Enzymatic Hydrolysis Does Not Reduce the Biological Reactivity of

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Enzymatic hydrolysis does not reduce the biological reactivity of soybean proteins for all allergic subjects Rakhi Panda, Afua O. Tetteh, Siddanakoppalu N. Pramod, and Richard E. Goodman J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b02927 • Publication Date (Web): 08 Oct 2015 Downloaded from http://pubs.acs.org on October 16, 2015

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Enzymatic hydrolysis does not reduce the biological reactivity of soybean proteins for all

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allergic subjects

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Rakhi Panda1, Afua O. Tetteh1, Siddanakoppalu. N. Pramod2 and Richard E. Goodman1*

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Food Allergy Research and Resource Program, University of Nebraska, 1901 North 21st Street,

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Food Innovation Center, Lincoln, NE 68588-6207, USA

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Department of Biochemistry, Kuvempu University, Shimoga-577203, Karnataka, India

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9 *

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Corresponding author.

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Richard E. Goodman, 1901 North 21st Street, Department of Food Science & Technology, Food

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Innovation Center, University of Nebraska, Lincoln, NE 68588-6207, TEL: +1 (402) 472-0452,

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E-mail address: [email protected]

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Current Affiliations: Rakhi Panda- Center for Food Safety and Applied Nutrition, Food and Drug

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Administration, 5100 Paint Branch Parkway, College Park, MD, 20740; Afua O. Tetteh,

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Stanford, CA 94305, USA

Department of Medicine, Stanford University, 300 Pasteur Dr., Grant Building, room S303,

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Abstract

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Many soybean protein products are processed by enzymatic hydrolysis to attain desirable

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functional food properties or in some cases to reduce allergenicity. However, few studies have

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investigated the effects of enzymatic hydrolysis on the allergenicity of soybean products. In this

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study the allergenicity of soybean protein isolates (SPI) hydrolyzed by Alcalase, trypsin,

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chymotrypsin, bromelain or papain were evaluated by IgE immunoblots using eight soybean

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allergic patient sera. The biological relevance of IgE binding was evaluated by a functional assay

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using a humanized rat basophilic leukemia (hRBL) cell line and serum from one subject. Results

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indicated that hydrolysis of SPI by the enzymes did not reduce the allergenicity and hydrolysis

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by chymotrypsin or bromelain has the potential to increase the allergenicity of SPI. Two

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dimensional (2D) immunoblot and liquid chromatography-tandem mass spectrometry (LC-

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MS/MS) analysis of the chymotrypsin hydrolyzed samples indicated fragments of β-conglycinin

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protein are responsible for the apparent higher allergenic potential of digested SPI.

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Key words: Soybean, enzymatic hydrolysis, IgE binding, functional assay

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INTRODUCTION

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Soybean is one of the eight allergenic foods or food groups (peanuts, soybeans, tree nuts, milk,

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egg, fish, crustaceans, and wheat) that are thought to cause nearly 90% of food-allergic reactions

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in the US.1 Soybean allergy affects approximately 0.4% of children worldwide, although this

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sensitivity resolves in a high percentage of children after five or more years.2 Clinical

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manifestations of soy allergy ranges from mild urticaria or atopic eczema and gastric upset to

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severe enterocolitis, facial or oropharyngeal angioedema to severe anaphylaxis.3 At least 16 IgE

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binding proteins of molecular weight ranging from 14 kDa to 70 kDa have been identified in

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soybean and the abundant seed storage proteins, the five glycinins and three β-conglycinins, are

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likely to be the major allergens based on the number of allergic subject with demonstrated IgE

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binding to the seed storage proteins and their abundance in soybeans.4, 5 Soybean proteins are

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modified by processing that may include heating, solvent extraction, enzymatic hydrolysis or a

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combination of processes to improve their functionality as food ingredients.6,7,8 The

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concentration of allergenic proteins present in soybean and the ability to induce allergic reactions

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in sensitized individuals is likely to depend on the type and degree of processing to produce

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soybean ingredients.5,9 Enzymatic hydrolysis of soybean proteins is a common process used by

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industry to improve functional properties and has been used to reduce allergenicity in making

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hypoallergenic soybean products.10,11 The reduced solubility of soybean proteins at acidic pH,

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close to the isoelectric point (4.5) of major seed storage protein glycinins and β-conglycinins,

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limits their use as functional ingredients in moderately acidic foods such as citric beverages and

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salad dressings. Hydrolysis of soybean proteins with proteases can increase protein solubility

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thereby providing functional properties that depends on protein solubility such as foaming and

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emulsifying properties.12,13 Several enzymes have been used by investigators to improve

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soybean protein functionality. Calderon de la Barca, et al. (2000) treated defatted soybean flour

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with chymotrypsin to improve protein solubility, emulsifying and foaming properties.14 The SPI

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have also been treated with enzymes such as Alcalase®, α-chymotrypsin, trypsin, Liquozyme®,

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papain, bromelain, and proteases from cucurbita and rennet to improve solubility, foaming

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properties, emulsifying capacity and the ability to undergo thermal aggregation and gel

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formation.15-17 Since allergic reactions occur when subjects produce IgE antibodies that bind to

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two or more sites on inhaled or ingested proteins and the IgE are bound to the surface of mast

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cells or basophils, causing them to release histamine and other mediators, scientists and food

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processors have attempted to cleave proteins with proteases to reduce their ability to elicit

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reactions. However, in practice enzyme hydrolysis has often not reduced the allergenicity of

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complex foods and can result in an increase in allergenicity because of exposure of new

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antigenic epitopes due to protein breakdown, it is essential to evaluate the allergenicity of

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various hydrolyzed soybean products. There are only a few studies, where the allergenicity of a

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few soybean proteins (Gly m Bd 30K, Gly m Bd 28K, 11s globulin, trypsin inhibitors) have been

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investigated after enzymatic hydrolysis by in vitro IgE binding tests.18-21 All of the studies have

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shown a reduction in soybean protein IgE immunoreactivity after enzymatic treatment except for

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the study by Sung et al. (2014), which showed a slight increase in IgE reactivity by soybean

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Kunitz trypsin inhibitor after sequential hydrolysis of the protein by pepsin and chymotrypsin.21

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However, in vitro IgE binding to individual proteins does not correlate well with the expression

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of clinical symptoms; which may be due to the presence of IgE binding cross-reactive

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carbohydrate determinants, low affinity binding, the presence of only one IgE binding epitope, or

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ineffective spatial orientation of the IgE epitopes 22. Therefore, positive IgE binding detected by

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in vitro tests should be evaluated further for clinical relevance, while reduced binding following

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treatment of proteins demonstrates potential reduced allergenicity, but that proof would require

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appropriate biological activity measurement, either using a basophil activation, degranulation

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assays or in vivo challenges.22 Until now, the effect of hydrolysis with several other enzymes

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commonly used in making functional soybean products including Alcalase, papain and

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bromelain on the overall allergenicity of soybean proteins has not been reported in literature.

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In this study, the overall allergenicity of SPI treated with Alcalase, trypsin,

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chymotrypsin, bromelain or papain were evaluated by both in vitro IgE binding as well as

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mediator release assay using soybean allergic patient sera. Further 2D immunoblots and LC-

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MS/MS analysis was conducted to identify protein fragments resulting from hydrolysis.

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MATERIALS AND METHODS

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Samples. Solubilized SPI was prepared according to previously published methods, 12, 23 were

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used as the starting material for enzyme hydrolysis. Soybean seeds were ground in a SPEX

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CertiPrep 6850 freezer mill under liquid nitrogen to make flour. Samples were defatted using a

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1:20 (w/v) ratio of flour to hexane in a shaking water bath at 50oC for 30 min. The process was

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repeated three more times to remove lipids, and then residual hexane was removed by vacuum

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filtration and aeration. Defatted soybean flour was extracted with alkaline water (1:10 w/v) at

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room temperature for 2 hours after adjusting the pH of water to 8.0 with 2N NaOH. The extract

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was clarified by centrifugation at 10,000 x g for 30 min at 4oC. The pellet was discarded and the

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supernatant was adjusted to pH 4.5 using 2N HCl. An isoelectric precipitate was formed, which

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was collected by centrifuging at 5000 x g for 10 min at 4oC. The precipitate was resuspended in

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0.01M Phosphate-Buffered Saline (PBS, Fisher Bioreagents, Pittsburg, PA, USA) at 5% w/v and

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the pH was adjusted to 8.0 using 2N NaOH. The precipitate was dissolved by vortexing and

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subsequently shaking at room temperature for 1 hour, and stored at -20oC. The protein content of

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solubilized SPI was determined by the Lowry method (BioRad, Hercules, CA, USA) using BSA

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(BioRad) as a standard.

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Human sera. Self-reported soybean allergic or peanut allergic donor sera with significant

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soybean specific IgE tested with soybean ImmunoCAP® (Phadia, now Thermo Scientific,

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Uppsala, Sweden) and the non-soybean allergic control serum sample, collected by PlasmaLab

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International, an FDA licensed blood collection company, were used in this study (Table 1). Use

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of these serum samples was approved by the UNL Institutional Review Board (IRB#

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2009059345EP). The allergic patients utilized in this study have soybean specific IgE level

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ranging from 3-68 kU/L as measured by ImmunoCAP® (Thermo Scientific,) allergen-specific

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IgE test systems. Most of the soybean allergic subjects also reported peanut specific allergic

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reactions and have significant peanut specific IgE levels ranging from 15 to100 kU/L. The

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control subject used in this study did not report any food allergies.

