1. Allergenic Protein Content in Apples Michelle Campeau Chem 4101 December 9, 2011

2. Background Oral allergy syndrome causes the body to react negatively to proteins in fresh fruits, vegetables, and nuts. The severity of reactions can range from an itchy mouth to anaphylactic shock. Mal d 1 is the primary protein responsible for causing allergic reactions.

3. Analyte MGVYTFENEFTSEIPPSRLFKAFVLDADN LIPKIAPQAIKQAEILEGNGGPGTIKKITFG EGSQYGYVKHRIDSIDEASYSYSYTLIEGD ALTDTIEKISYETKLVACGSGSTIKSISHY HTKGNIEIKEEHVKVGKEKAHGLFKLIES YLKDHPDAYN

4. Problem and Hypothesis

5. Separation Techniques Considered TechniqueProsCons Size-Exclusion Chromatography (SEC) Affordable (~$1000), able to separate the complex matrix by mass A protein could have different retention times whether or not it is denatured due to differences in volume Micellar electrokinetic chromatography (MEKC) Much faster separation than SEC

6. Detectors Considered DetectorProsCons Mass Spectroscopy (MALDI) Great for fragile proteins, high resolution, sensitive Not as quantitative; more sample prep for quantitative analysis (isotope labeling) UV-Vis Spectroscopy Inexpensive, fast analysis Complex matrix makes UV-Vis absorption not as practical Fluorescence Spectroscopy Very sensitive, fast, cost effective ($1500-$5000) Analysis requires derivatization

7. Sample Preparation Removal of the Apple Core Centrifuged at 4°C for 15 minutes at 5000g Incubation at room temperature for 4 hours Supernatant drawn off and frozen at -80°C for storage

8. Separation

9. Size-Exclusion Chromatography Separates analytes based on size, allowing for potential quantification of all four Mal d proteins Larger molecules elute quickly Smaller analytes spend more time trapped in the porous column

10. Analysis Specifications Sensitivity<1 ng/ml Dynamic range Resolution*0.06 nm S/N ratio*> 6000:1 Wavelength accuracy*+/- 0.2 nm *for the FLS920-s Spectrometer from Edinburgh Instruments LtdEdinburgh Instruments Ltd

11. Conclusion Best separation and analysis with size-exclusion chromatography and fluorescence spectroscopy Future possibilities: ▫Potential to breed apples with low concentrations of Mal d 1, causing it to be more hypoallergenic ▫Since little is known of Mal d 2, 3, and 4, this analysis could be applied to them as well.

12. References 1.Canadian Food Inspection Agency. “Food Allergens – Oral Allergy Syndrome.” Canadian Food Inspection Agency. 07-15-2009.. 2." FLS920-s Spectrometer (Spectro-Fluorimeter) from Edinburgh Instruments Ltd." LabCompare.. 3.Internet Symposium on Food Allergens. Matthias Besler. 2000, Vol 4, No 2. Internet Symposium on Food Allergens.. 4.J. Szamos et all. " Purification of natural Mal d 1 and Mal d 2 allergens and monitoring of their expression levels during ripening in Golden Delicious apple." Food Research International (2011): pgs 2674-2678. 5.Maria Puehringer, Helene et all. "MdAP, a novel protein in apple, is associated with the major allergen Mal d 1." Gene 321(2003): pgs. 173-183. 6.Matthes, Anne, Schmitz-Eiberger, Michaela. "Apple (Malus domestica) Allergen Mal d 1: Effect of Cultivar, Cultivation System, and Storage Conditions." J. Agric. Food Chem 57 (2009): 10548- 10553. 7.Mogensen, Jesper et all. “The Major Birch Allergen, Bet v 1, Shows Affinity for a Broad Spectrum of Physiological Ligands.” Journal of Biological Chemistry 3-1-02. 8.Size-Exclusion Chromatography. University of Adelaide.. 9.Skozo, Eva. “Protein and Peptide analysis by capillary zone electrophoresis and micellar electrokinetic chromatography." Electrophoresis 18 (1997): pgs. 74-81. 10.University of Texas Medical Branch. "Allergen Mal d 1." SDAP: Structural Database of Allergenic Proteins. 2011. University of Texas Medical Branch..