Improved Quantitation of Gluten in Wheat Starch for Celiac Disease

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Article

Improved quantitation of gluten in wheat starch for celiac disease patients by gel-permeation high-performance liquid chromatography with fluorescence detection (GP-HPLC-FLD) Katharina Anne Scherf, Herbert Wieser, and Peter Koehler J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02512 • Publication Date (Web): 16 Sep 2016 Downloaded from http://pubs.acs.org on September 20, 2016

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Journal of Agricultural and Food Chemistry

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Improved quantitation of gluten in wheat starch for celiac disease

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patients by gel-permeation high-performance liquid chromatography

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with fluorescence detection (GP-HPLC-FLD)

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Katharina Anne Scherf*, Herbert Wieser and Peter Koehler

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Deutsche Forschungsanstalt für Lebensmittelchemie, Leibniz Institut, Lise-Meitner-Straße 34,

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85354 Freising, Germany

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*Corresponding author

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phone +49 8161 712927; fax +49 8161 712970; e-mail: [email protected]

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1 Environment ACS Paragon Plus


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Abstract

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Purified wheat starch (WSt) is commonly used in gluten-free products for celiac disease (CD)

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patients. It is mostly well-tolerated, but doubts about its safety for CD patients persist. One

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reason may be that most ELISA kits primarily recognize the alcohol-soluble gliadin fraction of

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gluten, but insufficiently target the alcohol-insoluble glutenin fraction. To address this problem,

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a new sensitive method based on the sequential extraction of gliadins, glutenins and gluten

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from WSt followed by gel-permeation high-performance liquid chromatography with

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fluorescence detection (GP-HPLC-FLD) was developed. It revealed that considerable amounts of

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glutenins were present in most WSt. The gluten contents quantitated by GP-HPLC-FLD as sum

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of gliadins and glutenins were higher than those by R5 ELISA (gluten as gliadin content

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multiplied by a factor of 2) in 19 out of 26 WSt. Despite its limited selectivity, GP-HPLC-FLD may

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be applied as confirmatory method to ELISA to quantitate gluten in WSt.

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Keywords

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Celiac disease; gliadin; gluten analysis; glutenin; enzyme-linked immunosorbent assay (ELISA);

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gel-permeation HPLC fluorescence detection; wheat starch


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INTRODUCTION

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Gluten is a complex mixture of storage proteins found in the starchy endosperm of wheat, rye,

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barley and oats grains. Traditionally, gluten proteins may be separated into two fractions, the

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monomeric prolamins (called gliadins in wheat) soluble in aqueous alcohols and the polymeric

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glutelins (called glutenins in wheat) insoluble in aqueous alcohols, that are linked by

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intermolecular disulfide bonds and can only be solubilized with reducing and disaggregating

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agents.1 Wheat gliadin and glutenin fractions may be further subdivided into the gluten protein

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types ω5-, ω1,2-, α- and γ-gliadins as well as ωb-gliadins and high- and low-molecular weight

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glutenin subunits (HMW- and LMW-GS) that share similar amino acid sequences and molecular

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weights.2 One characteristic feature of gluten proteins is their exceptionally high content of

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glutamine (26-53 mol-%) and proline (10-29 mol-%).3 This makes them resistant to

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gastrointestinal enzymes, so that large peptides may reach the small intestine where they may

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initiate the dysregulated immune response in genetically predisposed persons known as celiac

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disease (CD).4 Once triggered, the essential treatment is a lifelong gluten-free (GF) diet with a

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maximal daily ingestion of 20 mg gluten.5 CD affects about 1% of the population,6 but wheat-

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allergic patients (estimated prevalence ≈ 1%)7 and a growing number of people suffering from

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non-celiac gluten sensitivity (estimated prevalence 0.6-6%)8 also adopt a GF diet. In order to

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comply with the labeling requirements set by Codex Alimentarius Standard 118-1979,9 GF

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dietary products must not contain more than 20 mg gluten per kg of the food. Enzyme-linked

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immunosorbent assays (ELISAs) are the only validated methods to determine the gluten

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content of supposedly GF products and are most commonly used in compliance testing.10,11

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Much research is going on to develop alternatives for gluten determination based on

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biosensors12 or liquid chromatography-mass spectrometry (LC-MS),13-16 but none of these

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methods has been validated so far. Gluten detection by ELISAs faces some challenges, because



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the results for the same product analyzed with different test kits showed systematic deviations

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depending on the specific characteristics (e.g., extraction procedure, reference material,

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antibody specificity) of each kit.11,17-21 Another important aspect is that most antibodies mainly

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target prolamins,20 although glutelins also harbor CD-active peptides22 and HMW-GS were

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reported to stimulate CD in vivo.23 The duplication of the prolamin content determined by

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ELISA tends to result in an overestimation of gluten contents, because gliadin-to-glutenin

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(gli/glu) ratios are typically not 1, but 1.5-3.1 in wheat flours.24 However, this duplication was

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shown to be inaccurate in wheat starches (WSt), because gli/glu ratios were down to 0.3,

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leading to a potential underestimation of gluten contents.25 The limited ability of antibodies to

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detect glutenins thus results in a considerable measurement uncertainty regarding gluten

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contents of WSt determined by ELISA, which contributes to the controversy concerning the

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safety of WSt as part of a GF diet, especially in the USA and Canada.26,27

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Because reversed-phase high-performance liquid chromatography with UV detection (RP-HPLC-

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UV) was not sensitive enough to detect gluten contents below 300 mg/kg,25 the present study

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aimed to use fluorescence detection (FLD) to increase the sensitivity of the HPLC method.

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HPLC-FLD without derivatization is based on detecting the UV-induced, intrinsic fluorescence of

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the aromatic side chains of the amino acids tryptophan, phenylalanine and tyrosine.28-30

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Intrinsic FLD of peptides/proteins was reported to be over 100-fold more sensitive compared to

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UV,31 offered good linearity and reproducibility32 and limits of detection (LOD) in the low

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femtomole range.33 Laser-induced fluorescence detection of peptides/proteins is also

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commonly used in capillary electrophoresis (CE-LIF).34 Furthermore, gluten proteins may be

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visualized in dough and bread by confocal laser scanning microscopy using their intrinsic

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fluorescence.35 However, to the best of our knowledge, HPLC-FLD has not been applied to

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gluten quantitation so far. Therefore, a new HPLC-FLD method was developed to improve the


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quantitation of gluten in WSt, with a special focus on detecting both gliadins and, especially,

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glutenins. Although not as specific and versatile as an LC-MS method, this HPLC-FLD method

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may be used by smaller laboratories that may be equipped with an HPLC, but not with an LC-

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MS instrument. In addition, the HPLC-FLD method directly yields quantitative values for gluten

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after appropriate calibration, whereas it is still difficult to calculate back to original gluten

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contents after quantitation of gluten-specific peptides by LC-MS.