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Enzyme hydrolysis of SPI. Five different enzymes including Alcalase® (Novozymes,

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Bagsvaerd, Denmark), papain, bromelain, trypsin and chymotrypsin (Sigma-Aldrich, Saint

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Louis, MO) were purchased and were used to hydrolyze the SPI sample. Enzyme concentrations

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and conditions (pH and temperature) used for hydrolysis are summarized in Table 2. These

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hydrolysis conditions were chosen based on previously published studies. 15, 16, 24 Hydrolysis was

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carried out for 5, 15, 30 or 60 min with each enzyme. After hydrolysis, the enzymes were

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inactivated by rapidly heating the samples at 95oC for 5 min. Samples were not clarified by

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centrifugation or filtration after the enzymatic treatment, rather they were aliquoted and stored at

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-20oC until thawing and diluting for the immunoblot and hRBL analysis. Two types of control

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samples (Unheated and heated SPI protein) were used in each assay along with the hydrolyzed

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samples for analysis. Heated SPI control samples were prepared by incubated SPI in buffer (no

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enzyme) at the respective incubation temperatures for each enzyme along with the test samples,

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and then heating at 95oC for 5 min. The unheated SPI was simply diluted to the same

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concentration and used directly without incubation or heating. An enzyme only control was also

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analyzed along with the treated and control extracts in the immunoblot and hRBL release assay.

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Electrophoresis and IgE immunoblotting. The hydrolyzed SPI and control samples were diluted

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using Laemmli SDS-sample buffer (Boston BioProducts, Ashland, MA) to allow loading 10 µg

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of soybean protein (10 µl /well) in SDS-PAGE gels. Samples were run under both reducing (2-

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mercaptoethanol, 5% by volume and heating at ~ 95°C for 5 min) and non-reducing conditions

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using Novex 10-20% tris-glycine gels (Invitrogen, Carlsbad, CA). Pre-stained Precision Plus

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molecular weight marker proteins (BioRad) were run in separate lanes to estimate protein size.

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Proteins were separated at a constant 125 V for 90 min, then fixed (7% acetic acid, 40%

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methanol in water) and stained with Brilliant Blue G-colloidal (Sigma-Aldrich)) for at least 2

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hours. Gels were then destained for one min in 10% acetic acid, 25% methanol in water followed

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by multiple changes of 25% methanol in water until the background was clear.

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For 2D electrophoresis, a BioRad PROTEAN IEF Cell (BioRad) was used for the first

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dimensional separation of the proteins based on their isoelectric points. Samples representing 25

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µg undigested, heated control SPI or SPI samples digested for 60 min with Alcalase®, trypsin or

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chymotrypsin were diluted to 125 µl with IEF sample buffer [8M urea, 2% CHAPS, 50 mM DTT

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(Fisher Bioreagents, Pittsburg, PA, USA) and 0.5% ampholyte (BioRad)] and then applied to

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individual troughs of the IEF focusing tray (BioRad). Individual pI 3-10 linear IEF strips

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(BioRad) were submerged in the trough of each sample well and covered with 4 ml of mineral oil

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(BioRad). Active rehydration was performed at 50 V for 12 hours followed by separation at 250 V

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for 15 min, 4000 V ramping for 2 hours with a final 4000 V limit step until 34,000 integrated Vhr

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was reached. Proteins were then maintained in position with a constant application of 500 V until

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morning. The strips were then equilibrated for 15 min in 2.5 ml of DTT equilibration buffer (6 M

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urea, 2% SDS, 0.375 M Tris-HCl, pH 8.8, 20% glycerol, 130 mM DTT) and then 15 min in 2.5

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ml iodoacetamide equilibration buffer (6 M urea, 2% SDS, 0.375 M Tris-HCl, pH 8.8, 20%

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glycerol, 135 mM iodoacetamide) for reduction and acetylation. Separation in the second

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dimension was carried out by placing the focused strips into the 7cm wide well and 4 µl of pre-

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stained Precision Plus molecular weight marker proteins into the small well of NuPAGE® Novex

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4-12% Bis-Tris ZOOM® Gels, Mini (8 cm x 8 cm) (Invitrogen). The wells were sealed with

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molten 0.5% agarose (Invitrogen). Electrophoresis was accomplished at a constant 150 V for 60

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min. Proteins in the gels were stained after electrophoresis using EZBlueTM gel stain (Sigma).

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For immunoblots, the separated proteins from unstained gels were electro-transferred to

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polyvinylidene difluoride (PVDF) membranes (Invitrogen) at 25 V for 90 min using Novex®

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transfer buffer (Invitrogen). The protein transfer was verified by staining the membranes with

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Ponceau S stain (Sigma). The membranes were then blocked with 5% non-fat dry milk (NFDM)

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in PBS containing 0.05% Tween 20 (PBST) for at least one hour. Individual human sera (eight

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individual soybean allergic and one control serum) were diluted 1:10, in 2.5% NFDM in PBST

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and allowed to bind to the NFDM for 1 hour before incubating with the blocked membrane for

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overnight at room temperature. Unbound antibodies were removed from the membranes by

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washing four times 5 min each with PBST. Bound IgE was detected using horse radish peroxidase

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(HRP) conjugated monoclonal anti-human IgE (SouthernBiotech, Birmingham, AL: clone

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B3102E8), diluted 1:1000 (v/v) with 2.5% NFDM in PBST. The unbound secondary antibodies

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were removed by washing the membranes four times with PBST. Detection was achieved using

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Supersignal West Dura Extended Duration substrate (Pierce, Rockford, IL, USA) and capturing

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emitted light with a Kodak Gel Logic 440 image station. A nitrocellulose membrane (Invitrogen)

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spotted with diluted purified IgE (Human IgE, monoclonal with kappa light chain, ABCAM, Inc.,

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Cambridge, MA), blocked with 5% NFDM in PBST and incubated with the secondary antibody

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and substrate as immunoblots was exposed along with the immunoblots to evaluate signal strength

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between immunoblots with different sera.

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Identification of proteins by mass spectrometry. The selected proteins or fragments from the

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digestion samples were identified by isolating protein spots from EZBlueTM (Sigma #G1041)

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stained 2D gels that correspond to IgE bound spots on 2D immunoblot. The Proteomics Core

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facility at the University of Nebraska (N. Madayiputhiya) performed an in-gel digestion of the

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proteins with trypsin after first reducing and alkylating the proteins. Following digestion, the

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peptides were extracted and separated and identified by LC-MS/MS using a 3000 Dionex nano

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LC system (Dionex) integrated with a LCQ Fleet Ion Trap mass spectrometer (Thermo Fischer

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Scientific) as described in Hasim et al. (2013) and analyzing results using Distiller software

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(Matrix Science) to produce peak lists for analysis.25 The data were analyzed using MASCOT

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(Matrix Science) using the NCBI non-redundant database with searching restricted to green

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plants (Viridiplantae).

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Mediator release assay using a humanized rat basophilic leukemia (hRBL) cell line.

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Humanized rat basophilic leukemia (hRBL) cells (RBL-703/21) developed by transfecting an

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immortalized RBL with the alpha-chain of the human FcεRI gene to present the high-affinity

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receptor for human IgE were used for mediator release assays as previously.26 Cultured adhered

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cells grown to stationary phase were dislodged by application of 0.01 M EDTA in MEM for 30-

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45 min. The cells were washed twice with fresh media and diluted to a cell density of 2 X 106

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cells/ml. Then 50 µl containing 1 X 105 cells was seeded into each well of a 96 well micro titer

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plate followed by sensitization adding 50 µl of individual human plasma diluted 1:10 (v/v) with

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the MEM and incubating the plates at 5% CO2 at 37°C for approximately 12 hours. Cells were

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then washed with Tyrode’s wash buffer twice and challenged with 100 µL of hydrolyzed SPI at

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one of five concentrations (10 µg/ml, 1 µg/ml, 0.1 µg/ml, 0.01 µg/ml and 0.001 µg/ml), or with

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control samples diluted in allergen challenge buffer. Plates were then incubated in a water bath

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(37°C) for 1 hour before withdrawing 30 µl of cell supernatant to add to 50 µl of substrate

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(0.13% β-nitro-N-acetyl-β-D-glucosamide) in untreated polystyrene 96-well micro plates

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(Thermo Scientific Nunc). The plates were incubated at 37°C in a water bath for one hour and

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reactions were stopped by adding 100 µl stopping solution (0.2 M glycine). The absorbance of

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each sample was measured at 405 nm. Absorbance values (OD) of samples challenged with

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antigen were adjusted to a baseline reference by subtracting readings from IgE sensitized cells

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that were not exposed to antigen (serum negative controls). Cells sensitized by human IgE

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(#ab65866 from Abcam Inc., Cambridge, MA) and cross-linked by anti-human IgE (Sigma)

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were used as positive controls. Additional cell samples sensitized with test serum and cross-

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linked by anti-IgE instead of the antigen were used as a positive control for total serum IgE

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release. The β-hexosaminidase release from the SPI and hydrolysate samples were expressed as a

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percentage of total serum IgE release by comparing the OD values of the samples with that of the

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positive control for total serum IgE release.

