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Food Safety and Toxicology

Targeted LC-MS/MS reveals similar contents of #amylase/trypsin-inhibitors as putative triggers of nonceliac gluten sensitivity in all wheat species except einkorn Sabrina Geisslitz, Christina Ludwig, Katharina Anne Scherf, and Peter Koehler J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04411 • Publication Date (Web): 26 Oct 2018 Downloaded from http://pubs.acs.org on October 27, 2018

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Journal of Agricultural and Food Chemistry

Targeted LC-MS/MS reveals similar contents of α-amylase/trypsininhibitors as putative triggers of non-celiac gluten sensitivity in all wheat species except einkorn

Sabrina Geisslitz1, Christina Ludwig2, Katharina Anne Scherf1, Peter Koehler3*

1 Leibniz-Institute

for Food Systems Biology at the Technical of University Munich, Lise-

Meitner-Strasse 34, 85354 Freising, Germany 2

Bavarian Center for Biomolecular Mass Spectrometry, Gregor-Mendel-Strasse 4, 85354 Freising, Germany

3

biotask AG, Schelztorstrasse 54-56, 73728 Esslingen am Neckar, Germany

* Corresponding author: E-Mail address: [email protected] (P. Koehler)

1 ACS Paragon Plus Environment


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ABSTRACT

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Amylase/trypsin-inhibitors (ATIs) are putative triggers of non-celiac gluten sensitivity

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(NCGS), but contents of ATIs in different wheat species were not available. Therefore,

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the predominant ATIs 0.19+0.53, 0.28, CM2, CM3 and CM16 in eight cultivars each of

5

common wheat, durum wheat, spelt, emmer and einkorn grown under the same

6

environmental conditions were quantitated by targeted liquid chromatography-tandem

7

mass spectrometry (LC-MS/MS) and stable isotope dilution assays (SIDA) using specific

8

marker peptides as internal standards. The results were compared to a label-free

9

untargeted LC-MS/MS analysis, in which protein concentrations were determined by

10

intensity based absolute quantitation (iBAQ). Both approaches yielded similar results.

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Spelt and emmer had higher ATI contents than common wheat, with durum wheat in

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between. Only three of eight einkorn cultivars contained ATIs in very low concentrations.

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The distribution of ATI types was characteristic for hexaploid, tetraploid and diploid

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wheat species and suitable as species-specific fingerprint. The results point to a better

15

tolerability of einkorn for NCGS patients, because of very low total ATI contents.

16 17 18

KEYWORDS

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α-Amylase/Trypsin-Inhibitor; Modern and ancient wheats; Mass Spectrometry; Stable

20

isotope dilution assay; non-celiac gluten sensitivity

21


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INTRODUCTION

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Wheat proteins are classically divided into four fractions, namely albumins, soluble in

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water, globulins, soluble in 0.5 mol/L sodium chloride, gliadins, soluble in 70% aqueous

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ethanol, and insoluble glutenins.1 Some wheat proteins contain amino acid sequences

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that may trigger celiac disease, wheat allergy and non-celiac gluten sensitivity (NCGS)

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in susceptible individuals.2 With a reported prevalence of 0.6 - 6%,3,4 NCGS is

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characterized by gastrointestinal complaints (e.g. abdominal pain, diarrhea, bloating)

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and extraintestinal symptoms (e.g. headache, chronic fatigue, depression), which

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disappear on a gluten-free or -reduced diet.3,5,6 Different constituents of gluten-

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containing cereals such as fermentable oligo-, di-, and monosaccharides and polyols

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(FODMAPs), gluten and α-amylase/trypsin inhibitors (ATIs) were suggested as triggers

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of NCGS. ATIs have been shown to activate the toll-like receptor 4 TLR4-MD2-CD14

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complex causing secretion of pro-inflammatory chemokines and cytokines.7,8 This dose-

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dependent activation of innate immunity may lead to the symptoms typical of NCGS as

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well as worsening of pre-existing inflammatory reactions.8,9

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Genome sequencing of common wheat revealed up to 19 different ATI-encoding

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genes,10 of which 13 types have evidence at protein level in common wheat (UniProtKB:

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0.19, 0.28, 0.53, CM1, CM2, CM3, CM16, CM17, CMX1/3, CMX2, wheat subtilisin