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MATERIALS AND METHODS

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Materials. The 30 WSt samples (GfW1-14 declared as GF and W1-16 without specification

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regarding gluten content) investigated in this study were either purchased or kindly donated

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from the sources reported previously.36 Wheat gluten was provided by Sonneveld

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(Papendrecht, The Netherlands), wheat kernels (cultivar Akteur, 2013) were donated by I.G.

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Pflanzenzucht (Munich, Germany), α-chymotrypsin from bovine pancreas (TLCK treated, ≥ 40

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units/mg enzyme) was from Sigma-Aldrich (Steinheim, Germany) and the reference material

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PWG-gliadin37 was made available by Prof. Dr. Peter Koehler, Chairman of the Working Group

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on Prolamin Analysis and Toxicity. Water for chromatographic separations and ELISA

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measurements was purified using a Milli-Q Gradient A10 system (Millipore, Schwalbach,

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Germany). The sandwich ELISA test kit RIDASCREEN® Gliadin (R-Biopharm, Darmstadt,

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Germany) approved by the AOAC International38 and the AACC International39 was used for

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immunological gluten determination as recommended by the Codex Alimentarius.9 The

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RIDASCREEN® Gliadin competitive kit (R-Biopharm) was used for additional confirmation.

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Methods. Fluorescence spectra. PWG-gliadin (0.5 mg/ml) was dissolved in 60% ethanol (v/v)

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and vital gluten (0.5 mg/ml) was dissolved in 0.067 mol/l K2HPO4/KH2PO4-buffer (pH 7.6)/2-



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propanol (1+1; v/v) containing 10 mg/ml dithiothreitol under nitrogen (gluten extraction

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solution). Fluorescence excitation and emission spectra of these solutions were recorded on a

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fluorescence spectrophotometer (Cary Eclipse, Agilent Technologies, Waldbronn, Germany) to

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determine excitation and emission maxima.

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HPLC-FLD at 277/345 nm and comparison to diode-array detection (DAD) at 210 nm. A Merck-

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Hitachi LaChrom Elite HPLC system (Tokyo, Japan) was used for all analyses with an L-2130

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pump, an L-2200 autosampler, an L-2480 fluorescence detector, an L-2450 DAD detector and

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the software EZChrom Elite (version 3.1.1) for instrument control and data analysis. For RP-

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HPLC, gluten proteins were separated on an AcclaimTM 300 C18 column (particle size 3 µm, pore

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size 30 nm, 2.1 × 150 mm, ThermoFisher Scientific, Braunschweig, Germany) at 60 °C and a flow

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rate of 0.3 ml/min with trifluoroacetic acid (TFA) (0.1%, v/v) in water (A) and TFA (0.1%, v/v) in

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acetonitrile (B) with the following linear gradient: 0 min 24% B, 20 min 56% B, 21-26 min 90% B,

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27-37 min 24% B. Gliadin and glutenin extracts of wheat flour (cv. Akteur, 2013)25 were injected

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(10 µl) to study the qualitative protein profiles using RP-HPLC. For gel-permeation (GP-)HPLC,

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gluten proteins were separated on a Biosep-SEC-S3000 column (4.6 x 300 mm, Phenomenex,

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Aschaffenburg, Germany) with a separation range from 5,000 to 100,000 under isocratic

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conditions with acetonitrile/water (1+1, v/v) containing 0.1% TFA (v/v) at 22 °C and a flow rate

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of 0.3 ml/min.40 The injection volume was 10 µL. FLD was carried out with 277/345 nm as

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excitation/emission wavelengths (FLD277/345) followed by DAD detection in the range from 200

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to 300 nm. The same gliadin and glutenin extracts of wheat flour (cv. Akteur, 2013) were

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injected (10 µl) to study the qualitative protein pattern using GP-HPLC. Linear dilutions of PWG-

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gliadin in 60% (v/v) ethanol (0.25 - 50 µg/ml) and vital gluten in gluten extraction solution


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(0.25 - 50 µg/ml) were injected (10 µl) to compare the sensitivity of FLD277/345 and DAD

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detection at 210 nm (DAD210).41

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Identification of the proteins within the peak (retention time 6.5-12.8 min) of a reduced

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protein extract of GfW4 by LC-MS. GfW4 (32 g) was pre-extracted twice with 160 ml salt

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solution (0.4 mol/l NaCl with 0.067 mol/l Na2HPO4/KH2PO4, pH 7.6), the supernatant discarded

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and the residue extracted three times with 160 ml gluten extraction solution under nitrogen by

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magnetic stirring at 60 °C. The combined supernatant was lyophilized and the sediment (1.04 g)

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redissolved in 5 ml of gluten extraction solution at 60 °C (20 min). After filtration (WhatmanTM

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Spartan 13/0.45 RC, GE Healthcare, Freiburg, Germany), 90 µl of this solution were injected into

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the GP-HPLC-FLD system and the peak between 6.5-12.8 min corresponding to a molecular

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weight range of approximately 30,000 - 100,000 was collected from 30 subsequent runs. The

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eluate was dried in a vacuum centrifuge (40 °C, 6 h, 800 Pa). Three times 1 mg of the dried

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eluate (yield of eluate: 5 mg in total) was reconstituted in 800 µl Tris-HCl buffer (0.1 mol/l, pH

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7.8, with 2 mol urea/l) followed by the addition of 200 µl α-chymotrypsin (0.2 mg/ml in Tris-HCl

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buffer) and incubation at 37 °C for 24 h.40 The digestion was stopped with 3 µl TFA and the

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peptide mixture purified by solid-phase extraction on Strata-X-C devices (Phenomenex,

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Aschaffenburg, Germany) as reported by Rombouts et al.42 After evaporation, the peptide

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digest was dissolved in 500 µl formic acid (FA) (0.1%), filtered (0.45 µm) and analyzed by LC-MS