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RESULTS

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1D-SDS-PAGE and immunoblots. The SDS-PAGE protein patterns of both control samples

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(heated and unheated) under reducing conditions showed multiple bands between molecular

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weight of 10 to 100 kDa (Figure 1). After hydrolysis most of the higher molecular weight bands

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disappeared rapidly while an increase was noted in stainable low molecular weight protein bands

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ranging from 6-10 kDa. Under non-reducing conditions SPI digested with Alcalase, trypsin and

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papain showed a reduced band intensities compared to the heated SPI control whereas SPI

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digested with bromelain and chymotrypsin showed a reduction in high MW bands, but little

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difference in low MW bands compared to the control in stained gels. Hydrolysis was most

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effective in the first five minutes, with only slight additional reduction in visible bands after 15

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min of hydrolysis time.

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In order to determine the impact of digestion on potential allergenic properties, the IgE

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binding patterns of protease treated samples were evaluated using eight individual soybean

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allergic patient sera and one non-soybean allergic control serum by immunoblotting. Binding

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patterns of the three subjects showing the highest intensity and diversity of band recognition are

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shown. Figure 2 A-C shows the IgE binding pattern of the hydrolyzed and control SPI samples

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with serum 19392-CS, 20431 and 20770-MH respectively. Serum IgE binding patterns of four

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other sera (18534-LN, 20197-BH, 23736-AM and 20247-LA) were similar to that of 20770-MH

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and serum 9735-RE demonstrated very faint IgE binding with all the hydrolyzed samples (not

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shown). The IgE binding patterns of the unheated and heated SPI control samples was similar

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with all the eight soybean allergic sera under both reducing and non-reducing conditions, except

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for slight signal intensity differences observed mostly under non-reducing condition. Serum

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19392-CS (Figure 2A) has strong IgE binding to a band of approximately 20 kDa appeared

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following digestion with Alcalase under reducing conditions. Under non-reducing conditions, the

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intense IgE binding observed to the higher molecular weight protein bands ranging from 35 to

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250 kDa in the heated SPI control sample were reduced in the digested samples. A marked

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reduction in IgE binding to the trypsin and papain digested SPI was observed under both

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reducing and non-reducing conditions with this serum. However, strong IgE binding to a 20 kDa

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band remained in the trypsin hydrolyzed SPI under reducing conditions. With bromelain and

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chymotrypsin most of the IgE binding observed in the heated SPI control remained in the

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hydrolyzed samples. Additionally a complex pattern of new IgE binding bands appeared in the

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bromelain hydrolyzed samples mostly under reducing conditions (30, 45 and 55 kDa bands).

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With chymotrypsin, IgE binding to a 20 kDa band was obviously stronger for the digested SPI

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compared to the heated SPI control.

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The IgE binding to Alcalase and papain hydrolyzed samples with serum 20431 (Figure

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2B) was markedly reduced compared to the heated SPI control except for a strong IgE binding

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band at 25 kDa. With trypsin hydrolysis, IgE binding to the higher molecular weight protein

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bands ranging from 50-150 kDa in the heated SPI control was eliminated or markedly reduced.

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Relatively faint IgE binding to a 50 kDa band remained in the hydrolyzed samples (mostly under

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reducing conditions) and faint IgE binding to a 25 kDa band that was observed in the control

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sample was strongly augmented. Additionally a new IgE binding protein band (15 kDa) was

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observed. The SPI digested with chymotrypsin also showed stronger IgE binding to a 25 kDa

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band compared to the heated SPI control with this serum. For bromelain hydrolyzed SPI, IgE

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binding to most of the higher molecular weight protein bands were eliminated except for a 50

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kDa band and two new IgE binding protein bands (25 and 30 kDa) appeared in all hydrolyzed

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samples.

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With serum 20770-MH (Figure 2C), both Alcalase and papain digestion resulted in the

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disappearance of most IgE binding bands observed in the heated SPI control. Although trypsin

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hydrolyzed samples showed similar IgE binding as control under reducing conditions, little IgE

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binding was observed under non-reducing conditions except for a 30 kDa band that showed

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higher intensity compared to the heated SPI control. Bromelain and chymotrypsin hydrolyzed

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samples retained most of the IgE binding that was observed in the heated SPI control.

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Interestingly a new IgE binding band appeared at approximately 10 kDa for the trypsin and

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bromelain hydrolyzed SPI samples. Immunoblots with the non-soybean allergic control serum

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did not show any IgE binding to any soybean samples (not shown).

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Mediator release assays. To evaluate whether the reductions in IgE binding as well as

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appearance of new IgE binding bands in the hydrolyzed samples as observed in the immunoblots

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have any biological significance, mediator release assays were performed using hRBL cell line

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703/21. Figure 3 shows the β-hexosaminidase release results expressed as a percent of total

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serum IgE (anti-IgE induced cross-linking) release of the hydrolyzed samples using serum

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19392-CS. The β- hexosaminidase release using 1µg/ml antigen concentration of the unheated

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SPI control was approximately 25-45%. The release was markedly reduced for the heated SPI

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control (10-20%) even though it showed an overall similar IgE binding as the unheated SPI

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control in immunoblots with the same sera (Figure 2A). The heated SPI control was used to

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evaluate the effects of the hydrolysates as they all underwent same treatment conditions. SPI

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samples treated with Alcalase, trypsin and papain resulted in a similar mediator release as the

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heated SPI control sample (except for the 60 min Alcalase hydrolyzed sample, which showed a

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slightly lower release) in spite of showing a strong reduction in IgE binding by immunoblot

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(Figure 2A). However, in accordance with the immunoblot results, mediator release from the 5

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min and 60 min bromelain hydrolyzed SPI revealed no reduction in β-hexosaminidase release

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relative to the heated SPI control. Interestingly the SPI sample treated with bromelain for 30 min

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showed an increase in β-hexosaminidase release (approximately 30%) compared to the heated

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SPI control. Similarly all the chymotrypsin hydrolyzed SPI samples resulted in a higher β-

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hexosaminidase release compared to the heated SPI control (approximately 30-40%). This higher

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release might be related to the strong IgE binding observed to a 20 kDa band for the

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chymotrypsin hydrolyzed sample in immunoblot. The mediator release assay was unsuccessful

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with the other sera used in the immunoblot assays as the total release using those sera was low,

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similar to the negative (non-soybean) control.

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2D immunoblots and LC-MS/MS. The possible identity of the 20-25 kDa proteins that showed

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strong IgE binding following hydrolysis with Alcalase, trypsin and chymotrypsin with serum

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19392-CS (Figure 2A) and for trypsin or chymotrypsin hydrolyzed samples with serum 20431

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(Figure 2B) was investigated using 2D-gel immunoblotting. Samples included both the heated

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SPI control and the SPI samples hydrolyzed for 60 min. From the stained gel (Figure 4A) most

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of the higher molecular weight protein spots from approximately 37 to 75 kDa that are observed

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in the control sample were no longer visible when the samples were treated with the three

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enzymes. With the Alcalase treated SPI, very faint protein spots were observed around 25 kDa

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and 12 kDa. A number of lower molecular weight protein spots ranging from 10-20 kDa were

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observed when the samples were treated with trypsin or chymotrypsin. A protein spot of

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approximately 25 kDa at a pI of 4 (Spot #1) and two spots of similar molecular weight and pI

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values (Spots # 2, 3) appeared only in the trypsin and chymotrypsin digested samples

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respectively. Furthermore, spots of approximately 50 kDa appeared in both the control and the

309

chymotrypsin hydrolyzed SPI (# 10) (Figure 4A). Immunoblots with serum 19392-CS (Figure

310

4B) showed strong IgE binding to protein spots ranging from 37-150 kDa in the heated SPI

311

control. These dominant IgE binding spots were absent when the samples were treated with

312

Alcalase or trypsin. However, there was a faintly visible spot at approximately 50 kDa (# 10) in

313

the chymotrypsin hydrolyzed sample. With both trypsin and chymotrypsin hydrolyzed samples,

314

relatively strong IgE binding was observed to the 25 kDa, pI 4 spots (# 1, 2, 3) that seem to

315

correspond to the similarly marked spots visible in the stained gels (Figure 4A). The Alcalase

316

hydrolyzed sample also showed strong IgE binding to a spot of similar MW (25 kDa) and pI 4

317

that is marked as spot # 9, which was not visible in the stained gel. Furthermore, the immunoblot

318

of the trypsin hydrolyzed sample showed modest IgE binding to three spots of approximately 23

319

kDa, with pI values between 6-7 (spots # 4, 5, 6). IgE binding to those spots was very faint in the

320

control SPI sample. Immunoblot with serum 20431 also showed strong IgE binding to the spots

321

at 25 kDa, pI=4 (#1, 2, 3) only in the trypsin and chymotrypsin hydrolyzed samples (not shown).

