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Detection of Cereal Proteins and DNA Using MS, ELISA, and PCR

Detection of Cereal Proteins and DNA Using MS, ELISA, and PCR. William J. Hurkman, Ph. D. USDA/ARS Western Regional Research Center Albany, CA. Dough. Wash out starch granules. Gluten. Mix with alcohol/water. Insoluble. Soluble. Gliadins. Glutenins. Wheat Flour Proteins.

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Detection of Cereal Proteins and DNA Using MS, ELISA, and PCR

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  1. Detection of Cereal Proteins and DNA Using MS, ELISA, and PCR William J. Hurkman, Ph. D. USDA/ARS Western Regional Research Center Albany, CA

  2. Dough Wash out starch granules Gluten Mix with alcohol/water Insoluble Soluble Gliadins Glutenins Wheat Flour Proteins

  3. CerealSpeciesName Wheat Triticum Gliadins Barley Hordeum Hordeins Rye Secale Secalins Oat Avena Avenins Gliadins and Glutenins Are Prolamins Barley, rye, and oat have proteins that are similar to wheat gliadins.

  4. Detection Methods Several analytical tools are currently available to detect the presence of cereal prolamins in food products: • Mass Spectrometry (MS) • Monoclonal Antibodies (ELISA) • Polymerase Chain Reaction (PCR)

  5. MS Protein Identification Endosperm proteins Gel Spots Digest Pro Wheat Grain 2-DE Tryptic fragments Data Sciex QSTAR Spot Matching, Quantification, Profiling Wheat Endosperm Protein Database O-MALDI MS:Peptide mass mapping Tandem MS:Peptide sequencing

  6. pI pI pI 4.5 9.0 Stds 4.5 9.0Stds MW (kD) 200.0 116.3 HMW-GS 97.4  Gliadins 66.3 55.4 α, γGliadins LMW-GS 36.5 31.0 21.5 α-Amylase/trypsin inhibitors 14.4 Albumins & Globulins (KCl-soluble) Gluten Proteins (KCl-Insoluble) Wheat Endosperm Proteins SERPIN GAPDH Peroxidase

  7. MS Protein Identification Pros • Many proteins can be separated simultaneously. • Protein identification is relatively rapid. • Proteins can be quantified. • Proteins useful for antibody production. Cons • Limited to most abundant proteins. • Technically demanding. • Equipment is expensive.

  8. ELISA (Enzyme-Linked ImmunoSorbant Assay) • Takes advantage of the ability of antibodies to recognize and bind to proteins (antigens). • More rapid and less expensive than mass spectrometry. • Highly specific through use of monoclonal antibodies.

  9. Antigen is injected into a mouse to stimulate production of antibodies. Myeloma cells Antibody Lymphocytes from spleen Fuse lymphocytes with myeloma cells to produce antibody producing hybrid cells. Screen for hybrid cell that produces specific antibodies and clone to produce unlimited quantities of monoclonal antibody. Monoclonal Antibody Production

  10. Reactive Protein R5 Antibody Conjugate Substrate Sandwich ELISA Sample & Stds Conjugate Inc. Inc. Wash Stopping Reagent Substrate + or Wash Inc. -

  11. R5 Monoclonal Antibody • Detects: • Wheat gliadins • Barley hordeins • Rye secalins • But not in oat avenins. Méndez et al. Eur. J. Gastroenterol. (2003) 15:465.

  12. R5 Monoclonal Antibody Recognizes QQPFP Repetitive sequence in gliadins, hordeins, and secalins, but not in avenins. MKTFLIFVLLAMAMKIATAARELNPSNKELQSPQQSFSYQQQPFPQQPYPQQPYPSQQPYPSQQPFPTPQQQFPEQSQQPFTQPQQPTPIQPQQPFPQQPQQPQQPFPQPQQPFPWQPQQPFPQTQQSFPLQPQQPFPQQPQQPFPQPQLPFPQQSEQIIPQQLQQPFPLQPQQPFPQQPQQPFPQPQQPIPVQPQQSFPQQSQQSQQPFAQPQQLFPELQQPIPQQPQQPFPLQPQQPFPQQPQQPFPQQPQQSFPQQPQQPYPQQQPYGSSLTSIGGQ • glutamine-glutamine-proline-phenylalanine-proline

  13. R5 ELISA Pros • Detects wheat, barley, and rye prolamins. • Works well for a variety of unprocessed and heat-processed foods. • Relatively rapid (1 h 30 min). • Sensitive (1.5 ppm). • Available in commercial kits. • Tested in 20 laboratories (results soon to be published). • Temporarily endorsed by the Committee on Methods of Analysis and Sampling of the Codex Alimentarius Commission (more data needed).

  14. R5 ELISA Cons • Does not distinguish between wheat, barley, and rye. • Detects only gliadins in wheat. • Celiac patients also react to high and low molecular weight glutenins. • Some ingredients have glutenins, but not gliadins.

  15. PCR (Polymerase Chain Reaction) Amplification Denature Template DNA Anneal Primers (Forward & Reverse Oligonucleotides) Extend DNA (DNA polymerase, DNTPs)

  16. Wheat Gliadin(AF280605m, 2315-2495) ....cagaaa gcgagtggaa agatgaaagc aagccatgcg atgaaaacta taaagacaca tgcaccacca aggccacctt ccatcatcca aacttcacac acctagacca caagcatcaa aggcaagcaa gcagtagtca ccacaaatcc aacatgaaga ccttcctcat ctttgtcctc cttgc..... Rye Secalin(X60295, 325-505) ....tttttc agaaagcgag ttcaatgatg aatgaatcca taccataaca actataaata cacatgcacc attatagtca ccttccatca tccaaacttc acgcaccaag atcagaaaca tcaattccaa gcaagcatta gtaaccacaa atccaacatg aagaccttcc tcatctttgt cctcg..... PCR Primers for Prolamins Sandberg et al. Eur. Food Res. Technol. (2003) 217: 344-349.

  17. Barley Hordein(X03103, 1618-1763) .......att aattcccaaa ctgaacgact acgtgaaaag acagtcacac catgtttgta catccacccc tttgctcgaa atggcgttct tttgctggac agccgagctt cagaatctgc cgtcaagttc ctgagatcca tccacagatg tcgttcacattgttcgccat g......... Oat Avenin(J05486 1097-1200) ......cgct cagtggcttc taagaacact acaagagcta tagtactaca taaataccat cagcgtttag ccgatggacc gatcttgtag cggtgacaaa taaaataaaa .......... PCR Primers for Prolamins Sandberg et al. Eur. Food Res. Technol. (2003) 217: 344-349.

  18. PCR Specificity Primer Pairs Species Wheat Rye Barley Oat Sandberg et al. Eur. Food Res. Technol. (2003) 217: 344-349.

  19. PCR Pros • Species specific: distinguishes between wheat, rye, barley, and oats. • Sensitive and rapid. • Compliments ELISA results. Cons • Detects DNA rather than actual protein.

  20. Summary • Mass spectrometry • Excellent tool for protein identification. • Sensitive, but not rapid. • Expensive and technically demanding. • ELISA • Works well for a variety of unprocessed and heat-processed foods. • Sensitive and rapid. • PCR • Species specific. • Compliments ELISA. • Sensitive and rapid.

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