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using an UltiMate 3000 HPLC system (Dionex, Idstein, Germany) linked to an HCTultra PTM ion

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trap MS (Bruker Daltonics, Bremen, Germany) with collision-induced dissociation (CID). The

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peptides were separated on an Aeris 3.6 µm PEPTIDE XB-C18 column (pore size 10 nm, 2.1 × 150

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mm, Phenomenex) with FA (0.1%, v/v) in water (A) and FA (0.1%, v/v) in acetonitrile (B) as

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elution solvents, an injection volume of 10 µl, a flow rate of 0.2 ml/min, a column temperature



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of 30 °C and the following gradient: 0-5 min 0% B, 45 min 30% B, 55-60 min 90% B, 62-77 min

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0% B. The ESI interface was operated in the positive mode with capillary voltage -4000 V,

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capillary exit voltage -1500 V, skimmer voltage 40 V, and nitrogen as drying (8.0 l/min, 325 °C)

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and nebulizing gas (207 kPa). The scan was standard enhanced mode with range m/z 500 -

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3000, speed 8.1 m/z/s, smart target value 300,000, maximum acquisition time 100 ms. The

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MS/MS settings were Auto-MS(n), absolute threshold 10,000, relative threshold 0.5%,

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fragmentation amplitude 0.4 V and helium as collision gas.

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The acquired MS/MS data files were analyzed using the Bruker Daltonics Data Analysis 3.4 and

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BioTools 3.2 software (Bruker Daltonics) to generate a Mascot Generic File (*.mgf), which was

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used in the MS/MS ions search module of the Mascot software (Matrix Science, London, UK)

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based on the National Center for Biotechnology Information non-redundant (NCBInr) database

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(U.S. National Library of Medicine, Bethesda, MD, USA) of February 2014 with taxonomy

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Viridiplantae, peptide mass tolerance ± 0.5%, fragment mass tolerance ± 0.5, monoisotopic

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mass values, peptide charges +1, +2, +3, enzyme chymotrypsin, maximum number of missed

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cleavages 1 and ammonia-loss as variable modification. Based on the peptide mass fingerprints,

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peptide ion scores were calculated as -10 × log(P), with P being the probability that the

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observed match is a random event. Peptide scores > 40 indicated identity or extensive

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homology (p < 0.05)42 and scores between 15 and 40 were additionally validated manually

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according to Chen et al.43 Protein scores are derived from peptide ion scores as a non-

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probabilistic basis for ranking protein hits and are the sum of the highest ions score for each

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distinct sequence excluding duplicate and low-scoring random matches (for searches with a

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small number of queries). The sequence of each detected peptide was additionally entered into

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the BLAST tool (E-threshold 10, hits 1000) of the UniProtKB database (The UniProt Consortium)

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and the number of protein hits which contained the peptide with a sequence identity of 100%


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within Triticeae was counted. No adjustment for multiple occurrences of one peptide within

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one protein sequence was made. To assess possible CD-toxicity/-immunogenicity of the

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identified peptides, the peptide sequences were entered into the Peptide Exact Match tool of

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the AllergenOnline database (Food Allergy Research and Resource Program, University of

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Nebraska-Lincoln, Lincoln, NE, USA).

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Extraction of gliadin, glutenin and gluten from wheat starches. WSt (1 g) were pre-extracted

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twice with 5 ml salt solution by vortex mixing for 15 min (multi-tube vortexer VX 2500, VWR,

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Darmstadt, Germany) followed by magnetic stirring for 30 min at 22 °C to remove residual

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proteins soluble in dilute saline (albumins/globulins). The samples were centrifuged (3,550 g,

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25 min, 22 °C) and the supernatants discarded. Then the residue was extracted once with 5 ml

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60% ethanol by vortex mixing for 15 min followed by magnetic stirring for 30 min at 22°C and

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centrifugation to yield the gliadin extract. Subsequently, this residue was further extracted once

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with 5 ml gluten extraction solution under nitrogen by vortex mixing for 15 min followed by

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magnetic stirring for 30 min at 60 °C in a water bath and centrifugation to yield the glutenin

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extract. The gluten extract was obtained in a separate experiment after saline pre-extraction by

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omitting the gliadin extract and direct extraction of the albumin/globulin-free residue with 5 ml

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gluten extraction solution, as described for the glutenin extract. All supernatants were filtered

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(0.45 µm) and analyzed by GP-HPLC-FLD-DAD. Three sample extracts (W8, W11, W15) had to be

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diluted appropriately to fall within the calibration range. All WSt were analyzed in triplicate, i.e.

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three experiments for gliadin and glutenin extraction and another three experiments for gluten

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extraction. The peak area of the peak between 6.5-12.8 min was used for quantitation, because

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of its similar retention timeframe to the one determined with pure PWG-gliadin and vital gluten

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solutions as well as the confirmatory LC-MS results. Glutenin contents were calculated both



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from the glutenin extraction itself and from the difference between gluten and gliadin contents.

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One-way analysis of variance (ANOVA) with Tukey’s test (p < 0.05) was used to assess whether

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significant differences existed between both procedures (glutenin extraction vs. difference

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between gluten and gliadin) for the same sample (SigmaPlot 12.0, Systat Software, San Jose,

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CA, USA).

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Matrix calibration for GP-HPLC-FLD. To obtain a definitely GF matrix, GfW4 (800 g) was

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extracted five times with gluten extraction solution as described above. The starch residue

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(GfWgf) was lyophilized and checked for any residual gluten using the RIDASCREEN Gliadin and

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RIDASCREEN Gliadin competitive ELISAs and LC-MS as described above. The albumin/globulin,

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gliadin, and glutenin contents of wheat flour (cv. Akteur, 2013) were determined by sequential

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extraction and RP-HPLC-UV as described earlier.25 One gram of wheat flour contained 13.0 ± 0.4

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mg albumins/globulins, 75.9 ± 2.6 mg gliadins, and 37.2 ± 0.8 mg glutenins on an as-is basis.