322

Spots were excised from a stained gel and identified by LC-MS/MS by the analytical core

323

facility at the University of Nebraska. The Mascot (Matrix Sciences) program output search for

324

mass identity matches indicated matches to Glycinin G1 protein (Gly m 6.0101) for spot # 4, 5

325

and 6 (12.5%, 19% and 29% coverage respectively) from the non-redundant NCBI database

326

showing coverage strictly in the basic chain of the protein. Spots # 1 and 3 showed a match with

327

the α subunit of β-conglycinin (Gly m 5.0101: 8.3% and 5.1% coverage) whereas spot # 2

328

showed a match with the α’ subunit of β-conglycinin (Gly m 5.0201: 4.8% coverage) indicating

329

that these spots in the trypsin and chymotrypsin hydrolyzed samples are fragments of the full-

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length α’ and α subunits of β-conglycinin. Spots # 7 and 8 that were visible in the heated SPI

331

control stained gel and showed intense IgE binding with both the serum used in immunoblot

332

appear to be full-length Gly m 5.0201 and Gly m 5.0101 respectively with 19.8% and 21.3%

333

coverage. Sequence maps and coverage are shown in supplemental figures (Figure S1, S2, S3).

334

DISCUSSION

335

Soybean is a popular and widely used food protein source that is often processed by hydrolysis

336

prior to addition to foods in order to increase nutritional quality, flavor and functionality of

337

soybean proteins.17 Protein hydrolysate formulas have also been developed with the aim to

338

produce hypoallergenic foods that have reduced eliciting capacity. Enzymatic hydrolysis of food

339

proteins can lead to alteration of epitope structure thereby reduce or increase allergenicity.20, 21,24,

340

28-32

341

enzymatically hydrolyzed food proteins has been the reliance on IgE binding to immunoblots or

342

ELISA to judge potential changes in allergenicity. Although in vitro IgE binding provides

343

information on the immunoreactivity of the proteins, clinical studies have demonstrated that IgE

344

binding does not always correlate with the expression of clinical symptoms.22 Therefore

345

functional tests should be performed for more accurate evaluation (basophil histamine release

346

assay, skin prick tests or double blind placebo controlled food challenges ( DBPCFC) of

347

allergenicity. Additionally, results from many studies including our own demonstrate that

348

individual allergic subjects often have IgE that binds different proteins and presumably clinical

349

reactivity may differ to those proteins as well.33

350 351

One of the major drawbacks of most of the studies evaluating the allergenicity of the

Functional assays with basophils can be performed either by incubating heparinized whole blood from an allergic individual with allergen or after stripping endogenous IgE from

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basophils of non-allergic donors prior to incubating with serum IgE from appropriately allergic

353

donors, followed by stimulating with allergen. 34, 35 However, the major disadvantage of these

354

assays is the requirement for fresh blood to perform the assay and additional controls to

355

demonstrate that the stripped basophils are not activated by non-allergen related signaling. The

356

use of a hRBL cell line, enables the evaluation of stored sera to identify those that binding to

357

multiple epitopes and would likely elicit an allergic reaction in the serum donor.

358

26, 36

In this study, the potential allergenicity of proteins in SPI were evaluated following

359

hydrolysis with five different enzymes. Results were compared to heated non-hydrolyzed SPI

360

extract treated under similar conditions. The IgE immunoblot results with individual soybean

361

allergic sera showed an overall reduction in IgE binding to proteins for the SPI samples

362

hydrolyzed with Alcalase, papain and trypsin compared to the heated SPI control. However, for

363

some proteins and some serum donors, IgE binding remained strong to the hydrolyzed samples.

364

Bromelain and chymotrypsin digested samples showed a comparable staining patterns, but

365

comparable IgE binding with the heated SPI control (observed with seven out of eight soybean

366

allergic sera used in 1D-immunoblot). The IgE binding patterns with sera 19392-CS and 20431

367

showed new IgE binding bands in the samples hydrolyzed with Alcalase, bromelain, trypsin and

368

chymotrypsin. These could represent an increase in immunoreactivity either due to uncovering

369

previously masked epitopes or simply fragmentation of full-length proteins with retention of

370

binding. The 2D-immunoblot results showed different spot patterns. The identity of spots at

371

approximately 25 kDa, pI=4 present in trypsin and chymotrypsin digested samples were

372

identified as fragments of α’ and α subunits of β-conglycinin by LC-MS/MS. These spots

373

showed strong IgE binding in immunoblot with two soybean allergic sera. The immunoblot of

374

trypsinized SPI using serum 19392-CS showed increased IgE binding compared to the heated

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375

SPI control with three spots at approximately 23 kDa and pI 6-7. The spots were also present in

376

the control sample as intensely staining spots, but IgE binding was much stronger in the

377

trypsinized sample suggesting likely uncovering of masked epitopes in the undigested sample.

378

The IgE binding observed in immunoblots of the native and enzyme hydrolyzed samples

379

may represent IgE binding to sequential or conformational epitopes. While researchers often

380

assume the denaturing properties of SDS and reducing agents will eliminate conformational

381

epitopes Zhou et al. (2007) showed that some conformational epitopes are reconstituted in

382

western blots.37 The major seed storage proteins β-conglycinins and glycinins are large proteins

383

with naturally tightly packed complex structures (hetero- or homo-trimers and hexamers).

384

Although preparation of SPI may denature or change conformation of these proteins, significant

385

structural integrity should remain. Further denaturation is likely due to SDS and reducing agents

386

in SDS-PAGE, but that has not been objectively demonstrated. While there have been studies

387

evaluating epitopes of both types of these proteins in soybeans and peanuts, it is clear that

388

antibodies are formed against both sequential and conformational structures and furthermore,

389

that the state of aggregation, denaturation or proteolytic cleavage may positively or negatively

390

alter the binding efficiency.38-41 Few studies have been done to evaluate the biological relevance

391

of IgE binding even when specific epitopes are studied. However, the mediator release assay

392

does allow evaluation of the ability of both the digested and undigested proteins present in the

393

hydrolyzed SPI without having to map epitopes. Despite the overall reduction in IgE binding to

394

protein bands observed in immunoblot by the SPI hydrolyzed with papain, Alcalase and trypsin,

395

the extracts showed a similar or slightly reduced mediator release compared to the heated SPI

396

control in the hRBL assay. Interestingly, there was still positive mediator release in the hRBL

397

assay even when immunoblot binding was markedly reduced. Whether the digestion resistant

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398

proteins contain sequential or conformational epitopes, the important point is that they were able

399

to cross-link IgE antibodies on basophils and induce mediator secretion, although not as well as

400

the unheated control. Based on the hRBL data, we assume that digested and heated samples

401

would be able to elicit allergic reactions in vivo. Chymotrypsin hydrolysis of SPI at all-time

402

points led to an increase in mediator release compared to the heated SPI control. Results of the

403

2D-immunoblot and LC-MS/MS demonstrate that there are digestion resistant fragments of the

404

α’- , α- and β- subunits of β-conglycinin that could contribute to the mediator release with

405

soybean- chymotrypsin hydrolysates. The SPI treated with bromelain for 30 min also showed a

406

higher mediator release compared to the heated SPI control. It is possible that similar to

407

chymotrypsin hydrolysates, protein fragments appearing after 30 min of hydrolysis with

408

bromelain as well as the digestion resistant proteins are enough to cross-link IgE antibodies on

409

the hRBL cells.

410

Limitations of this study: Only one soybean allergic serum was used to perform the

411

mediator release assay due to the relatively low level of IgE and hRBL activity of other study

412

subjects. Thus conclusions regarding the potential allergenicity of the hydrolysates, may not be

413

representative for a large population of soybean allergic subjects. However, this data

414

demonstrates that some soybean allergic subjects are not likely to tolerate foods containing

415

soybean proteins that are enzymatically treated to reduce allergenicity. confirmation of our study

416

results would should be evaluated further by DBPCFC if a sufficient number of consenting

417

soybean allergic subjects can be identified and challenged with similarly prepared samples. We

418

tested sera from other subjects in this study, but they did not result in mediator release, which

419

could be due to a low level of soybean specific IgE, low affinities of IgE or the sub-optimal

420

spacing of epitopes on individual soybean protein fragments. Another limitation is the

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421

uncertainty of defining differences in IgE binding or basophil activity based on uncertain

422

relevance of dose. It is difficult to evaluate dose equivalence across sample digested with

423

different enzymes. We chose to estimate sample concentrations based on starting protein

424

concentrations. In conclusion, results from this study showed that the hydrolysis of soybean proteins by

425 426

enzymes such as Alcalase, papain, trypsin, chymotrypsin or bromelain do not remove IgE

427

binding to all soy proteins for many soybean allergic subjects. More importantly, at least some

428

individuals would likely still experience elicitation of food allergy when consuming SPI

429

hydrolysates as demonstrated by basophil activation for at least one subject out of eight. These

430

results should provoke caution in those claiming to have produced “hypoallergenic” soybean

431

food ingredients and stimulate more biologically relevant testing.

432 433

ACKNOWLEDGEMENTS

434

The authors thank Dr. Nandakumar Madayiputhiya of Proteomics and Metabolomics Core

435

Facility at University of Nebraska for performing LC-MS/MS analysis of the SPI samples. We

436

also thank the soybean allergic donors. Funds were provided by the US Environmental

437

Protection Agency STAR grants RD83313501 and RD83406501. We thank Drs. Steve Taylor

438

and Joe Baumert of FARRP, University of Nebraska-Lincoln for technical suggestions and

439

additional financial support.

440

Corresponding Author

441

*Telephone: +1-402-472-0452. E-mail: [email protected].

442

Notes

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443

The authors declare no financial conflict of interest.