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inhibitor, Bowman-Birk type trypsin inhibitor and chymotrypsin inhibitor WCI). Known as

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wheat allergens, ATIs account for as much as 2 - 4% of the protein in common wheat

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(Triticum aestivum L., hexaploid) with a relative distribution of 50% of CM-types, 33% of

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0.19 and 17% of 0.28.11 ATIs are salt-soluble plant-defense proteins located in the seed

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endosperm and the content of CM3 was influenced both by genetic and environmental

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factors.12 The nine to ten cysteine residues form four to five intramolecular disulfide 3 ACS Paragon Plus Environment


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bonds, leading to a compact three dimensional structure that is responsible for the

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resistance of ATIs to digestive enzymes of insects and mammals.13,14 Within ATIs, 0.19

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and CM3 from common wheat had the highest bioactivity on TLR4-bearing monocytes,

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whereas ATI extracts from the “ancient” (hulled) wheat species spelt (T. spelta L.,

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hexaploid), emmer (T. dicoccum L., tetraploid) and einkorn (T. monococcum L., diploid)

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only reached 30 - 70% of the activity of common wheat.7 Furthermore, liquid

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chromatography-tandem

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contained very low amounts of ATIs or even none.15 Although ancient wheats are only

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produced in small quantities due to low grain yields,16 they have gained increasing

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popularity in the last decades due to plant resilience, promotion of biodiversity and

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positive consumer perceptions of regional, organic and healthy.

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The low bioactivity of ancient wheats and the proposed absence of ATIs in einkorn lead

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to the hypothesis that the ancient wheats spelt, emmer and einkorn may be better

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tolerated by NCGS patients, because of lower ATI contents compared to the “modern”

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(naked) species common and durum wheat (T. durum L., tetraploid). To confirm this

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hypothesis, we aimed to develop a targeted LC-MS/MS method using stable isotope

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labeled peptides as internal standards (stable isotope dilution assay, SIDA) to quantitate

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the predominant ATIs 0.19, 0.28, 0.53, CM2, CM3 and CM16 in grains of a set of eight

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well-characterized common wheat, durum wheat, spelt, emmer and einkorn cultivars,

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respectively, grown under the same environmental conditions. To confirm the absolute

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ATI concentrations determined by SIDA, we also performed a global, label-free and

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untargeted LC-MS/MS analysis and applied the intensity based absolute quantitation

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algorithm (iBAQ) to estimate absolute protein abundances for the ATIs.

mass

spectrometry

(LC-MS/MS)

69 4 ACS Paragon Plus Environment

showed

that

einkorn

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MATERIALS AND METHODS

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Reagents. All reagents were of analytical grade or higher and purchased from VWR

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Merck (Darmstadt, Germany), Serva (Heidelberg, Germany), LECO (Kirchheim,

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Germany) or Sigma-Aldrich (Steinheim, Germany). Water was deionized by a water

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purification system Arium 611VF (Sartorius, Goettingen, Germany). Trypsin (TPCK-

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treated) and the ATI reference were from Sigma-Aldrich (α-Amylase Inhibitor from

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Triticum aestivum (wheat seed), type I). Unlabeled (marker peptides P1-P5) and stable

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isotope labeled (internal standards IS1-IS5) peptides (Table S-1) were synthesized by

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GenScript (Piscataway, NJ, USA). Each heavy peptide contained at least two [13C]- and

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[15N]-labeled amino acids (Table S-1). For stock solutions (1 mg/mL in water or dimethyl

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sulfoxide), the peptides were solubilized according to the manufacturer’s guidelines and

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stored at -80 °C prior to use. The puritie of IS1-IS5 were analyzed by

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nuclear magnetic resonance spectroscopy (13C qNMR).17 The purities of P1-P5 was

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analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) and

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UV-detection at 210 nm after alkylation. HPLC peak areas of IS1-IS5 were used as

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calibrants for P1-P5, respectively.