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Then GfWgf (100 g) was spiked with the appropriate amount of wheat flour to obtain 1000 mg

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gliadin/kg and homogenized by shaking upside down for 24 h. This stock mixture was further

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diluted with GfWgf to obtain 10, 20, 35, 50, 100, and 200 mg gliadin/kg (GfWgf + 10/20/35/50/

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100/200), which corresponded to 16, 31, 55, 79, 157, and 315 mg gluten/kg (GfWgf +

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16/31/55/79/157/315). Homogeneity of the spiked sample (GfWgf + 50) was checked by

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analyzing 10 replicates from different parts of the container44 by GP-HPLC-FLD. The GfWgf

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matrix and the five spiked samples (GfWgf + 16/31/79/157/315) were analyzed in triplicates on

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the same day as described above for the wheat starch samples. The additional spiked sample

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(GfWgf + 55) was used to check the recovery of the method.

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Limits of detection and quantitation, precision and recovery for GP-HPLC-FLD. The signals of

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the gliadin, glutenin and gluten extracts of GfWgf plus 3 and 10 times their respective standard

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deviations (n = 10) were considered to be the limits of detection (LODs) and LOQs,

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respectively.44,45 Interassay precision was determined three times over the course of six weeks

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by triplicate analyses of two WSt samples (W7, W12) that contained gliadin, glutenin and gluten

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all above the determined LOQ. The recovery was calculated from the peak areas of an

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additional GfWgf sample spiked at a level of 55 mg gluten/kg using the matrix calibration and

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expressed as percentage of the spiking level.

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Sandwich R5 ELISA. The gluten contents obtained after GP-HPLC-FLD analysis were compared

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to those measured by RIDASCREEN Gliadin (R-Biopharm). All WSt samples were extracted with

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Cocktail (patented)46 strictly according to the manufacturer’s instructions and the following test

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procedure was also performed exactly as described. WSt were extracted in triplicates and each

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extract was applied into two cavities of the 96-well plate (n = 6). Additional dilutions of sample

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extracts were made, if necessary. The standard provided in the kit was used for calibration and

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the gliadin contents were multiplied by a factor of 2 to yield gluten contents.9 To see whether

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significant differences existed between gliadin or gluten contents determined by R5 ELISA vs.

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those by GP-HPLC-FLD within one WSt sample, one-way ANOVA with Tukey’s test (p < 0.05) was

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used. Pearson product moment correlations were calculated between gliadin and gluten

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contents quantitated by R5 ELISA and GP-HPLC-FLD (SigmaPlot 12.0).

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RESULTS AND DISCUSSION

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Excitation/emission maxima of gluten proteins. The fluorescence spectra recorded for the

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gluten solution indicated an excitation maximum at 277 nm. When the wavelength 277 nm was



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used for excitation, the corresponding emission maximum was at 345 nm (Figure 1). The same

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maxima were observed for PWG-gliadin solutions (not shown), so that the excitation/emission

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wavelengths 277/345 nm were used as optimal combination for the HPLC-FLD method. An

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excitation maximum around 280 nm is typical for proteins and due to both tyrosine (quantum

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yield 0.14) and tryptophan (quantum yield 0.13). Fluorescence originating from phenylalanine is

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rarely observed for proteins, because the excitation/emission maxima are at 260/295 nm and

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the quantum yield is low (0.03).28 Gluten proteins contain very low amounts of tryptophan (0.4-

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0.6 mol-%),47 so that the observed fluorescence appears to come mostly from tyrosine, which is

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present in its tyrosinate form at neutral or alkaline pH and then emits at 345 nm instead of 303

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nm. Tyrosinate emission, as seen here for gluten proteins, has been reported for other proteins

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with little or no tryptophan, e.g., β-purothionin.28

255 256

Qualitative detection of gluten proteins by FLD compared to DAD. Wheat gliadins and

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glutenins show characteristic qualitative elution patterns when analyzed by RP- and GP-HPLC

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combined with UV detection at 210 nm.25,41,48-50 Therefore, the first step was to compare the

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qualitative protein profiles of gliadin and glutenin extracts from wheat flour detected by DAD210

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to those detected by FLD277/345, using both RP- and GP-HPLC for protein separation. Figure 2A

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shows the typical RP-HPLC pattern for gliadins detected by DAD210, with 9.1% ω5-, 6.8% ω1,2-,

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57.0% α- and 27.1% γ-gliadins (given as percentages referred to the total area). Using FLD277/345,

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the relative amounts of gliadin protein types shifted, resulting in 2.3% ω5-, 6.1% ω1,2-, 49.4%

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α- and 42.2% γ-gliadins (Figure 2B). The small peak area for ω5-gliadins may be explained by

265

their low tyrosine content (0.6-0.7 mol-%).51 α-Gliadins were clearly detected by FLD277/345 (2.3-

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3.2 mol-% tyrosine),51 but γ-gliadins were detected with the highest sensitivity, although their

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average tyrosine content (0.6-1.4 mol-%)51 is lower than that of α-gliadins. This may be


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explained by the fact that the spectral properties of proteins are hard to predict due to effects

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of the secondary and tertiary structure.28 For glutenins, the DAD210 chromatogram (Figure 2C)

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showed 3.7% ωb-gliadins, 33.8% HMW- and 62.5% LMW-GS. In comparison, FLD277/345 was

271

particularly suitable for the detection of HMW-GS, resulting in 2.2% ωb-gliadins, 55.2% HMW-

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and 36.4% LMW-GS (Figure 2D). HMW-GS contain the highest tyrosine amounts (5.1-6.4 mol-

273

%)51 of all gluten protein types and were thus detected with high sensitivity. Corresponding

274

observations were made using GP-HPLC. The shape of the gliadin peak was similar both by

275

DAD210 and FLD277/345 (Figure 3), but HMW-GS (first peak in glutenin extract, retention time 6.5-

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7.8 min) were detected with higher sensitivity than LMW-GS (second peak in glutenin extract,

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retention time 7.8-12.8 min) by FLD277/345 compared to DAD210. The DAD210 chromatogram

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yielded 17.8% HMW- and 82.2% LMW-GS (Figure 3A), whereas the distribution changed to

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36.0% HMW- and 64.0% LMW-GS by FLD277/345 (Figure 3B). Because interfering peaks from the

280

starch matrix were detected in RP-HPLC-FLD in the retention time window of 18-23 min, thus

281

co-eluting with gluten proteins (not shown), GP-HPLC-FLD was chosen for all further analyses of

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WSt samples. To compare the sensitivity of FLD277/345 vs. DAD210, linear dilutions of PWG-gliadin