444

SUPPORTING INFORMATION AVAILABLE

445

Figure S1- Peptides detected (shown in grey) by LCMS/MS with coverage in the identified

446

protein for the spots excised from the 2D-gel of SPI treated with trypsin for 60 min

447

Figure S2- Peptides detected (shown in grey) by LCMS/MS with coverage in the identified

448

protein from the spots excised from the 2D-gel of SPI treated with chymotrypsin for 60 min

449

Figure S3- Peptides detected (shown in grey) by LCMS/MS with coverage in the identified

450

protein from the spots excised from the 2D-gel of the heated SPI control

451

REFERENCES

452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474

1. Goodman, R. E.; Hefle, S. L.; Taylor, S. L.; van Ree, R. Assessing genetically modified crops to minimize the risk of increased food allergy: a review. Int. Arch. Allergy Immunol. 2005, 137, 153-166. 2. Savage, J. H.; Kaeding, A. J.; Matsui, E. C.; Wood, R. A. The natural history of soy allergy. J. Allergy Clin. Immunol. 2010, 125, 683-686. 3. Sicherer, S. H.; Sampson, H. A.; Burks, A. W. Peanut and soy allergy: a clinical and therapeutic dilemma. Allergy 2000, 55, 515-521. 4. Ladics, G. S.; Budzizewski, G. J.; Herman, R. A.; Herouet-Guicheney, C.; Joshi, S.; Lipscomb, E. A.; McClain, S.; Ward, J. J. Measurement of endogenous allergens in genetically modified soybeans-Short communication. Reg. Toxicol. Pharmacol. 2014, 70, 75-79. 5. Holzhauser, T.; Wackermann, O.; Ballmer-Weber, B. K.; Bindslev-Jensen, C.; Scibilia, J.; Perono-Garoffo, L.; Utsumi, S.; Poulsen, L. K.; Vieths, S. Soybean (Glycine max) allergy in Europe: Gly m 5 (beta-conglycinin) and Gly m 6 (glycinin) are potential diagnostic markers for severe allergic reactions to soy. J. Allergy Clin. Immunol. 2009, 123, 452458. 6. Singh, P.; Kumar, R.; Sabapathy, S.N.; Bawa, A.S. Functional and edible uses of soy protein products. Comp. Rev. food sci. food safety 2008, 7, 14-28. 7. Jiang, J.; Xiong, Y. L.; Chen, J. Role of β-conglycinin and glycinin subunits in the pH shifting-induced structural and physicochemical changes of soy protein isolate. J. Food Sci. 2011, 76, C293-C302. 8. Keerati-U-Rai, M.; Corredig, M. Heat-induced changes occurring in oil/water emulsions stabilized by soy glycinin and β−conglycinin. J. Agric. Food Chem. 2010, 58, 9171-9180.

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475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519

9. Kosma, P.; Sjolander, S.; Landgren, E.; Borres, M. P.; Hedlin, G. Severe reactions after the intake of soy drink in birch pollen-allergic children sensitized to Gly m 4. Acta Paediatr. 2011, 100, 305-306. 10. Host, A.; Halken, S. Hypoallergenic formulas--when, to whom and how long: after more than 15 years we know the right indication! Allergy 2004, 59 Suppl 78, 45-52. 11. L'Hocine, L.; Boye, J. I. Allergenicity of soybean: new developments in identification of allergenic proteins, cross-reactivities and hypoallergenization technologies. Crit. Rev. Food Sci. and Nutr. 2007, 47, 127-143. 12. Moline Ortiz, S.; Wagner, J. R. Hydrolysates of native and modified soy protein isolates: structural characteristics, solubility and foaming properties. Food Res. Intern. 2002, 35, 511-518. 13. Adler-Nissen, J. Enzymatic hydrolysis of proteins for increased solubility. J Agric. Food Chem. 1976, 24, 1090-1093. 14. Calderon de la Barca, A. M.; Ruiz-Salazar, R. A.; Jara-Marini, M. E. Enzymatic hydrolysis and synthesis of soy proteins to improve its amino acid composition and functional properties. J. Food Sci. 2000, 65, 246-253. 15. Kim, S. Y.; Park, P. S. W.; Rhee, K.C. Functional properties of proteolytic enzyme modified soy protein isolate. . J. Agric. Food Chem. 1990, 38, 651-656. 16. Moline Ortiz, S.; Cristina Anon, M. Analysis of products, mechanisms of reaction and some functional properties of soy protein hydrolysates. JAOCS. 2000, 77, 1293-1301. 17. Sun, X.D. Enzymatic hydrolysis of soy proteins and the hydrolysates utilization. Int. J. Food Sci. Tech. 2011, 46, 2447-2459. 18. Tsumura, K.; Kugimiya, W.; Bando, N.; Hiemori, M.; Ogawa, T. Preparation of hypoallergenic soybean protein with processing functionality by selective enzyme hydrolysis. Food Sci. Technol. Res. 1999, 5, 171-175. 19. Lee, H. W.; Keum, E. H.; Lee, S. J.; Sung, D. E.; Chung, D. H.; Lee, S. I.; Oh, S., Allergenicity of proteolytic hydrolysates of the soybean 11S globulin. J. Food Sci. 2007, 72, C168-172. 20. Wang, Z.; Li, L.; Yuan, D.; Zhao, X.; Cui, S.; Hu, J.; Wang, J. Reduction of the allergenic protein in soybean meal by enzymatic hydrolysis. Food Agric. Immunol. 2014, 25(3), 1-5. 21. Sung, D.; Ahn, K.M.; Lim, S.; Oh, S. Allergenicity of an enzymatic hydrolysate of soybean 2S protein. J. Sci. Food Agric. 2014, 94, 2482-2487. 22. Ladics, G. S.; van Bilsen, J. H.; Brouwer, H. M.; Vogel, L.; Vieths, S.; Knippels, L. M., Assessment of three human Fc epsilon RI-transfected RBL cell-lines for identifying IgE induced degranulation utilizing peanut-allergic patient sera and peanut protein extract. Regul. Toxicol. Pharmacol. 2008, 51, 288-94. 23. Sorgentini, D. A.; Wagner, J. R.; Cristina Anon, M. Effects of thermal treatment of soy protein isolates on the characteristics and structure- function relationship of soluble and insoluble fractions. J. Agric. Food Chem. 1995, 43, 2471-2479. 24. Cabanillas, B.; Pedrosa, M. M.; Rodriguez, J.; Gonzalez, A.; Muzquiz, M.; Cuadrado, C.; Crespo, J. F.; Burbano, C. Effects of enzymatic hydrolysis on lentil allergenicity. Mol. Nutr. Food Res. 2010, 54, 1266-1272. 25. Hasim, S.; Tati, S.; Madayiputhiya, N; Nandakumar R.; Nickerson, K. W. Histone biotinylation in Candida albicans. FEMS Yeast Res. 2013, 13, 529-539.

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26. Vogel, L.; Luttkopf, D.; Hatahet, L.; Haustein, D.; Vieths, S. Development of a functional in vitro assay as a novel tool for the standardization of allergen extracts in the human system. Allergy 2005, 60, 1021-1028. 27. Clemente, A.; Vioque, J.; Sanchez-Vioque, R.; Pedroche, J.; Millan, F. Production of extensive chickpea (Cicer arietinum L.) protein hydrolysates with reduced antigenic activity. J. Agric. Food Chem. 1999, 47, 3776-3781. 28. Szymkiewicz, A.; Jedrychowski, L. Reduction of immunoreactive properties of pean globulins as the result of enzymatic modification. Acta. Alimentaria 2005, 34, 295-306. 29. Wigotzki, M.; Schubert, S.; Steinhart, H.; Paschke, H. Effects of in vitro digestion on the IgE-binding activity of proteins from Hazelnuts. Internet Symposium on Food Allergens 2000, 2, (1), 1-8. 30. Kasera, R.; Singh, A.B.; Lavasa, S.; Prasad, K.N.; Arora, N. Enzymatic hydrolysis: a method in alleviating legume allergenicity. Food. Chem. Toxicol. 2015, 76, 54-60. 31. Cabanillas, B.; Pedrosa, M.M.; Rodriguez, J.; Muzquiz, M.; Maleki, S.J.; Cuadrado, C.; Burbano, C.; Crespo, J.F. Influence of enzymatic hydrolysis on the allergenicity of roasted peanut protein extract. Int. Arch. Allergy Immunol. 2012, 157, 41-50. 32. Shi, X.; Guo, R.; White, B.L.; Yancey, A.; Sanders, T.H.; Davis, J.P.; Burks, A.W.; Kulis, M. Allergenic properties of enzymatically hydrolyzed peanut flour extracts. Int. Arch. Allergy Immunol. 2013, 162, 123-130. 33. Panda, R.; Ariyarathna, H.; Tetteh, A.; Pramod, S.N.; Taylor, S.L.; Ballmer-Weber, B. K.; Goodman, R. E. Challenges in testing genetically modified crops for potential increases in endogenous allergen expression for safety. Allergy 2013, 68, 142-151. 34. Hamilton, R.G.; Franklin Adkinson, N. Jr. In vitro assays for the diagnosis of IgE-mediated disorders. J. Allergy Clin. Immunol. 2004, 114, 213-225. 35. Kleine Budde, I.; de Heer, P.G.; van der Zee, J.S.; Aalberse, R.C. The stripped basophil histamine release bioassay as a tool for the detection of allergen-specific IgE in serum. Int. Arch. Allergy Immunol. 2001, 126, 277-285. 36. Dibbern, D.A., Jr.; Palmer, G.W.; Williams, P.B.; Bock, S.A.; Dreskin, S.C. RBL cells expressing human Fc epsilon RI are a sensitive tool for exploring functional IgE-allergen interactions: studies with sera from peanut-sensitive patients. J. Immunol. Methods 2003, 274, 37-45.