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Grain samples. Eight cultivars each of common wheat, spelt, durum wheat, emmer and

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einkorn were cultivated by the State Plant Breeding Institute, University of Hohenheim

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(Stuttgart, Germany) at Seligenstadt, Germany, and harvested in 2013 (Table S-2).16

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The grains were milled into wholemeal flours using a cross-beater mill (Perten

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Instruments, Hamburg, Germany). To prepare the common wheat flour mix, grains of 15

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cultivars (the eight cultivars above plus seven cultivars from the same growing area)

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were mixed in equal weights prior to milling. Contents of water, ash, crude protein and

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albumins/globulins (ALGL) were already reported by Geisslitz et al.18 5 ACS Paragon Plus Environment

13C

quantitative


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Identification of ATI marker peptides. The extraction of ATIs from five flours of

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common wheat and one flour of durum wheat, spelt, emmer and einkorn, respectively,

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was based on Zevallos et al.7 and Junker et al.8 followed by reduction, alkylation, tryptic

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digestion and untargeted LC-MS/MS-Iontrap analysis as described by Rombouts et al.19

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(for details, see Supporting Information).

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Sample preparation for targeted LC-MS/MS and SIDA. The parameters extraction

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time, temperature and solvent, as well as conditions for reduction, alkylation and

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digestion were optimized using the common wheat flour mix (see Supporting

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Information). This resulted in the following final procedure: Flour (50 mg) was stirred

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twice with ammonium bicarbonate (Abic) solution (0.5 mL, 50 mmol/L, pH 7.8) for 30 min

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at 22 °C. After every extraction step the suspensions were centrifuged for 25 min at

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3,750 g and the supernatants combined. The extracts were evaporated to dryness and

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the residue was dissolved in Tris-HCl (320 µL, 0.5 mol/L, pH 8.5) and 1-propanol

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(320 µL). The IS (50 µL of the mixed solution, 20 µg/mL) were added and reduction was

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performed by adding tris(2-carboxyethyl)phosphine (TCEP) (50 µL, 0.05 mol/L TCEP in

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0.5 mol/L Tris-HCl, pH 8.5) and incubation for 30 min at 60 °C. Cysteine residues were

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alkylated with chloroacetamide (CAA) (100 µL, 0.5 mol/L CAA in 0.5 mol/L Tris-HCl,

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pH 8.5) for 45 min at 37 °C in the dark followed by lyophilization. Tryptic hydrolysis

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(0.5 mL, enzyme-to-substrate ratio 1:50, 0.04 mol/L urea in 0.1 mol/L Tris-HCl, pH 7.8)

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was performed for 24 h at 37 °C in the dark. The reaction was stopped with 2 µL

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trifluoroacetic acid. The solution was evaporated to dryness. The residue was dissolved

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in 1 mL of 0.1 % formic acid (FA) and the solution filtered through a 0.45 mm membrane.

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Response lines. Two solutions (100 µg/mL of each peptide), solution 1 with P1-P5 and

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solution 2 with IS1-IS5, were prepared from the stock solutions. An aliquot of each 6 ACS Paragon Plus Environment

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solution 1 and 2 was reduced with TCEP and alkylated with CAA as described for grain

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samples. Alkylated solutions 1 and 2 (20 µg/mL of each peptide) were mixed in molar

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ratios n(P)/n(IS) between 9.1 and 0.1 (9 + 1, 4 + 1, 3 + 1, 1 + 1, 1 + 3, 1 + 4 and 1 + 9)

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for calibration.

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Targeted LC-MS/MS and SIDA. An UltiMate 3000 HPLC system (Dionex, Idstein,

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Germany) coupled to a triple-stage quadrupole mass spectrometer (TSQ Vantage,

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ThermoFisher Scientific, Bremen, Germany) was used. An Aqua®-C18 column

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(50 × 2 mm, 5 µm, 12.5 nm, Phenomenex, Aschaffenburg, Germany) was used for

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peptide separation with the following LC conditions: solvent A, FA (0.1%, v/v) in water,

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solvent B, FA (0.1%, v/v) in acetonitrile; gradient, 0 - 5 min 10% B, 5 - 20 min 10 - 90%

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B, 20 - 23 min 90% B, 23 - 25 min 90 - 10% B, 25 - 40 min 10% B; flow rate, 0.2 mL/min;

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injection volume, 10 μL; column temperature, 22 °C. The ion source was operated in the

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ESI positive mode and the following source parameters were set: spray voltage, 4500 V;

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vaporizer temperature, 50 °C; sheath gas pressure, 40 arbitrary units (au); aux gas

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pressure, 5 au; capillary temperature, 300 °C.20 Selected reaction monitoring (SRM) was

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used to analyze the transitions from precursor to product ions using experimentally

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optimized collision energies (Table S-1).