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and gluten solutions (0.0025-0.5 µg) were injected (n = 3), detected by both FLD and DAD in

284

one run, and the peak areas compared using the most sensitive photomultiplier voltage setting

285

(super-high) of the FLD. While the LOD of the FLD was at 0.005 µg PWG-gliadin or 0.0025 µg

286

gluten, the LOD of the DAD was much higher at 0.1 µg PWG-gliadin or gluten. The comparison

287

of peak areas at 0.25 µg, where both detectors showed a clear signal, revealed a 36-fold (PWG-

288

gliadin) and 113-fold (gluten) higher sensitivity of FLD compared to DAD. The difference in

289

sensitivity between PWG-gliadin and gluten may also be explained by the highly sensitive

290

detection of HMW-GS within gluten due to their high tyrosine content. Having ascertained the

291

general suitability of the GP-HPLC-FLD method to detect gluten proteins with better sensitivity



292

compared to UV detection, it was subsequently applied to the quantitation of gliadin, glutenin

293

and gluten contents of WSt.

294 295

Identification of the proteins within the peak (retention time 6.5-12.8 min) of a reduced

296

protein extract of GfW4 by LC-MS. GfW4 was GF by sandwich R5 ELISA (gluten content

297

12.2 ± 1.1 mg/kg), but the GP-HPLC-FLD method yielded a higher gluten content of 44.9 ± 1.0

298

mg/kg (see below). To ascertain that the “gluten” peak (6.5-12.8 min) used for quantitation

299

really contained gluten, not only inferred from similarity of retention times (Figures 3 and 4),

300

the proteins contained within this peak were collected from several GP-HPLC runs and

301

identified by LC-MS after chymotryptic digestion.40 All compounds with retention times beyond

302

12.8 min were not considered to be relevant for gluten quantitation, because of low molecular

303

weights (Mr < 30,000) and non-dose-dependent behavior (Figure 4). The majority of peptides in

304

the gluten extract of GfW4 were derived from LMW-GS, but peptides from α-gliadins, HMW-GS

305

and starch synthases were identified as well (Table 1). LMW-GS were assigned as first protein

306

hit by the database in most cases, but many of these peptides may be found both in LMW-GS

307

and γ-gliadins (e.g., SIILQEQQQGF also in γ-gliadin P04729.1), because these gluten protein

308

types share similar sequence sections.2 The proteins corresponding to the identified peptides

309

had Mr in the range from ≈30,000 to ≈86,000 as expected on the basis of GP-HPLC retention

310

times. Some peptide sequences with single amino acid exchanges (e.g., GQQPQQQKL,

311

GKQPQQQQL, GQQPEQQQL or LQPHKIAQL or VQQQLPVVQPSIL or SQQQQPVIPQQPSF;

312

exchanged position in bold) were detected as well. Most of the peptides seemed to be typical

313

of HMW-GS, LMW-GS/γ-gliadins and α-gliadins, because they matched 4 up to 572 other

314

UniProtKB database entries and had high protein scores calculated by the Mascot software.

315

Several protein sequences had two or more matching peptides (e.g., the peptides LQPHQIAQL,


Page 14 of 37

Page 15 of 37


316

GQQPQQQQL, VLPQQQIPF and SHHQQQQPIQQQPQPF were all found within LMW-

317

GS ACA63873.1). Four peptides contained HLA-DQ2-mediated CD-immunogenic epitopes

318

(underlined or in italics in Table 1), but since the LC-MS method employed only provided

319

qualitative results, no estimation of peptide quantities was attempted. These findings are in

320

accordance with earlier studies that reported the presence of a multitude of proteins in

321

commercial starches, including gluten proteins, with glutenins being more frequent than

322

gliadins.52 The peptide RPQQPYPQPQPQY has already been used as a marker peptide to detect

323

wheat flour contaminations in oat flour by targeted LC-MS13 and may thus also be suitable for

324

gliadin detection in WSt. However, the problem with gluten quantitation in WSt seems to be

325

the higher relative abundance of glutenins compared to gliadins,25 because the R5 and G12

326

monoclonal antibodies used in gluten ELISA test kits show low reactivities with glutenins20 and

327

may thus underestimate gluten contents. The presence of starch synthases within the GP-HPLC

328

“gluten” peak could not be avoided, because their Mr of ≈66,300 and ≈67,700 was in between

329

those of HMW-GS and LMW-GS/gliadins. Because they are located within the starch granules,52

330

they must have been co-extracted with glutenins during heating to 60 °C, which leads to starch

331

gelatinization. Unfortunately, it was impossible to eliminate these starch synthases either

332

through pre-extraction or chromatographic separation, neither by RP- nor by GP-HPLC, so that

333

the quantitative GP-HPLC-FLD results had to be corrected for these internal proteins by matrix

334

calibration.

335 336

Matrix calibration for GP-HPLC-FLD. A definitely GF WSt matrix was obtained after extracting

337

GfW4 five times with gluten extraction solution and lyophilization. The starch residue (GfWgf)

338

contained gliadin/gluten below the LOQs of the R5 sandwich (2.5/5 mg/kg) and R5 competitive

339

(5/10 mg/kg) ELISAs. For confirmation, the “gluten” peak (6.5-12.8 min) from GfWgf (Figure 4C)



340

was collected again from several GP-HPLC runs, digested with chymotrypsin and the peptide

341

spectrum detected by LC-MS. Overall, only one peptide was assigned within Triticeae,

342

ELGVEGSEPGVIGEEIAPL derived from starch synthase with a comparatively low peptide score of

343

21. Peptides derived from gluten proteins could not be detected anymore, so that GfWgf was

344

deemed suitable as matrix for spiking purposes. Five different levels of wheat flour were spiked

345

for the calibration to yield gluten contents of 16, 31, 79, 157 and 315 mg/kg, composed of 10,

346

20, 50, 100 and 200 mg gliadin/kg and 6, 11, 29, 57 and 115 mg glutenin/kg. The gliadin,

347

glutenin and gluten extracts of GfWgf and the five spiked levels were analyzed by GP-HPLC-FLD

348

(n = 10 for GfWgf + 50, n = 3 for all others). The coefficient of variation (CV) was 3.1% for the

349

GfWgf + 50 sample and, therefore, acceptable for homogeneity.53 All calibration lines

350

constructed using the peak areas and the calculated gliadin, glutenin and gluten contents

351

(Figure 5) had R² values >0.995. The peak area of the unspiked GfWgf matrix was smallest for

352

the gliadin extract (Figure 4A) and highest for the gluten extract (Figure 4C), which was

353

reflected in the y-intercepts of the linear equations that allowed the correction for non-gluten

354

proteins.

