Allergenic Protein Content in Apples

Michelle CampeauChem 4101December 9, 2011

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.

Analyte

• Mal d 1 is produced in apples when the fruit is under environmental stres.

• Breaks down when exposed to heat (60° C) or during digestion due to pepsi.

• Protein Sequence:

Figure 1. 3D model of the Mal d 1 protei.

MGVYTFENEFTSEIPPSRLFKAFVLDADNLIPKIAPQAIKQAEILEGNGGPGTIKKITFGEGSQYGYVKHRIDSIDEASYSYSYTLIEGDALTDTIEKISYETKLVACGSGSTIKSISHYHTKGNIEIKEEHVKVGKEKAHGLFKLIESYLKDHPDAYN

Problem and Hypothesis•Problem:

▫In apples, the protein Mal d 1 is the protein primarily responsible for triggering allergic reactions in OAS patients.

•The amount of Mal d 1 present in an apple varies from species to specie.

•Hypothesis:▫Mal d 1 content in a crossbred apple,

such as the Honeycrisp, is proportional to that of the parent species.

Separation Techniques Considered

Technique Pros Cons

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

Not as efficient for large

Detectors ConsideredDetector Pros Cons

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

Sample PreparationRemoval of the Apple Core

Homogenization of pulp in potassium phosphate buffer (10 mM HP, 10 mM P, pH 7) using a grinde

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

Separation•The four Mal d proteins

vary in molecular weight and can be easily separated with size-exclusion chromatograph.

•Eluent: phosphate-buffered saline (pH 7.4

Figure 2. Chromatograph of separation of Mal d 1 and

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

Figure 3. Diagram of a size exclusion chromatography colum

Analysis•Mal d 1 can be derivatized

with SYBR Green I in order to be selectively detecte▫Excitation λ = 497 nm▫Emission λ = 520 nm

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 Ltd

FLS920-s Spectrometer from Edinburgh Instruments Lt

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.

References1. Canadian Food Inspection Agency. “Food Allergens – Oral Allergy Syndrome.” Canadian Food

Inspection Agency. 07-15-2009. < http://www.inspection.gc.ca/english/fssa/concen/tipcon/orale.shtml >.

2. " FLS920-s Spectrometer (Spectro-Fluorimeter) from Edinburgh Instruments Ltd." LabCompare. <http://www.labcompare.com/407-UV-VIS-NIR-Spectrometer/42977-FLS920-s-Spectrometer-Spectro-Fluorimeter/>.

3. Internet Symposium on Food Allergens. Matthias Besler. 2000, Vol 4, No 2. Internet Symposium on Food Allergens. <www.food-allergens.de>.

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 <http://www.jbc.org/content/277/26/23684.full>.

8. Size-Exclusion Chromatography. University of Adelaide. <http://www.chemistry.adelaide.edu.au/external/soc-rel/content/size-exc.htm>.

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. <http://fermi.utmb.edu/cgi-bin/SDAP/sdap_02?dB_Type=0&allid=304>.