37. Zhou, Y.-H.; Chen, Z.; Purcell, R. H.; Emerson, S. U. Positive reactions on western blots do not necessarily indicate the epitopes on antigens are continuous. Immunl. Cell Biol. 2007, 85, 73-78. 38. Huang, L.; Mills, E. N. C.; Carter, J. M.; Morgan, M. R. A. Analysis of thermal stability of soya globulins using monoclonal antibodies. Biochim. Biophys. Acta 1998. 1388, 215226. 39. L’Hocine, L.; Boye, J.; Jouve, S. Ionic strength and pH-induced changes in the immunoreactivity of purified soybean glycinin and its relation to protein molecular structure. J. Agric. Food Chem. 2007, 55, 5819-5826. 40. Adachi, M.; Kanamori, J.; Masuda, T.; Yagasaki, K; Kitamura, K.; Mikami, B.; Utsumi, S. Ctrystal structure of soybean 11s globulin: glycinin A3B4 homohexamer. Proc. Natl. Acad. Sci. USA 2003, 100, 7395-7400. 41. Jin, T.; Guo, F; Chen, Y.-W.; Howard, A; Zhang, Y.-Z. Crystal structure of Ara h 3, a major allergen in peanut. Mol. Immunol. 2009, 46, 1796-1804.

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567

FIGURE CAPTIONS

568

Figure 1. SDS-PAGE stained gels of SPI hydrolyzed with Alcalase, trypsin, chymotrypsin,

569

bromelain or papain. Samples (10 µg/lane) were run under both reduced and non-reduced

570

conditions. Lane information for bromelain, Alcalase and papain treated samples: Lane 1-

571

Enzyme only control, 2- Unheated SPI control, 3- Heated SPI control, 4-7- SPI treated with

572

enzymes for 5, 15, 30 and 60 minutes respectively. Lane information for trypsin or chymotrypsin

573

hydrolyzed samples: Lane 1- Trypsin only control, 2- Chymotrypsin only control, 3- Unheated

574

SPI control, 4- Heated SPI control, 5-8- SPI treated with Tryspin for 5, 15, 30 and 60 minutes

575

respectively; 9-12- SPI treated with chymotrypsin for 5, 15, 30 and 60 minutes respectively.

576

Figure 2A. IgE immunoblots of SPI treated with Alcalase, trypsin, chymotrypsin, bromelain or

577

papain using serum 19392-CS. Samples (10 µg pre-digested protein) were hydrolyzed with

578

respective enzymes, then separated by SDS-PAGE in both non-reduced and reduced gels prior to

579

transfer onto PVDF membranes.. Lane information for bromelain, Alcalase and papain treated

580

samples: Lane 1- Enzyme only control, 2- Unheated SPI control, 3- Heated SPI control, 4-6- SPI

581

treated with enzymes for 5, 30 and 60 minutes respectively. Lane information for trypsin or

582

chymotrypsin hydrolyzed samples: Lane 1- Trypsin only control, 2- Chymotrypsin only control,

583

3- Unheated SPI control, 4- Heated SPI control, 5-8- SPI treated with Trypsin for 5, 15, 30 and

584

60 minutes respectively, 9-12- SPI treated with chymotrypsin for 5, 15, 30 and 60 minutes

585

respectively.

586

Figure 2B. IgE immunoblots of SPI treated with Alcalase, trypsin, chymotrypsin, bromelain or

587

papain using serum 20431. Samples (10 µg pre-treatment protein) were treated as indicated 5,

588

15, 30 or 60 minutes , then separated by SDS-PAGE in both non-reduced and reduced gels prior

589

to transfer onto PVDF membranes. Lanes were as in Figure 2A.

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590

Figure 2C. IgE immunoblots of SPI treated with Alcalase, trypsin, chymotrypsin, bromelain or

591

papain using serum 20770-MH. Samples were treated for 5, 20 or 60 min with protease as

592

indicated for each lane, then separated by SDS-PAGE in both non-reduced and reduced gels

593

prior to transfer onto PVDF membranes. Lanes were as in Figure 2A.

594

Figure 3. β-hexosaminidase release from humanized RBL-703/21 cells sensitized with IgE from

595

serum 19392-CS and challenged withhydrolyzed or control SPI samples. The percent release is

596

relative to maximum release caused by cross-linking IgE of the serum 19392-CS with anti-IgE.

597

Cells of humanized RBL-clone 703/21 were sensitized with IgE from serum 19392-CS (diluted

598

1: 10) and were challenged with 100 µl of hydrolyzed and control SPI samples Representing

599

original protein concentrations of 0.001 µg/ml to 10 µg/ml of antigen. Absrobance values were

600

measure at 405 nm and β-hexosaminidase release was expressed as percentage of total serum IgE

601

release (cells sensitized with serum and challenged with anti-IgE). Each data point represents the

602

mean of three assays done on separate days with separate batches of cells.

603

Figure 4A. Two dimensional gel electrophoresis of heated control SPI and SPI treated with

604

Alcalase, trypsin or chymotrypsin then stained with Brilliant Blue G Colloidal. Samples

605

representing 25 µg of protein from the original undigested extracts were separated Gels were

606

fixed, stained and images were captured under white light illumination.

607

Figure 4B. Immunoblot of heated control SPI, Alcalase, trypsin or chymotrypsin treated SPI

608

separated by two dimensional gel electrophoresis using serum 19392-CS. Samples representing

609

25 µg of protein from the original undigested extracts were separated. A 1:10 diluted sample of

610

serum was incubated with each membrane and bound IgE was detected using monoclonal anti-

611

human IgE-HRP conjugate with detection by ECL.

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TABLES Table 1. Soybean allergic and control human serum samples from PlasmaLab International, Everett, WA were used to analyze the hydrolyzed soybean proteins.

Serum

Reported food allergies

18534-LN

Nuts, beans and seeds; no symptoms specified Anaphylaxis to peanut; soybean, causes sore throat, itchy mouth, queasy stomach Itchy throat with nuts and raw veggies Angioedema, vomit, EOS G; milk, egg, meat, fruit, peaches, pears Throat swelling with peanut Peas, peanut, soy, lentil, sulfur drugs, garbanzo beans; anaphylactic shock from peanut, eczema, hives

9735-RE

20197-BH 19392-CS

20770-MH 24033/20431

23736all trees, grass, peanuts, cats, AM/20300 rabbits 20247-LA/20160 buckwheat, rice, rye, celery, lettuce, orange, crab, parsley, tomato, almond, coconut, peanut, pecan, sesame, corn, pea, white bean, carrot, potato, wheat, oat, soybean Control serum No known allergies (RP)

Soybean-specific PeanutIgE specific IgE (ImmunoCAP) (ImmunoCAP) 17.30 NA 5

58

3

95

68

15

38 NA

43 NA

15.3

>100

14.9

15.6

NA

NA

EOS G= eosinophilic gastroenteritis NA= not analyzed

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Table 2. Hydrolysis conditions of SPI with different enzymes Enzyme

Concentration

Hydrolysis condition

Alcalase® (Protease from Bacillus licheniformis, Sigma # P4860), Activity ≥ 2.4 Anson Units (U)/g

0.2 U of enzyme/gm of SPI

50oC and pH 8.00

Papain from papaya latex (Sigma 4 parts of SPI mixed with 1 part # P4762). Activity ≥ 10 units/mg of 0.168 mg/ml of enzyme solid). One unit will hydrolyze solution 1.0 micromole of N-AlphaBenzoyl-L-Arginine ethyl ester (BAEE) per min at pH 6.2 at 25 °C.

40oC and pH 8.00

Bromelain from pineaapple stem (Sigma # B4882), Activity= 37 units/mg protein. One unit will release 1.0 micromole of PNitrophenol from N-AlphaCBZ-L-Lysine P-Nitrophenyl Ester per min at pH 4.6 at 25 °C.

4 parts of SPI mixed with 1 part of 0.168 mg/ml of enzyme solution

40oC and pH 8.00

Trypsin (Sigma, Trypsin from bovine pancreas # T8003), Activity ~10,000 BAEE units/mg protein. One BAEE unit will produce a delta A253 of 0.001 per min in a reaction volume of 3.2 mL at pH 7.6 at 25 °C (1 CM light path).

2 mg of enzyme per 100 mg of SPI

37oC and pH 8.00

Chymotrypsin (Sigma, αchymotrypsin from bovine pancreas # C4129), Activity ≥40 units/mg protein. One unit will hydrolyze 1.0 µmole of benzoyl-L-tyrosine ethyl ester (BTEE) per min at pH 7.8 at 25 °C.