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Data analysis of SIDA. SRM peak area integration was performed using Skyline 4.1

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(MacCoss Lab Software, University of Washington, Seattle, WA, USA)21 with manual

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verification of automated peak integration. The data are publicly available on Panorama

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Public22 (https://panoramaweb.org/V8iLUY.url). The ratios between the three transitions

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(precursor-product ion pairs) were confirmed to be the same for the response lines and

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the flour samples (± 5% absolute deviation, Table S-3), allowing the use of averaged

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peak ratios from the three transitions, respectively. These constant ratios were 7 ACS Paragon Plus Environment


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additionally used as identification criteria for the marker peptide signals. Response lines

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(Table S-3) were plotted by linear regression of the peak area ratios A(P1-P5)/A(IS1-

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IS5) against the molar ratios n(P1-P5)/n(IS1-IS5). All quantitations were performed from

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three biological replicates with one injection. Contents of ATIs were calculated by

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multiplying the peptide content with the respective factor (MProtein/MPeptide) using the

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molecular mass of the protein without the signal peptide (Table S-1). For CM2 and

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CM16, the protein contents were calculated separately from P3/P3c and P5/P5c and

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summed up. Both ratios were constant at about 95/5 for P3/P3c and 50/50 for P5/P5c.

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P3c and P5c occurred because of water loss at N-terminal glutamine or glutamic acid

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after dissolving P3 and P5.

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Precision. Repeatability precision was evaluated by analysis of six biological replicates

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(n = 6) of the common wheat flour mix and intermediate precision with six biological

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replicates on three days (same analyst, same instrument), respectively (n = 18).23

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Limit of detection and limit of quantitation. To determine the limits of detection (LOD)

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and quantitation (LOQ), P1-P5 and IS1-IS5 (500 ng, 250 ng, 50 ng, and 25 ng absolute)

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were added to cassava starch (100 mg) as analyte-free matrix (n = 6). Sample

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preparation and analysis were the same as described above (extraction volume

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2 × 1 mL). The last spike-on step, where the identification criteria (correct ratios of the

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three transitions) were fulfilled, was used to calculate LOD and LOQ. LOD was defined

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as three times the standard deviation and LOQ as ten times the standard deviation of

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this last step.24 LOD and LOQ were verified by adding P1-P5 and IS1-IS5 six times to

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cassava starch in the concentrations calculated for LOD and LOQ and calculation of

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recovery near LOQ (Table 1). In a second experiment, the common wheat flour mix was


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diluted 1 + 9, 1 + 49 and 1 + 99 with cassava starch in triplicates and each sample

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(n = 9) was analyzed and evaluated as described above.

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Recovery. Recovery was determined by means of a) spiking experiments of P1-P5 to

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the common wheat flour mix and b) dilution of the common wheat flour mix with cassava

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starch. For the spiking experiments, a mixed solution of P1-P5 was prepared to spike to

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+75% of P1-P5. Twelve samples of the common wheat flour mix (50 mg) were extracted

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two times with Abic buffer and the supernatants were lyophilized. Then, IS1-IS5 were

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added to all samples and the mixed solution of P1-P5 (100 µL) was added to six of the

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twelve samples followed by sample treatment as described. Recovery was calculated by

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comparison of the theoretical amounts with the difference between samples with and

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without addition of P1-P5. For the dilution experiments, the recovery was calculated from

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the common wheat flour/cassava starch sample (1 + 9).

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Sample preparation of ATI reference and flour samples for untargeted nanoLC-

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MS/MS (iBAQ). The protein content of the ATI reference was determined by RP-HPLC-

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UV according to Geisslitz et al.18 For in-gel digestion, the ATI reference was dissolved in

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ultrapure water, incubated with sample buffer, reduced, alkylated and run on a 4 - 12%

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NuPAGE gel prior to in-gel tryptic digestion following standard procedures.25 The

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resulting peptides were lyophilized, dissolved in 0.1% FA and analyzed by nanoLC-

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MS/MS-Orbitrap. Flours (50 mg) of two cultivars of each wheat species were treated as

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described for targeted LC-MS/MS and SIDA. The peptide concentrations were

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determined by Nanodrop (One, Thermo Scientific, Madison, USA) and 0.5 µg total

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peptide amount was injected per nanoLC-MS/MS-Orbitrap run.

