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Michelle Campeau-Analytical Problem-Proteins and Fruit Allergies

Food allergies are a serious medical concern for some people and a mere annoyance to others. Unfortunately, food allergies, particularly those to fruits and vegetables, can be a side effect of another common allergy: seasonal pollen allergies, or hay fever. This condition is called oral allergy syndrome, in which the body reacts to proteins in fruits and vegetables in the same way it can react to pollen, causing an allergic reaction (1). Fortunately, the type of pollen allergy a person has can determine what kind of fruits and vegetables they're allergic to. As someone who suffers from this, this is an important issue to me. The allergic reactions I've experienced to fruits have gotten steadily worse over time, starting as just watery eyes while eating an apple about 7 years ago to having my lips swell up for over a day after eating a peach last week. I personally do not want to accidentally ingest a fruit that I am allergic to and have my throat close on me. Consequently, one thing that concerns me is how genetics can affect the fruit's proteins. Genetically altered foods are showing up on the market now (2), but how do these foods differ from "natural" or organic foods? Are there any different proteins? Are the allergen proteins even there?

My hypothesis is that any minor genetic altering of a plant will not affect the proteins that cause the allergic reactions. A conclusion can be reached by analyzing fruit samples of genetically altered fruits, "organic" fruits, and fruits that are neither genetically altered or "organic." The analyte could be a variety of things. For example, one could test the skin of the fruit for protein content as well as juices from the meat of the fruit. The matrix of the juice would also contain carbohydrates and other organic molecules.

1. Mayo Clinic. Food Allergy: Symptoms. http://www.mayoclinic.com/health/food-allergy/DS00082/DSECTION=symptoms

2. Harry A. Kuiper, Gijs A. Kleter, Hub P. J. M. Noteborn, Esther J. Kok. Assessment of the food safety issues related to genetically modified foods. The Plant Journal 2001, vol. 27, issue 6, p. 503-528.

UV-Vis Absorption Spectrometry
With fruit allergies stemming from oral allergy syndrome, different proteins in different fruits can trigger reactions. Also, the proteins allergic reactions will result from are due to an individual's allergy to a specific kind of pollen. Consequently, the wavelengths and extinction coefficient depend on which allergy and protein one is analyzing. For example, the proteins Art v 1 and Act c 1 are the proteins in kiwi that cause allergic reactions due to complications from mugwort pollen allergies. In order to determine the concentration of these proteins, HPLC-UV is used, analyzing the separated proteins at 280 nm. The solvent used in analysis for Art v 1 and Act c 1 were acetate and NaCl, and Tris and citrate, respectively.

In the analysis of allergen content of the kiwi and mugwort samples, the extinction coefficients used were found to me 0.59 +/- 0.06 for Art v 1 and 1.72 +/- 0.02 for Act c 1.

As stated earlier, the values for both the absorption and the extinction coefficient should differ, depending on which allergy and protein are being examined.

2. Milan Blanusa, Iva Perovic, Milica Popovic, Natalija Polovic, Lidija Burazer, Mina Milovanovic, Marija Gavrovic-Jankulovic, Ratko Jankov, Tanja Cirkovic Velickovic. Quantification of Art v 1 and Act c 1 being major allergens of mugwort pollen and kiwi fruit extracts in mass-units by ion-exchange HPLC-UV method. Journal of Chromatography B 2007, vol. 857, issue 2, p. 188-194.

Blog 6: Chemical Structure and Standards
2. For the mal d 1 protein, the amino acid sequence is (1):


3. I was unable to find a company that sells the mal d 1 protein, or the mal d 2,3, and 4 proteins as well. However, the proteins can be extracted from an apple and purified (2). The concentration could be calculated by using Beer's law and a spectrometer (as mentioned in a previous entry) if the extinction coefficient is known.