2 mg of enzyme per 100 mg of SPI

37oC and pH 8.00

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FIGURES Figure 1 Bromelain Reduced

Non-Reduced

Reduced 1 2 3 4 5 6 7

Trypsin/Chymotrypsin

1 2 3 4 5 6 7

M

M

15

25 20 15

25 20 15

10 5 2

10 5 2

10 5 2

25 20 15 10 5 2

25 20

50 37

Papain Reduced

Non-Reduced M

1

1 2 3 4 5 6

2 3 4 5 6 7

Non-Reduced M

250 150 100 75

250 150 100 75

50

50 37

37

M 250 150 100 75 50 37

250 150 100 75

Reduced

1 2 3 4 5 6 7 8 9 1011 12

M 250 150 100 75 50 37

250 150 100 75 50 37

Alcalase 1 2 3 4 5 6

Non-Reduced

1 2 3 4 5 6 7 8 9 10 11 12

25 20

1

2 3 4 5 6 7

M 250 150 100 75 50 37

25 20 15

15

25 20 15

10 5 2

10 5 2

10 5 2

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Figure 2A. Trypsin/Chymotrypsin

Bromelain Non-reduced

Reduced 1 2 3 4 5 6

102

Reduced

101

1 2 3 4 5 6

1

250 150 100 75 50 37

250 150 100 75 50 37

25 20 15 10 5

25 20 15 10 5

10-1

250 150 100 75 50 37

102

10-2 10-3

102

101

101

250 150 100 75 50 37

250 150 100 75 50 37

25 20 15 10 5

25 20 15 10 5

10-1

10-1

1

Reduced

1 2 3 4 5 6

1

250 150 100 75 50 37

10-2 10-3

25 20 15 10 5

102

101

Papain

Non-reduced

Reduced

1 2 3 4 5 6 7 8 9 10 11 12

25 20 15 10 5

Alcalase 1 2 3 4 5 6

Non-reduced

1 2 3 4 5 6 7 8 9 10 11 12

10-2 10-3

Non-reduced

1 2 3 4 5 6

102

101

1 2 3 4 5 6 250 150 100 75 50 37

250 150 100 75 50 37

25 20 15 10 5

25 20 15 10 5

1

10-1

10-2 10-3

ng IgE/spot

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10-1

10-2 10-3 ng IgE/spot

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Figure 2B Trypsin/Chymotrypsin

Bromelain Reduced

101

250 150 100 75 50 37

250 150 100 75 50 37

250 150 100 75 50 37

25 20 15 10 5

25 20 15 10 5

25 20 15 10 5

10-1

1

102

10-2 10-3

101

Non-reduced

1 2 3 4 5 6

1 2 3 4 5 6

Reduced

250 150 100 75 50 37

250 150 100 75 50 37

25 20 15 10 5

25 20 15 10 5

1

10-1

10-2 10-3

250 150 100 75 50 37

102

101

Papain

Reduced

101

10-1

1

Alcalase

102

1 2 3 4 5 6 7 8 9 10 1112

1 2 3 4 5 6 7 8 9 10 11 12

1 2 3 4 5 6

1 2 3 4 5 6

102

Non-reduced

Non-reduced

Reduced

10-2 10-3

Non-reduced 1 2 3 4 5 6

1 2 3 4 5 6

102

101

250 150 100 75 50 37

250 150 100 75 50 37

25 20 15 10 5

25 20 15 10 5

1

10-1

10-2 10-3 ng IgE/spot

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10-1

10-2

25 20 15 10 5 10-3 ng IgE/spot

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Figure 2C Trypsin/Chymotrypsin

Bromelain Reduced Reduced

Non-reduced

1 2 3 4 5 6

102

101

250 150 100 75 50 37

1 2 3 4 5 6 250 150 100 75 50 37

250 150 100 75 50 37

25 20 15 10 5

25 20 15 10 5

10-1

1

10-2 10-3

102

101

101

10-1

1

250 150 100 75 50 37

25 20 15 10 5

25 20 15 10 5 10-1

10-2 10-3

Reduced

1 2 3 4 5 6 250 150 100 75 50 37

1

250 150 100 75 50 37 25 20 15 10 5 102

101

1

Papain

Non-reduced

Reduced 1 2 3 4 5 6

1 2 3 4 5 6 7 8 9 10 1112

25 20 15 10 5

Alcalase

102

Non-reduced

1 2 3 4 5 6 7 8 9 10 11 12

10-2 10-3

Non-reduced

1 2 3 4 5 6

102

101

1 2 3 4 5 6 250 150 100 75 50 37

250 150 100 75 50 37

25 20 15 10 5

25 20 15 10 5

1

10-1

10-2 10-3 ng IgE/spot

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10-2 10-3 ng IgE/spot

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Figure 3

β-hexosaminidase % release of +ve control

100

60 40 20 0 0.001 -20

100

β-hexosaminidase % release of +ve control

SPI treated with bromelain

0.01

0.1

1

10

80 60 40 20 0 0.001 -20

60 40 20 0 0.001 -20

60

SPI treated with chymotrypsin

0.01

0.1

1

SPI treated with trypsin

80

Antigen concentration (µg/ml)

β-hexosaminidase % release of +ve control

β-hexosaminidase % release of +ve control

80

0.01

0.1

1

10

Antigen concentration (µg/ml)

SPI treated with Alcalase

40

20

0 0.001

0.01

0.1

1

10 -20

Antigen concentration (µg/ml)

Antigen concentration (µg/ml)

β-hexosaminidase % release of +ve control

70

SPI treated with papain

Unheated control

50

Heated control 5 min

30

30 min 10

60 min 0.001 -10

0.01

0.1

1

10

Antigen concentration (µg/ml)

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Figure 4A Trypsin treated for 60 minutes

Heated control SPI PI = 3

PI = 10

8

PI = 3

PI = 10

MW (kDa) 250 150 100 75

7 10

MW (kDa) 250 150 100 75

50 37 4 5

50 1

6

25 20 15

37 45

6

25 20 15 10

10 5 2

Chymotrypsin treated for 60 minutes

Alcalase treated for 60 minutes PI = 3

5 2

PI = 3

PI = 10

PI = 10 MW (kDa) 250 150 100 75

MW (kDa) 250 150 100 75 50 37

10

50 37 2

3

25 20 15

25 20 15

10 5 2

10 5 2

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Figure 4B

Trypsin treated for 60 minutes

Heated control SPI PI = 3

7 8

10

6

4, 5

102

PI = 10 MW (kDa) 250 150 100 75 50 37 25 20 15 10 5

101

1

10-1

10-2

10-3

PI = 10 MW (kDa) 250 150 100 75

1

102

PI = 10 MW (kDa) 250 150 100 75

101

102

101

10-1

10-2

10-3

10-1

10-2

10-3

PI = 10 MW (kDa) 250 150 100 75 10

2

25 20 15 10 5 1

1

6

PI = 3

50 37 9

4, 5

50 37 25 20 15 10 5 ng IgE/spot

Chymotrypsin treated for 60 minutes

Alcalase treated for 60 minutes PI = 3

PI = 3

102

101

50 37

3

25 20 15

1

10-1

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10-3

10 5 ng IgE/spot

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Supplementary Figure S1 Spot 1 (β-conglycinin α subunit) GI:15425633 MRARFPLLLL KVEKEEECEE REEQEWPRKE QEEDEDEEQQ RSPQLQNLRD DALRVPSGTT EASYDTKFEE DKPFNLRSRD LVINEGDANI NFFAIGINAE PKKKEEGNKG

GVVFLASVSV GEIPRPRPRP EKRGEKGSEE RESEESESSE YRILEFNSKP YYVVNPDNNE INKVLFSREE PIYSNKLGKF ELVGLKEQQQ NNQRNFLAGS RKGPLSSILR

SFGIAYWEKQ QHPEREPQQP EQDGREHPRP SQRELRRHKN NTLLLPNHAD NLRLITLAIP GQQQGEQRLQ FEITPEKNPQ EEQQEEQPLE QDNVISQIPS AFY

NPKHNKCLQS GEKEEDEDEQ HQPHDEDEEQ KNPFHFGSNR ADYLIAILNG VNKPGRFESF ESVIVEISKE LRDLDIFLSI VRKYRAELSE QVQELAFLGS

CNSERDSYRN PRPIPFPRPR DERQFPFPRP FETLFKNQYG TAILSLVNND FLSSTEAQQS QIRALSKRAK VDMNEGALLL QDIFVIPAGY AQAVEKLLKN

QACHARCNLL QPRQEEEHEQ PHQKESEERK RIRVLQRFNQ DRDSYRLQSG YLQGFSRNIL SSSRKTISSE PHFNSKAIVI PVVVNATSNL QRESYFVDAQ

PDNRIESEGG GMIYPGCPST VAVSIIDTNS LSGFTLEFLE EEEEDEKPQC VTTATSLDFP VFDGELQEGR QHTFNLKSQQ

LIETWNPNNK FEEPQQPQQR LENQLDQMPR HAFSVDKQIA KGKDKHCQRP ALSWLRLSAE VLIVPQNFVV ARQIKNNNPF

PDNRIESEGG GMIYPGCPST VAVSIIDTNS LSGFTLEFLE EEEEDEKPQC VTTATSLDFP VFDGELQEGR QHTFNLKSQQ

LIETWNPNNK FEEPQQPQQR LENQLDQMPR HAFSVDKQIA KGKDKHCQRP ALSWLRLSAE VLIVPQNFVV ARQIKNNNPF

PDNRIESEGG GMIYPGCPST VAVSIIDTNS LSGFTLEFLE EEEEDEKPQC VTTATSLDFP VFDGELQEGR QHTFNLKSQQ

LIETWNPNNK FEEPQQPQQR LENQLDQMPR HAFSVDKQIA KGKDKHCQRP ALSWLRLSAE VLIVPQNFVV ARQIKNNNPF

Spot 4 (Glycinin G1 protein) GI:255221 MAKLVFSLCF PFQCAGVALS GQSSRPQDRH RFYLAGNQEQ KNLQGENEGE RGSQSKSRRN FGSLRKNAMF AARSQSDNFE KFLVPPQESQ