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Untargeted nanoLC-MS/MS (iBAQ). An Eksigent nanoLC-Ultra 1D+ system (Eksigent

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Technologies, Dublin, CA, USA) was coupled online to a LTQ-Orbitrap Velos mass 9 ACS Paragon Plus Environment


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spectrometer (ThermoFisher Scientific). Using a flow rate of 5 µl/min of solvent A1 (0.1

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% FA in water), peptides were loaded onto a trap column (ReproSil-Pur 120 ODS-3, 5

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µm, 2 cm × 75 µm, Dr. Maisch, Ammerbuch-Entringen, Germany). Subsequently,

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peptides were separated on an analytical column (ReproSil-Gold 120 C18, 3 µm, 40 cm

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× 75 µm, Dr. Maisch) using a flow rate of 300 nl/min and a gradient from 2 % to 32 %

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solvent B (solvent A: 0.1 % FA and 5 % DMSO in water, solvent B: 0.1 % FA and 5 %

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DMSO in acetonitrile) in 110 min. The eluate from the analytical column was sprayed via

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a stainless steel emitter (ThermoFisher Scientific) into the MS at a source voltage of 2.2

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kV. The transfer capillary was heated to 275 °C. The Orbitrap Velos was set to data-

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dependent acquisition in positive ion mode, automatically selecting the 20 most intense

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precursor ions from the preceding full MS (MS1) spectrum with an isolation width of 2.0

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m/z for fragmentation using CID at 35% normalized collision energy and subsequent

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identification by MS/MS (MS2). MS1 (360 - 1300 m/z) spectra were acquired in the

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Orbitrap using a resolution of 60,000 (at 400 m/z) and MS2 spectra in the ion trap.

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Dynamic exclusion was set to 60 s. The peptides and proteins in ATI reference and flour

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samples were identified and relatively quantitated using MaxQuant (version 1.5.3.30) by

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searching the MS data against a wheat protein reference database derived from

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UniprotKB (004565_triticum_aestivum_uniprot-proteome%3AUP000019116, download

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11.05.17, protein entries 136867) using the search engine Andromeda.26 Variable

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modifications included oxidation of methionine and N-terminal protein acetylation.

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Carbamidomethylation on cysteines was specified as fixed modification and trypsin as

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proteolytic enzyme with up to two allowed missed cleavage sites. Match-between-runs

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(matching time window 0.7 min, alignment time window 20 min) was enabled and results

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were filtered for a minimal length of seven amino acids and 1% peptide and protein false 10 ACS Paragon Plus Environment

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discovery rate. To investigate the differences in concentration for the identified proteins

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within one sample, we estimated absolute protein intensities using the iBAQ algorithm

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implemented within MaxQuant.27 A total sum normalization of iBAQ protein intensities

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between samples was performed to correct for different total protein injection amounts.

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Statistics. Principal component analysis (PCA), Grubbs’ test for outliers and one-way

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analysis of variance (ANOVA) with Tukey’s test were performed with Origin 2017

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(OriginLab, Northampton, Massachusetts, USA).

220 221

RESULTS AND DISCUSSION

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Identification of ATI marker peptides. The first step to develop a targeted LC-MS/MS

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SIDA method (abbreviated as SIDA in the following) to quantitate the five major ATIs in

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common wheat, spelt, durum wheat, emmer and einkorn was to identify ATI marker

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peptides that are present in all wheat species. ATIs were extracted from wholemeal

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flours of each wheat species, reduced, alkylated and hydrolyzed with trypsin followed by

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untargeted LC-MS/MS-Iontrap analysis. In total, 19 peptides were identified for the ATIs

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0.19, 0.28, 0.53, CM2, CM3, CM16 and CM17 in common wheat, durum wheat, spelt

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and emmer, but not in einkorn (Table S-4). The peptides were unique for each different

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ATI type, except for P1 that occurred in 0.19 and 0.53. Furthermore, the database

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search showed that some of the marker peptides also occur in other cereal species such

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as rye and barley. No unique peptide was identified for 0.19, because the peptides all

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belonged to uncharacterized ATI-like proteins. One peptide that was only present in 0.19

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and not in 0.53 was not suitable either, because it was not present in durum wheat and

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emmer. Thus, 0.19 and 0.53 could not be differentiated and the content of P1 represents

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the sum of both. The marker peptide for CM17, which was found in common wheat and 11 ACS Paragon Plus Environment


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spelt, could not be synthesized. The final five marker peptides P1-P5 (Table S-1) were

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chosen, because of high scores in all wheat species, reproducible tryptic cleavage, good

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MS response and high stability during storage at -80 °C.

