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

3. Gliadins and Glutenins Are Prolamins Barley, rye, and oat have proteins that are similar to wheat gliadins. CerealSpecies Name WheatTriticumGliadins BarleyHordeumHordeins RyeSecaleSecalins OatAvenaAvenins

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

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

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

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

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

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

10. Sandwich ELISA Sample & Stds Conjugate Substrate Inc. Wash Stopping Reagent + - Reactive ProteinConjugateR5 AntibodySubstrate or

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

12. MKTFLIFVLLAMAMKIATAARELNPSNKELQSPQQSFSYQQQPFP QQPYPQQPYPSQQPYPSQQPFPTPQQQFPEQSQQPFTQPQQPTPI QPQQPFPQQPQQPQQPFPQPQQPFPWQPQQPFPQTQQSFPLQPQQ PFPQQPQQPFPQPQLPFPQQSEQIIPQQLQQPFPLQPQQPFPQQP QQPFPQPQQPIPVQPQQSFPQQSQQSQQPFAQPQQLFPELQQPIP QQPQQPFPLQPQQPFPQQPQQPFPQQPQQSFPQQPQQPYPQQQPY GSSLTSIGGQ R5 Monoclonal Antibody Recognizes QQPFP Repetitive sequence in gliadins, hordeins, and secalins, but not in avenins. 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. PCR Primers for Prolamins 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..... Sandberg et al. Eur. Food Res. Technol. (2003) 217: 344-349.

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

18. PCR Specificity Winter Wheat +--- Spring Wheat +--- Durum Wheat +--- Spelt+--- Kamut+--- Triticale+--- Rye-+-- Barley--+- Oat---+ Primer Pairs Species Wheat Rye Barley Oat Sandberg et al. Eur. Food Res. Technol. (2003) 217: 344-349.

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

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