEEFLPMLQAAANNKDQGTFEDFVEGLRVFDKEGN 100 LGNPSKEEMNAKKITFEEFLPMLQAAANNKDQGTYEDFVEGLRVFDKEGN GTVMGAELRHVLATLGEKMTEEEVEELMKGQEDSNGCINYEAFVKHIMSV 150 GTVMGAELRHVLATLGEKMTEEEVEELMKGQEDSNGCINYEAFVKHIMSV Myosin Light Chain 3 High sequence homology between chicken and turkey muscle proteins

31 Discrimination between closely related species OFFGEL separation of chicken and turkey proteins MLC-3 Trypsin digestion + MALDI-TOF Intens. [a.u.] x M+H Position Sequence EAFLLFDR EAFLLFDK Origin Gallus gallus Meleagris gallopavo offgel10\0_H1\1\1SRef Chicken myosin light chain 3 Yes, we can! Intens. [a.u.] D1 (Fr 9)\0_D1\1\1SRef Turkey myosin light chain m/z

32 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 Differentiation of closely related species More robustness with respect to actual major limitations of DNA analysis: - Food processing Can be applied for both fresh and cooked meats - Standardized extraction procedures Possibility to develop reliable quantitative determinations - Possibility to use routine, user-friendly, mass spectrometry equipment

33 GOOD, GOOD CHAPS! Prof. Peter Bramley Dr. Paul Fraser Dr. Kaisa Koistinen Mr. Chris Gerrish Dr. Quique Sentandreu

34 Thank you Valencia, Spain

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