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Targeted LC-MS/MS and SIDA. To develop the SIDA, full-scan mass spectra of P1-P5

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and IS1-IS5 were acquired to define suitable precursor ions for targeted LC-MS/MS. In

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general the 2+ charge states of the precursor ions had the highest intensity and were

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therefore selected except for P3 and IS3 (3+). Next, the selected precursor ions were

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fragmented to define the three most abundant transitions for SRM, including at least one

245

m/z higher than m/z of the precursor ion for improved selectivity. The collision energies

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of the three selected SRM transitions were experimentally optimized to obtain the

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highest peak area of the product ions (Table S-1). The purities of the unalkylated IS

248

were determined by

249

the other IS (IS1, 80.6%; IS2, 69.3 %; IS4, 68.5 %; IS5, 72.0 %) (Table S-3). The

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purities of alkylated P1 to P5 were 90.1 %, 91.5 %, 28.7 %, 81.0 % and 83.2 %,

251

respectively. These values were taken into account for all calculations. The intercepts of

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all response lines were close to 0.0 and the slopes were between 0.7 and 1.2, with the

253

exception of P3c/IS3c and P5/IS5 (0.6). The response lines were re-analyzed weekly

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together with the samples and only slight variations were observed (Table S-3). Ideally,

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the slope should be 1.0 in SIDA, but the ranges observed here were consistent with

256

earlier reports.20

257

Optimization of sample preparation for SIDA. Details of the performed sample

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optimization can be found in Supporting Information. Each parameter was systematically

259

varied and the procedure resulting in higher absolute peak areas of marker peptides was

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chosen. A common wheat flour mix consisting of 15 cultivars was used to even out

13C

qNMR. The purity of IS3 was very low (13.0 %) in contrast to


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cultivar-specific variations. First, the ATI extraction overnight at 4 °C7 was shortened and

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simplified to two times 30 min at 22 °C without significant changes in peak areas. A third

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extraction step was not necessary, because over 97% of marker peptides were

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extracted in the first and second step. Reducing the sample weight from 500 mg to

265

50 mg led to higher peak areas (+100%) and smaller standard deviations (-50 %) due to

266

better homogenization by the stirring bar in the sample vessels. The sample weight of

267

50 mg appeared to be the best compromise between a homogenous distribution of ATIs

268

in the flour and low amounts of IS required, which is why no lower sample weight was

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tested. Instead of the Abic buffer, solutions based on phosphate buffer, 60% ethanol

270

(v/v) and 50% 1-propanol (pH 7.6, v/v) under reducing conditions (glutenin solution)

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were tested. The peak areas were similar for P1 and P4 with both Abic and phosphate

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buffer, but the Abic buffer resulted in higher peak areas for P2 (+20 %), P3 (+15 %) and

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P5 (+70%). Stepwise extraction of the flour first with Abic buffer, followed by 60%

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ethanol and then glutenin solution showed that ≥ 90% of P1-P5 were detected in the

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Abic solution, < 8% in 60% ethanol and < 2% in the glutenin solution. This confirmed the

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exhaustive extraction of ATIs using Abic buffer.7 The use of CAA for alkylation gave

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slightly higher peak areas (≤ +10%) compared to iodoacetamide, so that CAA was

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chosen. The average efficiency of alkylation was >99%. To ensure complete tryptic

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hydrolysis, different incubation times (2, 4, 6, 18, 21 and 24 h) were tested. Peak areas

280

increased ( 70%) between 2 and 18 h, but then a plateau was reached (≤ ±8%)

281

between 18 and 24 h. An incubation time of 24 h was chosen.19

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Precision, LOD, LOQ and recovery. Precision, LOD, LOQ and recovery of the SIDA

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were evaluated (Table 1) using the final method. Except for P3c, very good values for