1. http://www.uniprot.org/uniprot/P43211

blog 7:
View image

blog 11:

1. All four electrophoresis methods seem like they would work just fine.
2. CGE seems like it would be the best choice. Although I could not find an example where the mal d proteins were analyzed via a CE method, CGE was used as a means to analyze the concentrations of other proteins in apples.
3. Since I was not able to find information directly on the mal d proteins, the following information is based off other proteins found in apples (proteins analyzed not known):
A coated fused-silica capillary is used with an internal diameter of 100um and a total length of 32.5 cm. TRIS, aspartic acid, SDS, and acrylamide were used as the buffer (pH 8). The voltage was 7kV, and the separation took less than 15 minutes. (1)
4. In the above example, they used a UV-vis spectrometer with two wavelengths analyzed (215 and 280). They had very low concentrations of analyte in the end (20 ug/ml). Although this would probably work for my problem, I think using a fluorimeter might be a better choice since fluorimeters are more sensitive, especially since the analyte concentration is so low. Also, the fluorescence excitation and emission wavelengths are known for my analyte.

1. Blanco Gomiz, Domingo et all., Size-based separations of proteins by capillary
electrophoresis using linear polyacrylamide as a sieving medium: Model studies and analysis of cider proteins. Electrophoresis, vol 24, issue 9, pg. 1391-1396.


Blog 13? - 1 pt

I am now not sure the place you are getting your information, but great topic. I must spend some time finding out much more or working out more. Thank you for excellent info I used to be in search of this info for my mission.

Blog 9. Instead of an enzyme, an antibody would be needed to bind to your analyte if you were to use affinity chromatography. The pH to use is determined by the pI of your analyte proteins. You may use tools such as those found at www.expasy.org to find/calculate the pI of our analytes.
UV detection is not the best detector in your case. Proteins usually have very similar UV spectra. Allergens are likely to be present with many more proteins that are highly abundant. The only way that you could use UV detection is if you selectively purify your sample to eliminate other proteins. This would require an affinity column with an antibody (-0.1 pt).

Blog 10. See comments for blog 9.

Blog 11. Good answers.

Blog Post 10:

1. Ion-exchange chromatography with UV-VIS absorption spectroscopy as the detection method is best for my analytical problem.

2. The other problems that chose this method (generalizing to chromatography + UV-VIS absorption) are the problems on arbutin and
tranexamic acid in skin whitening
products (Chuxin Chen)and Nitric oxide and muscle growth (Andrew Xayamongkhon). Although only two other problems used chromatography and absorption spectroscopy, most others used RPLC in conjunction with fluorimetry or mass spec.

Blog Post 9:

1. GC is not useful since it does not work with large molecules. Affinity chromatography would work well if an enzyme that binds to the proteins exists. Reverse phase, as well as ion exchange and size-exclusion, would also work very well. Chiral chromatography does not work for large molecules.
2. I would use ion-exchange chromatography. It is frequently used for protein separations (1) and has been used to purify the mal d proteins in the past (2).
particle size: 50 micrometers
column length:4.8 cm
column diameter:1.6 cm
back pressure: max 3 bars
company name: Pall

4. I would need to use a buffered solution as the mobile phase. Although I was not able to find an exact number, I would guess it needs to be a lower pH (2-5) due to the possibility of the proteins binding to elements of the matrix (4).

5. I would use a uv-vis absorption spectrometer. Since there are multiple allergenic proteins in apples (mal d 1-4), as well as a host of other molecules in the matrix, if they absorb at different wavelengths, I could select a wavelength that would be optimal for one specific protein.

1.Elbert A. Peterson, Herbert A. Sober. Chromatography of Proteins. I. Cellulose Ion-exchange Adsorbents. J. Am. Chem. Soc. 1956, 78 (4), pp 751–755.
2. Ursula Smole et all. Mal d 2, the Thaumatin-Like Allergen from Apple, Is Highly Resistant to Gastrointestinal Digestion and Thermal Processing. International Archives of Allergy and Immunology. Vol 147, No 4, 2008.
4. J. Szamos, K. Takács, E.E. Szab, E. Kovács, É. Gelencsér. 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, Vol 44, issue 9, Nov 2011, p2674-2678.

Something is missing here.

If you are proposing to use preparative HPLC to clean up (or isolate) a fraction that contains the Mal d allergen, this fraction will need to be stored and then reconstituted in a different solvent for analysis. What are these conditions?

If the HPCL is the final analysis, how do you address the complexity of the sample? There will be other highly abundant compounds extracted in the citrate-phosphate buffer? (-0.5 pt).