355 356

LODs, LOQs, precision and recovery for GP-HPLC-FLD. The calculation of LODs and LOQs was

357

based on the signals of the gliadin, glutenin and gluten extracts of GfWgf plus 3 and 10 times

358

their respective standard deviations (n = 10).44,45 The LODs and LOQs were 3.0 and 5.4 mg/kg

359

for gliadins, 3.3 and 10.2 mg/kg for glutenins and 9.0 and 17.2 mg/kg for gluten, respectively.

360

As such, the GP-HPLC-FLD method fulfilled the requirement for an LOD of 10 mg gluten/kg or

361

below, as stated in Codex Standard 118-1979.9 Interassay precision (n = 3 × 3) was determined

362

as CV of 2.6% for gliadins (30.3 ± 0.8 mg/kg), 6.3% for glutenins (25.5 ± 1.6 mg/kg) and 8.1% for

363

gluten (55.8 ± 4.5 mg/kg) extracted from W7. For W12, the CVs were 7.2% for gliadins


Page 16 of 37

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364

(20.9 ± 1.5 mg/kg), 4.7% for glutenins (37.9 ± 1.8 mg/kg) and 6.3% for gluten (58.8 ± 3.7 mg/kg).

365

The overall values for CV ranged from 2.6 to 8.1%, which was comparable to the 5.5% (range

366

2.7-11.0%) recommended for an analyte concentration of 55 mg/kg,54 so that the precision of

367

the GP-HPLC-FLD method was considered satisfactory. Recovery was calculated from triplicate

368

measurements of one additional GfWgf sample (GfWgf + 35) spiked to a gliadin content of 35

369

mg/kg, corresponding to a glutenin content of 20 mg/kg and a gluten content of 55 mg/kg. For

370

gliadin, recovery was 92 ± 4%, for glutenin 101 ± 5% and for gluten 103 ± 7%. All recovery

371

values lay within the recommended 85-110% limits (100 mg/kg).54 For glutenins, the contents

372

were calculated both from the glutenin extraction itself and from the difference between

373

gluten and gliadin contents. Both ways agreed well and showed no significant differences

374

(p > 0.05) for the W7 and W12 samples. Further on, glutenin contents are given derived from

375

the glutenin extraction (Table 2). Having established that the GP-HPLC-FLD method met the

376

required performance criteria, it was subsequently applied to the quantitation of gliadin,

377

glutenin and gluten in 30 WSt samples, 14 of them declared as GF.

378 379

Analysis of gliadin and glutenin in WSt using GP-HPLC-FLD. The gliadin contents of GfW1-14

380

(declared as GF) analyzed by GP-HPLC-FLD ranged from < 5.4 mg/kg to 17.7 mg/kg (GfW12).

381

Looking at the W1-16 samples without specification regarding the gluten content, the range of

382

gliadin contents was much broader from < 5.4 mg/kg to 7757.3 mg/kg (W8) (Table 2). In total, 6

383

out of 30 WSt contained gliadin below the LOQ (5.4 mg/kg). The glutenin contents of GfW1-14

384

were between < 10.2 mg/kg and 58.9 mg/kg (GfW6) and those of W1-16 ranged from < 10.2

385

mg/kg to 2614.5 mg/kg (W8). Overall, 5 out of 30 WSt contained glutenin below the LOQ (10.2

386

mg/kg) and 3 out of 30 WSt contained both gliadin and glutenin below the respective LOQs

387

(GfW2, W2, W9). W8 and W15 had exceptionally high gliadin and glutenin contents compared



388

to the other 28 samples. The resulting gli/glu ratios lay between 0.19 and 0.52 (mean: 0.40 ±

389

0.07, median: 0.40) for GfW1-14 and between 0.39 and 2.97 (mean: 1.14 ± 0.75, median: 0.91)

390

for W1-16. From the 22 WSt samples that allowed the calculation of gli/glu ratios, 16 ratios

391

were below 1.0, indicating that gliadins had been removed more extensively than glutenins

392

during industrial processing, as has been described before for WSt prepared on a laboratory

393

scale25 and also industrial WSt that were not destined for the production of GF foods.24 Here,

394

these low gli/glu ratios (≤ 0.52) were also shown for industrial GfW that were declared as GF.

395 396

Comparison of GP-HPLC-FLD and R5 ELISA results for gliadin and gluten contents of WSt. The

397

gliadin and gluten contents determined by GP-HPLC-FLD were also compared to those by R5

398

ELISA. The gliadin contents showed a medium correlation49 (r = 0.752, p < 0.001) between both

399

methods based on 21 WSt samples, excluding W8, W11 and W15 with exceptionally high gliadin

400

contents (due to their disproportionate influence on correlation analyses) and the 6 samples

401

with values below the LOQ of either or both methods. The R5 ELISA resulted in significantly

402

lower gliadin contents compared to GP-HPLC-FLD in 15 out of 24 cases with values above the

403

LOQ of both methods, in higher contents in 5 cases and there were no significant differences

404

(p > 0.05) in 4 cases. Looking at the gluten contents, the comparison of GP-HPLC-FLD and R5

405

ELISA also resulted in a medium correlation (r = 0.688, p < 0.001) between both methods based

406

on 22 WSt samples, again excluding W8, W11 and W15 and the 5 samples with gluten contents

407

below the LOQs. In comparison to the R5 ELISA (gluten = gliadin × 2), the gluten contents

408

quantitated by GP-HPLC-FLD (gluten = gliadin + glutenin) were significantly higher in 18 out of

409

25 cases with results above the LOQs. There were no significant differences in the remaining 7

410

cases, including the W8, W11 and W15 samples with high gluten contents (Figures 6 and 7).