284

repeatability (1.8 - 5.3%) and intermediate precision (2.2 - 6.8%) were obtained. The 13 ACS Paragon Plus Environment


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comparatively poor precision of P3c was due to contents close to the LOQ. Wheat

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starch, vital gluten and cassava starch were considered as possible matrices to

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determine LOD and LOQ. Targeted LC-MS/MS showed the absence of marker peptides

288

and signal interferences only in cassava starch and excluded wheat starch and vital

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gluten. The marker peptides were detected with high sensitivity, resulting in an LOD

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between 0.1 and 1.6 µg/g and an LOQ between 0.3 and 4.7 µg/g. The only outlier was

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A3c with an LOD of 6.8 µg/g and an LOQ of 20.5 µg/g. These limits agreed well with

292

those reported for gluten peptides from common wheat.20 LOD and LOQ were

293

additionally confirmed by spiking the calculated concentrations of LOD and LOQ to

294

cassava starch. The concentrations at LOD still fulfilled the identification criteria,

295

because the three ratios of precursor to product ions were constant. Furthermore,

296

concentrations at LOQ also showed acceptable recoveries (78.3 - 113.6%, Table 1).

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Additionally, the dilution experiments of common wheat flour with cassava starch

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confirmed the detection limits. It was possible to detect P1, P2 and P4 in the 1+99 (flour

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+ starch) mix and the other marker peptides in the 1+9 mix. The concentrations of P1,

300

P2 and P4 matched the LOD in the 1+99 mix and those of P3 and P5 matched the LOQ

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in the 1+9 mix. The recovery in the 1+9 mix (92.3 - 101.9%, Table 1) substantiated the

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good performance of the extraction and the developed SIDA. The recovery, proven by

303

spiking experiments to common wheat, revealed good ranges for the majority of marker

304

peptides (76.3 - 120.9%, Table 1). The poor recovery (29.1%) of A3c was again due to

305

very low concentrations in common wheat.

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Comparison of SIDA with untargeted nanoLC-MS/MS-Orbitrap (iBAQ). To confirm

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the quantitative accuracy of the SIDA results, we performed a label-free and untargeted

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nanoLC-MS/MS analysis and applied the iBAQ algorithm to estimate absolute protein 14 ACS Paragon Plus Environment

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abundances. A key advantage of this algorithm, which is implemented within MaxQuant,

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is that iBAQ intensities of different proteins can directly be compared to each other

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within one sample.27 Because MaxQuant uses protein masses with signal peptide from

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the UniProtKB database, both SIDA and iBAQ peptide contents were converted to

313

protein concentrations with the appropriate factor including the signal peptide for better

314

comparability. First, a commercially available ATI reference, which consisted of different

315

ATIs and other proteins, was analyzed by the established SIDA. The ATI reference was

316

directly dissolved, reduced, alkylated and hydrolyzed with trypsin. The IS were added

317

prior to reduction. The advantage of SIDA is that losses during the preparation, e.g.

318

filtration, are considered, but the usage of labeled peptides does not compensate for

319

incomplete digestion or peptide modifications. The efficiency of digestion was checked

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by LC-QTOF-MS and SDS-PAGE. In both cases no molecular masses typical of ATIs

321

were detected after enzymatic hydrolysis (data not shown). The total ATI content (sum

322

of 0.19+0.53, 0.28, CM2, CM3 and CM16) was 188.2 mg/g based on SIDA. The most

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abundant ATIs were 0.19+0.53 with 79.3 mg/g and the least abundant one was CM2

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with 8.5 mg/g (0.28: 48.4 mg/g; CM3: 31.3 mg/g and CM16: 20.7 mg/g). The ATI

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reference consisted of 38% protein and 62 % other ingredients such as starch, minerals

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and other small molecules. Considering the total protein content in the ATI reference of

327

380.0 mg/g determined by RP-HPLC-UV, 49.5% of proteins belonged to the analyzed

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ATIs (188.2 mg/g of 380.0 mg/g). The distribution of the different ATIs (20.9%

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0.19+0.53, 12.7% 0.28, 2.2% CM2, 8.2% CM3 and 5.5% CM16; 50.5 % other proteins)

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in the ATI reference was hard to compare to that in real grain samples,7,13 because the

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preparation procedure is unknown. Nevertheless, the contents of 0.19+0.53 and CM3