Blog 8:

To extract the Mal d 1 protein,
1. The apple is cut up into small pieces and frozen. These small pieces are then ground to a pulp.
2. The pulp is extracted with cold citrate-phosphate buffer (pH=3).
3. The larger pulp particles are removed by filtering the solution through a nylon cloth and centrifuging (10000xg) for 10 minutes.
4. Chromatography is used to further purify and separate the Mal d allergens in apples.

J. Szamos, K. Takács, E.E. Szaba, E. Kovács, É. Gelencsér. 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, Vol 44, issue 9, Nov 2011, p2674-2678.

Good answers for blogs 6 and 7.

Blog 7:
4. Mass spectrometry is a better choice for the large proteins than aas/aes/icp-aes.

I would choose to analyze the mal d 1 protein, as that is the one that most frequently causes allergic reactions. Its mass is 17kDa. TOF and electrospray ionization ms are the two best choices for analysis. However, I was unable to find a spectrum, due to every search site turning "mal d 1" into MALDI. I did find a mass spectrum mal d 3, a lesser allergen in apples. They used esi q-tof ms/ms/ms for their analysis. A study on an allergen protein similar to mal d 1 used esi-TOF ms/ms for their analysis.

image source:
Anita Herndl1 et all. Electrophoresis, vol 28, issue 3, pp. 437–448.

alternate study source:
Hanna Berkner et all. Protein Expression and Purification, vol 56, issue 2, pp 197-204.

Blog 4. The proposed studies do not seem to test the hypothesis. How do they help you determine that genetically altered varieties (e.g. apples) have similar capabilities to release the allergen (e.g., Mal d3)? (-0.75 pt).

Blog 5. The answer is good. However, when working with proteins, it is better to derivatize them to enhance the sensitivity of the assays. This is particularly important when the levels of the compound of interest are very low.

Blog Post 5:

Since apples are a big part of the U of M, I decided to focus on differences in apple varieties.

The mal d 2 protein, one of 4 that cause allergies in apples, is fluorescent. It fluoresces at a wavelength range of 310-390 nm, and excitation wavelength is 295 nm. A phosphate buffer of pH 7 is used for analysis. Mal d 2 fluoresces when the protein itself unfolds when heat is applied. A spectrofluorometer would work for this analysis.

1. Gorji Marzban et all. Conformational changes of Mal d 2, a thaumatin-like apple allergen, induced by food processing. Food Chemistry, vol 112, issue 4, 2-15-09, p. 803-811.

My source for blog 4 is:

1. Jerónimo Carnés PhD, Angel Ferrer PhD, Enrique Fernández-Caldas PhD. Allergenicity of 10 different apple varieties. Annals of Allergy, Asthma & Immunology, p. 564-570.


Blog Post 4:

a. Determine which portion of the fruit has the highest concentration of the protein in question (ie, the seeds, skin, or the meat of the fruit)
b. Determine if there is a link to geographic regions of high pollen levels, where the fruit is grown, and the allergy prevalence.
c. Determine protein contents of variations of a fruit (ie, gala apples vs granny smith vs honeycrisp vs etc.)
d. Determine protein content relative to how ripe the fruit is.

2. I was unable to find information on the protein content of specific fruits. However, I was able to find relative amounts in apple species. For the Mal d 3 protein responsible for the allergic reaction in apples, Starking apples have a very high protein content, while Royal Gala apples have a lower Mal d 3 content.

BLOG 2. Give units for 3b. (-0.15 pt)

Posting for BLOG 2 is too long. Shorten and provide only the necessary information that is relevant to the question being asked. Grade will be released when you notify Chad that this change has been done.

BLOG 3? (1.0 pt)

My Analytical problem: "Pesticides and toxins in fragrances and natural flavors" shares a few points in common with this topic.

There is potential for natural protein toxins to eventually contaminate a natural flavoring, so a similar analyte is not out of the question. Additionally, both problems may need to employ trace analysis and separation, depending on the protein that is being analyzed in fruit and the dosage that a person would react to. There is high potential for similar separation and detection methods as well, considering the complex food matrices. The shared potential for cross contamination is also prevalent. Both problems also share potential to significant acute impact on the consumer of any contaminated product.

Great posting!