LLFSGCCFAF RCTLNRNALR QKIYNFREGD EFLKYQQEQG DKGAIVTVKG GIDETICTMR VPHYNLNANS YVSFKTNDTP KRAVA

SSREQPQQNE RPSYTNGPQE LIAVPTGVAW GHQSQKGKHQ GLSVIKPPTD LRHNIGQTSS IIYALNGRAL MIGTLAGANS

CQIQKLNALK IYIQQGKGIF WMYNNEDTPV QEEENEGGSI EQQQRPQEEE PDIYNPQAGS IQVVNCNGER LLNALPEEVI

Spot 5 (Glycinin G1 protein) GI:255221 MAKLVFSLCF PFQCAGVALS GQSSRPQDRH RFYLAGNQEQ KNLQGENEGE RGSQSKSRRN FGSLRKNAMF AARSQSDNFE KFLVPPQESQ

LLFSGCCFAF RCTLNRNALR QKIYNFREGD EFLKYQQEQG DKGAIVTVKG GIDETICTMR VPHYNLNANS YVSFKTNDTP KRAVA

SSREQPQQNE RPSYTNGPQE LIAVPTGVAW GHQSQKGKHQ GLSVIKPPTD LRHNIGQTSS IIYALNGRAL MIGTLAGANS

CQIQKLNALK IYIQQGKGIF WMYNNEDTPV QEEENEGGSI EQQQRPQEEE PDIYNPQAGS IQVVNCNGER LLNALPEEVI

Spot 6 (Glycinin G1 protein) GI:255221 MAKLVFSLCF PFQCAGVALS GQSSRPQDRH RFYLAGNQEQ KNLQGENEGE RGSQSKSRRN FGSLRKNAMF AARSQSDNFE KFLVPPQESQ

LLFSGCCFAF RCTLNRNALR QKIYNFREGD EFLKYQQEQG DKGAIVTVKG GIDETICTMR VPHYNLNANS YVSFKTNDTP KRAVA

SSREQPQQNE RPSYTNGPQE LIAVPTGVAW GHQSQKGKHQ GLSVIKPPTD LRHNIGQTSS IIYALNGRAL MIGTLAGANS

CQIQKLNALK IYIQQGKGIF WMYNNEDTPV QEEENEGGSI EQQQRPQEEE PDIYNPQAGS IQVVNCNGER LLNALPEEVI

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Supplementary Figure S2

Spot 2 (β-conglycinin α'subunit) GI:341603991 MMRARFPLLL LKVEEEEECE EEHEWHRKEE QDEDEEQDKE QQLQNLRDYR LRVPAGTTYY SYDTKFEEIN PFNLRSRDPI INEGEANIEL FAFGINAENN QKEEGNKGRK

LGVVFLASVS EGQIPRPRPQ KHGGKGSEEE SQESEGSESQ ILEFNSKPNT VVNPDNDENL KVLFGREEGQ YSNKLGKLFE VGIKEQQQRQ QRNFLAGSKD GPLSSILRAF

VSFGIAYWEK HPERERQQHG QDEREHPRPH REPRRHKNKN LLLPHHADAD RMITLAIPVN QQGEERLQES ITPEKNPQLR QQEEQPLEVR NVISQIPSQV Y

QNPSHNKCLR EKEEDEGEQP QPHQKEEEKH PFHFNSKRFQ YLIVILNGTA KPGRFESFFL VIVEISKKQI DLDVFLSVVD KYRAELSEQD QELAFPGSAK

SCNSEKDSYR RPFPFPRPRQ EWQHKQEKHQ TLFKNQYGHV ILTLVNNDDR SSTQAQQSYL RELSKHAKSS MNEGALFLPH IFVIPAGYPV DIENLIKSQS

NQACHARCNL PHQEEEHEQK GKESEEEEED RVLQRFNKRS DSYNLQSGDA QGFSKNILEA SRKTISSEDK FNSKAIVVLV VVNATSDLNF ESYFVDAQPQ

CNSERDSYRN PRPIPFPRPR DERQFPFPRP FETLFKNQYG TAILSLVNND FLSSTEAQQS QIRALSKRAK VDMNEGALLL QDIFVIPAGY AQAVEKLLKN

QACHARCNLL QPRQEEEHEQ PHQKESEERK RIRVLQRFNQ DRDSYRLQSG YLQGFSRNIL SSSRKTISSE PHFNSKAIVI PVVVNATSNL QRESYFVDAQ

Spot 3 (β-conglycinin α subunit) GI:15425633 MRARFPLLLL KVEKEEECEE REEQEWPRKE QEEDEDEEQQ RSPQLQNLRD DALRVPSGTT EASYDTKFEE DKPFNLRSRD LVINEGDANI NFFAIGINAE PKKKEEGNKG

GVVFLASVSV GEIPRPRPRP EKRGEKGSEE RESEESESSE YRILEFNSKP YYVVNPDNNE INKVLFSREE PIYSNKLGKF ELVGLKEQQQ NNQRNFLAGS RKGPLSSILR

SFGIAYWEKQ QHPEREPQQP EQDGREHPRP SQRELRRHKN NTLLLPNHAD NLRLITLAIP GQQQGEQRLQ FEITPEKNPQ EEQQEEQPLE QDNVISQIPS AFY

NPKHNKCLQS GEKEEDEDEQ HQPHDEDEEQ KNPFHFGSNR ADYLIAILNG VNKPGRFESF ESVIVEISKE LRDLDIFLSI VRKYRAELSE QVQELAFLGS

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Supplementary Figure S3

Spot 7 (β-conglycinin α'subunit) GI:341603991 MMRARFPLLL LKVEEEEECE EEHEWHRKEE QDEDEEQDKE QQLQNLRDYR LRVPAGTTYY SYDTKFEEIN PFNLRSRDPI INEGEANIEL FAFGINAENN QKEEGNKGRK

LGVVFLASVS EGQIPRPRPQ KHGGKGSEEE SQESEGSESQ ILEFNSKPNT VVNPDNDENL KVLFGREEGQ YSNKLGKLFE VGIKEQQQRQ QRNFLAGSKD GPLSSILRAF

VSFGIAYWEK HPERERQQHG QDEREHPRPH REPRRHKNKN LLLPHHADAD RMITLAIPVN QQGEERLQES ITPEKNPQLR QQEEQPLEVR NVISQIPSQV Y

QNPSHNKCLR EKEEDEGEQP QPHQKEEEKH PFHFNSKRFQ YLIVILNGTA KPGRFESFFL VIVEISKKQI DLDVFLSVVD KYRAELSEQD QELAFPGSAK

SCNSEKDSYR RPFPFPRPRQ EWQHKQEKHQ TLFKNQYGHV ILTLVNNDDR SSTQAQQSYL RELSKHAKSS MNEGALFLPH IFVIPAGYPV DIENLIKSQS

NQACHARCNL PHQEEEHEQK GKESEEEEED RVLQRFNKRS DSYNLQSGDA QGFSKNILEA SRKTISSEDK FNSKAIVVLV VVNATSDLNF ESYFVDAQPQ

CNSERDSYRN PRPIPFPRPR DERQFPFPRP FETLFKNQYG TAILSLVNND FLSSTEAQQS QIRALSKRAK VDMNEGALLL QDIFVIPAGY AQAVEKLLKN

QACHARCNLL QPRQEEEHEQ PHQKESEERK RIRVLQRFNQ DRDSYRLQSG YLQGFSRNIL SSSRKTISSE PHFNSKAIVI PVVVNATSNL QRESYFVDAQ

Spot 8 (β-conglycinin α subunit) GI:15425633 MRARFPLLLL KVEKEEECEE REEQEWPRKE QEEDEDEEQQ RSPQLQNLRD DALRVPSGTT EASYDTKFEE DKPFNLRSRD LVINEGDANI NFFAIGINAE PKKKEEGNKG

GVVFLASVSV GEIPRPRPRP EKRGEKGSEE RESEESESSE YRILEFNSKP YYVVNPDNNE INKVLFSREE PIYSNKLGKF ELVGLKEQQQ NNQRNFLAGS RKGPLSSILR

SFGIAYWEKQ QHPEREPQQP EQDGREHPRP SQRELRRHKN NTLLLPNHAD NLRLITLAIP GQQQGEQRLQ FEITPEKNPQ EEQQEEQPLE QDNVISQIPS AFY

NPKHNKCLQS GEKEEDEDEQ HQPHDEDEEQ KNPFHFGSNR ADYLIAILNG VNKPGRFESF ESVIVEISKE LRDLDIFLSI VRKYRAELSE QVQELAFLGS

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