411

Among the 14 GfW, 12 were GF (gluten content < 20 mg/kg)9 by R5 ELISA and 2 had gluten


Page 18 of 37

Page 19 of 37


412

contents above 20 mg/kg (GfW8: 29.7 ± 2.7 mg/kg and GfW11: 21.2 ± 2.1 mg/kg). According to

413

GP-HPLC-FLD, only 2 out of the 14 GfW were GF (GfW1 and GfW2, that were also GF by R5

414

ELISA), but the other 12 samples contained gluten in the range between 25.6 mg/kg to 69.0

415

mg/kg. The higher gluten contents found by GP-HPLC-FLD were mostly attributable to the

416

occurrence of glutenins with quantities in the range of 23.1 mg/kg to 58.9 mg/kg (Table 2).

417

Glutenins are recognized with low sensitivity by the R5 antibody and thus largely escape

418

detection by R5 (and also G12) antibody-based ELISAs.20 Out of the 16 W, 3 were GF both by R5

419

ELISA and GP-HPLC-FLD (W2, W9, W16), 2 were GF by R5 ELISA, but not by GP-HPLC-FLD (W1

420

and W5) and the other 11 samples were gluten-containing according to both methods. Taken

421

together, these findings highlight the importance of using methods for gluten quantitation that

422

are capable of detecting both gliadins and glutenins. The GP-HPLC-FLD method revealed that

423

considerable amounts of glutenins were detectable in most WSt. Gluten contents expressed as

424

sum of gliadins and glutenins were higher in 19 out of 26 cases than gluten contents given by

425

ELISA quantitation of gliadins followed by duplication, especially in GfW. Due to its rather low

426

selectivity, GP-HPLC-FLD for gluten detection appears to be limited to raw materials such as

427

starches or flours, because proteins from other sources (e.g., milk, egg) commonly present in

428

GF products may interfere. Despite this disadvantage, the new HPLC method described here

429

may be applied in smaller laboratories without highly sophisticated LC-MS equipment, e.g., as a

430

confirmatory method to ELISA to check for the presence of glutenins within WSt.

431 432

ABBREVIATIONS USED

433

CD, celiac disease; CID, collision-induced dissociation; DAD, diode-array detection; ELISA,

434

enzyme-linked immunosorbent assay; ESI, electrospray ionization; FA, formic acid; GF, gluten-

435

free; GP-HPLC-FLD, gel-permeation high-performance liquid chromatography with fluorescence



436

detection; HMW-GS, high-molecular weight glutenin subunits; LC-MS, liquid chromatography

437

mass spectrometry; LMW-GS, low-molecular weight glutenin subunits; LOD, limit of detection;

438

LOQ, limit of quantitation; PWG, Prolamin Working Group; RP, reversed-phase; TFA,

439

trifluoroacetic acid, WSt, wheat starch

440 441

ACKNOWLEDGMENT

442

The authors would like to thank Ms. Angelika Grassl and Ms. Ines Otte for excellent technical

443

assistance.

444 445

Funding

446

This research was funded by the German Federal Ministry of Education and Research via the

447

VDI Technologiezentrum GmbH, grant number 13GW0042 (GLUTEVIS: Optical fluorescent rapid

448

test system for sensitive gluten detection).

449 450

Notes

451

The authors declare no competing financial interest.


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52. Kasarda, D.D.; Dupont, F.M.; Vensel, W.H.; Altenbach, S.B.; Lopez, R.; Tanaka, C.K.; Hurkman, W.J. Surface-associated proteins of wheat starch granules: suitability of wheat starch for celiac patients. J. Agric. Food Chem. 2008, 56, 10292-10302.

603 604 605

53. Thompson, M.; Ellison, S.L.R.; Wood, R. The international harmonized protocol for the proficiency testing of analytical chemistry laboratories. Pure Appl. Chem. 2006, 78, 145196.

606 607

54. AOAC International. AOAC Official Methods of Analysis. Appendix K: Guidelines for dietary supplements and botanicals. AOAC, Gaithersburg, MD, USA. 2012.

608 609 610 611

55. Vader, W.; Kooy, Y.; Van Veelen, P.; De Ru, A.; Harris, D.; Benckhuijsen, W.; Pena, S.; Mearin, L.; Drijfhout, J.W.; Koning, F. The gluten response in children with celiac disease is directed toward multiple gliadin and glutenin peptides. Gastroenterology. 2002, 122, 17291737.

612 613

56. Koning, F.; Gilissen, L.; Wijmenga, C. Gluten: a two-edged sword. Immunopathogenesis of celiac disease. Springer Semin Immunopathol. 2005, 27, 217-232.

614 615 616 617

57. Vader, W.; Stepniak, D.; Kooy, Y.; Mearin, L.; Thompson, A.; van Rood, J.J.; Spaenij, L.; Koning, F. The HLA-DQ2 gene dose effect in celiac disease is directly related to the magnitude and breadth of gluten-specific T cell responses. Proc. Natl. Acad. Sci. USA. 2003, 100, 12390-12395.



618

Figure Captions

619

Figure 1. Fluorescence spectra of gluten solutions (0.5 mg/ml). (A) Excitation maximum of

620

gluten. (B) Emission maximum of gluten after excitation at 277 nm.

621 622

Figure 2. Reversed-phase-HPLC chromatograms of gluten fractions extracted from wheat flour.

623

Gliadins, detected by diode-array detection at 210 nm (DAD210) (A) and by fluorescence

624

detection at 277/345 nm (FLD277/345) (B). Glutenins, detected by DAD210 (C) and by FLD277/345 (D).

625

ω5, ω5-gliadins; ω1,2, ω1,2-gliadins; α, α-gliadins; γ, γ-gliadins; ωb, ωb-gliadins; HMW, high-

626

molecular-weight glutenin subunits; LMW, low-molecular-weight glutenin subunits; AU,

627

absorbance units; FLU, fluorescence units.

628 629

Figure 3. Gel-permeation-HPLC chromatograms of gluten fractions extracted from wheat flour.

630

Gliadins and glutenins, detected by diode-array detection at 210 nm (DAD210) (A) and by

631

fluorescence detection at 277/345 nm (B). The peak at 14 min in the glutenin extract detected

632

by DAD210 consists of the reducing agent DTT. AU, absorbance units; FLU, fluorescence units.