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(58.8%) made up more than 50% of the total ATI content, as has been reported earlier 15 ACS Paragon Plus Environment


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(Figure 1).7 The proportion of the other ATIs were lower (0.28: 25.6%; CM2: 4.6%;

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CM16: 11.0%). Then, all iBAQ protein intensities of all identified proteins were summed

335

up and the percentages of single ATIs were calculated considering this normalized total

336

iBAQ intensity. The predominant ATIs were 0.19+0.53 with 17.7% followed by 0.28

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(14.9%), CM3 (9.0%), CM16 (4.8%) and CM2 (4.2%). In total, 50.6% of identified

338

proteins belonged to the ATIs of interest by iBAQ (other identified proteins: non-specific

339

lipid transfer protein, ß-amylase, thioredoxin H, and others), which agreed perfectly with

340

the 49.5% determined by SIDA. Some differences in the distribution of ATIs were

341

detected (Figure 1), especially for CM2 (SIDA: 4.6%, iBAQ 8.3%). Small differences

342

were obtained for 0.19+0.53, which was higher by SIDA (+15%), and for 0.28, which

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was higher by iBAQ (+14%), but the contents of CM3 and CM16 agreed very well.

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Reasons for the differences could be the low accuracy of the iBAQ algorithm, because it

345

is assumed that the distribution of peptide intensities per protein is constant between all

346

proteins, which is not true for all proteins.

347

All in all, the results of iBAQ and SIDA appeared to be comparable for the ATI reference

348

and so this comparison was expanded to two cultivars per wheat species (Figure 2).

349

Einkorn is not shown, because the iBAQ intensities were at least 1000fold lower than

350

those of other wheat species, SIDA results were below the LOD and no peptides were

351

identified in Skyline. The percentages of 0.19+0.53 were perfectly comparable for both

352

methods in all samples, but some differences between iBAQ and SIDA were detected

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for the other ATIs. Higher percentages of CM2 and lower percentages of CM16 were

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detected in durum wheat and emmer by iBAQ compared to SIDA, while the values for

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0.28 were higher in all wheat species. The values for CM2 and CM16 matched well for

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common wheat and spelt, and those of CM3 for durum wheat and emmer. Compared to 16 ACS Paragon Plus Environment

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untargeted LC-MS/MS, SIDA is less affected by sample complexity and background and

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typically has lower LODs, wider dynamic range and excellent repeatability and

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reproducibility, because of nonredundant targeted data acquisition.28 However, one

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major drawback is that everything except the predefined marker peptides is filtered

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out,29 even if there may be naturally occurring variations resulting in amino acid

362

substitutions or deletions. These are in turn picked up by iBAQ, so that both methods

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are complementary, even though iBAQ also has drawbacks, e.g., due to irreproducible

364

ion selection during data-dependent acquisition.30 Due to the overall good agreement of

365

SIDA and iBAQ, the quantitative accuracy of SIDA was substantiated.

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SIDA quantitation of ATIs in different wheat species. The ATI contents of flours of

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eight cultivars per wheat species (40 samples grown at the same location), which had

368

already been characterized in detail for protein composition,18 were analyzed by SIDA

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(Figure 3, Table S-5).

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The contents of ATIs 0.19+0.53 were significantly higher in common wheat (1.47 -

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1.89 mg/g) and spelt (1.51- 2.08 mg/g) than in durum wheat (0.84 - 1.15 mg/g) and

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emmer (0.90 - 1.25 mg/g) and they were detectable in three out of eight einkorn

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cultivars, but with low contents (0.12 - 0.29 mg/g). Common wheat (0.31 - 0.40 mg/g),

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spelt (0.30 - 0.55 mg/g) and emmer (0.24 - 0.33 mg/g) had similar contents of ATI 0.28.

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Within durum wheat, two out of eight cultivars (LUN and WIN, 0.21 mg/g and 0.26 mg/g)

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had comparable contents of ATI 0.28 to common wheat, spelt and emmer, but the

377

content was near the LOD or even below (< 6.6 µg/g) in the other six cultivars. The

378

analysis of two of these six durum wheat cultivars (AUR and LOG) by iBAQ (Figure 2)

379

confirmed the same low content of 0.28. Therefore, all peptides of 0.28 were not

380

detectable (

trypsin

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