633 634

Figure 4. GP-HPLC-FLD chromatograms of gliadin (A), glutenin (B) and gluten (C) extracts of

635

gluten-free GfW4 (GfWgf) and the GfWgf samples spiked with wheat flour for matrix calibration

636

to obtain 10, 20, 50 and 100 mg gliadin/kg (GfWgf + 10/20/50/100 mg/kg) (A), corresponding to

637

6, 11, 29 and 57 mg glutenin/kg (GfWgf + 6/11/29/57 mg/kg) (B) and to 16, 31, 79 and 157 mg

638

gluten/kg (GfWgf + 16/31/79/157 mg/kg) (C). The peak with retention times between 6.5-12.8

639

min was used for quantitation of gliadins, glutenins and gluten. FLU, fluorescence units.

640


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641

Figure 5. Matrix calibration lines obtained for gliadin (A), glutenin (B) and gluten extracts (C) of

642

GfWgf and GfWgf + 10/20/50/100/200 mg/kg for gliadins (A), GfWgf and GfWgf + 6/11/29/57/115

643

mg/kg for glutenins (B) and GfWgf and GfWgf + 16/31/79/157/315 mg/kg for gluten (C). FLU,

644

fluorescence units.

645 646

Figure 6. Gluten contents of wheat starches declared as gluten-free (GfW1-14) determined by

647

GP-HPLC-FLD (gluten as sum of gliadin and glutenin contents) and R5 ELISA (gluten as gliadin

648

content multiplied by 2). Data are presented as mean value + standard deviation (n = 3).

649

Asterisks indicate significant differences between gluten contents determined by GP-HPLC-FLD

650

and R5 ELISA. “40 are considered to indicate identity or extensive similarity (p < 0.05) and scores 15-40 were additionally validated manually43; b accession.version number in database National Center for Biotechnology Information non-redundant (NCBInr) of the protein with the highest protein score c protein scores are derived from peptide ion scores as a non-probabilistic basis for ranking protein hits d number of hits with a sequence identity of 100 % (BLAST search within Triticeae in database UniProtKB for each peptide, numbers not adjusted for multiple occurrences within one protein) e contains two HLA-DQ2-mediated immunogenic sequences, one underlined, one in italics (entry IDs 677, 693, allergenonline.org)55,56 f contains 7 out of 9 amino acid residues of an HLA-DQ2-mediated immunogenic sequence, underlined (entry IDs 701, 706, allergenonline.org)55,57 g contains one HLA-DQ2-mediated immunogenic sequence, underlined (entry ID 138, allergenonline.org)55 Peptide

m/z (charge state) 645.83 (+2)

28 ACS Paragon Plus Environment

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Table 2. Gliadin content quantitated by R5 ELISA, gliadin and glutenin contents quantitated by GP-HPLC-FLD and resulting gliadin/glutenin ratios. Data are presented as mean value ± standard deviation with n = 3 for gliadin and glutenin contents, respectively. Sample

Gliadin

Gliadin

Glutenin

Gliadin/ Glutenin

R5 ELISA GP-HPLC-FLD [mg/kg] [mg/kg] [mg/kg] ratio a GfW1 4.3 ± 0.5 a 6.6 ± 1.3 a < 10.2 -b GfW2 7.4 ± 1.0 < 5.4c < 10.2 GfW3 7.5 ± 0.6 a 13.5 ± 0.5 b 29.9 ± 1.3 0.45 GfW4 6.1 ± 0.5 a 15.3 ± 0.3 b 29.6 ± 0.6 0.52 d GfW5 < 2.5 < 5.4 23.2 ± 0.5 GfW6 4.4 ± 0.4 < 5.4 58.9 ± 1.9 GfW7 7.3 ± 0.6 a 9.7 ± 1.2 a 32.6 ± 0.2 0.30 GfW8 14.9 ± 1.3 a 10.6 ± 0.4 b 31.2 ± 0.3 0.34 GfW9 7.7 ± 0.6 a 14.6 ± 0.2 b 36.7 ± 0.9 0.40 GfW10 5.2 ± 0.5 < 5.4 23.1 ± 1.2 GfW11 10.6 ± 1.1 a 5.6 ± 0.2 b 30.1 ± 1.2 0.19 GfW12 6.4 ± 0.6 a 17.7 ± 1.1 b 40.8 ± 1.0 0.43 GfW13 8.9 ± 0.9 a 17.6 ± 1.1 b 51.4 ± 1.0 0.34 GfW14 8.2 ± 0.8 a 12.5 ± 0.9 b 33.5 ± 1.9 0.37 W1 8.1 ± 0.5 a 13.3 ± 0.4 b 13.1 ± 1.9 1.02 W2 3.1 ± 0.1 < 5.4 < 10.2 W3 10.1 ± 0.3 a 12.3 ± 0.2 a 13.6 ± 0.3 0.91 W4 23.4 ± 0.8 a 68.8 ± 1.1 b 89.9 ± 1.4 0.76 W5 8.2 ± 0.5 a 14.7 ± 0.6 b 16.2 ± 1.8 0.91 W6 41.3 ± 1.1 a 51.8 ± 1.1 b 51.8 ± 4.5 1.00 W7 33.0 ± 2.0 a 30.3 ± 0.8 a 25.5 ± 1.6 1.19 W8 5951.9 ± 570.7 a 7757.3 ± 176.5 b 2614.5 ± 52.4 2.97 W9 < 2.5 < 5.4 < 10.2 W10 24.2 ± 0.8 a 10.7 ± 1.0 b 27.2 ± 1.0 0.39 W11 212.2 ± 3.0 a 139.4 ± 10.8 b 303.3 ± 3.5 0.46 W12 34.1 ± 1.2 a 20.9 ± 1.5 b 37.9 ± 1.8 0.55 W13 44.2 ± 2.2 a 68.3 ± 1.6 b 127.6 ± 2.7 0.54 W14 26.8 ± 1.6 a 56.8 ± 1.6 b 30.4 ± 3.3 1.87 W15 3511.0 ± 29.9 a 4571.7 ± 307.2 b 1971.7 ± 99.2 2.32 W16 4.6 ± 0.1 a 10.7 ± 1.5 b < 10.2 a b Limit of quantitation (LOQ) for glutenins; gliadin/glutenin ratio could not be calculated, because either the gliadin or glutenin content or both were below their respective LOQ; c LOQ for gliadins; d LOQ for gliadins (R5 ELISA); different lower case letters indicate significant differences (p < 0.05) between gliadin contents determined by R5 ELISA vs. GP-HPLC-FLD 29 Environment ACS Paragon Plus


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TOC Graphic


Improved Quantitation of Gluten in Wheat Starch for Celiac Disease

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