Catcher of the Rye: Detection of Rye, a Gluten-Containing Grain, by

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Catcher of the rye – detection of rye, a gluten-containing grain, by LC-MS/MS Daniel Pasquali, Malcolm Blundell, Crispin A. Howitt, and Michelle L Colgrave J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.9b00314 • Publication Date (Web): 23 Jul 2019 Downloaded from pubs.acs.org on July 23, 2019

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Journal of Proteome Research

Catcher of the rye – detection of rye, a gluten-containing grain, by LC-MS/MS Daniel Pasquali1, Malcolm Blundell2, Crispin A. Howitt2 and Michelle L. Colgrave1* 1. CSIRO Agriculture and Food, 306 Carmody Rd, St Lucia QLD 4067, Australia; 2. CSIRO Agriculture and Food, GPO Box 1700, Canberra ACT 2601, Australia Corresponding author: Michelle L. Colgrave, CSIRO Agriculture and Food, 306 Carmody Rd, St Lucia QLD 4067; Phone: +61 (0)7 3214 2697; Email: [email protected]

Abstract Rye, wheat and barley contain gluten, proteins that trigger immune-mediated inflammation of the small intestine in people with coeliac disease (CD). The only treatment for CD is a lifelong gluten-free diet. To be classified as gluten-free by the World Health Organisation the gluten content must be below 20 mg/kg, but Australia has a more rigorous standard of no detectable gluten and not made from wheat, barley, rye or oats. The purpose of this study was to devise an LC-MS/MS method to detect rye in food. An MS-based assay could overcome some of the limitations of immunoassays, wherein antibodies often show cross-reactivity and lack specificity due to the diversity of gluten proteins in commercial food and the homology between rye and wheat gluten isoforms. Comprehensive proteomic analysis of 20 rye cultivars originating from 12 countries enabled the identification of a panel of candidate rye-specific peptide markers. The peptide markers were assessed in 16 cereal and pseudo-cereal grains, and in 10 breakfast cereals and 7 snacks foods. One of two spelt flours assessed was contaminated with rye at a level of 2% and trace levels of rye were found in a breakfast cereal that should be gluten-free based on its labelled ingredients. Keywords: gluten, Coeliac disease (CD), proteomics, LC-MS, rye, Secale cereale

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Introduction Rye (Secale cereale) is a member of the grass family Poaceae, which is one of the largest families of monocotyledonous flowering plants with wide global distribution. Rye was first domesticated and cultivated in southern Anatolia (now known as Syria) by the early Neolithic and it slowly spread in Europe as a weed among other cereals (1). At the beginning of the pre-Roman Iron Age the harvesting methods changed and rye turned from weed to cultivar, due to its ability to grow in poor soils and hostile climates (2). It is widely used as a grain, as a fermentable material and to a limited extent as fodder (1). Nowadays, rye is consumed worldwide, but there is higher consumption in Eastern Europe where it is used in bread, crackers, beer and whisky. Bread made exclusively from rye flour has a dense texture and rich flavour, and for this reason is often mixed with wheat to lighten the loaf in terms of both colour and texture. Rye flour contains high levels of soluble dietary fibre (3.2% on a daily basis, db) in comparison to wheat (1.1% db), barley (1.1% db), oats (1.8% db) and millet (1.9% db) (3). High levels of dietary fibre provide beneficial health effects including appetite reduction, decreasing the risk of intestinal disease, serum cholesterol reduction and regulating blood glucose levels (4). Rye, like the closely related cereals, wheat and barley, contains gluten. The dominant proteins of rye can be classified into the four Osborne fractions: (1) water-soluble albumins; (2) salt-soluble globulins; (3) the alcohol-soluble prolamins; and (4) insoluble glutelins. The latter glutelins are characteristically polymerised by interchain disulfide bonds, and are only soluble in alcohol in the presence of reducing agents (5). The last two groups comprise the secalins. The secalins share homology with the glutenins and gliadins from wheat and the hordeins from barley. Collectively these proteins are called gluten (from Latin gluten, "glue") (6). Gluten proteins play a critical role in the baking process through the creation of a viscoelastic dough defined by four properties: extensibility, resistance to stretch, mixing tolerance and gas holding ability (7). These properties are important in the food industry in the production of baked goods. However the gluten proteins are also the causative agent of coeliac disease (CD) which affects ~1% of the global population (8). The high proportion of proline and glutamine in these proteins results in


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incomplete digestion producing protein fragments (peptides) that can trigger an immunological reaction. Consequently, the ingestion of gluten in genetically-susceptible individuals results in damage to the small intestine and a diverse range of gastrointestinal symptoms. With no cure, CD is treated by a lifelong gluten-free diet (9). Most jurisdictions, including Europe and North America allow the use of a gluten-free label if the product contains no more than 20 mg gluten/kg as endorsed by the Codex Alimentarius (10) and the US FDA (11), but Australia has a more rigorous standard of no detectable gluten. Accurate quantitation of the gluten content in foods is critical for those who need to avoid gluten in their diets. Current commercial test kits based on the use of antibodies in the enzyme-linked immunosorbent assay (ELISA) are available, but questions remain as to the accuracy of these assays, most concerning is the possibility of false negatives (12). To date no single antibody exists that reacts with all the gluten protein types from wheat, rye and barley (13). There are more than 30 different ELISA kits to detect gluten in foods (14) and they are mainly used to detect wheat prolamins (gliadins) (15). These kits are based on the R5, G12, α20 and Skerritt monoclonal antibodies (mAbs) or several polyclonal antibodies (pAbs). Three of them (Skerritt, G12 and α20) were raised against wheat gliadin or gliadin peptides, while the R5 was raised against rye secalins (16). These antibodies are semi-specific to the prolamin fraction and as such a conversion factor of 2 is used based on the assumption of a 1:1 ratio of prolamins to glutelins (10). This assumption is a broad generalisation and it is known that: (1) the ratio varies in different cultivars of wheat (17, 18); (2) the ratio is impacted by environmental conditions during grain development (19) and references therein; (3) the ratio differs in rye and barley (16), and (4) the ratio can be highly skewed in processed food products. All of these assumptions provide the potential to introduce errors into the results. Mass spectrometry (MS) is a complementary method that can be used for gluten detection and quantitation. LC-MS has been used for disease biomarker elucidation and verification, owing to its sensitivity, precision, accuracy, and robust quantitative ability (20, 21). Multiple reaction monitoring (MRM) is a targeted proteomics approach that can be used for protein quantitation and validation of peptide markers for clinical applications (22), but also shows great promise in food testing applications 3 ACS Paragon Plus Environment


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(23). LC-MRM-MS has been applied to gluten detection in food and beverages (24-31). Previously, cereal-specific methods for wheat (26), barley (24) and oats (32) have been devised, yet the paucity of rye protein sequences in protein databases has slowed the development of similar methods for rye.

Experimental Plant material Twenty cultivars of rye (Table 1) were obtained from the Australian Winter Cereals Collection (AWCC, Tamworth, Australia). Additionally, grains of barley cv Sloop, wheat cv Chara, rye cv Sunrye, oats cv Jumbo, millet, maize cv W22 and rice cv Nippon were also obtained from AWCC. Grains of green wheat (Freekeh™), amaranth, chia, quinoa, sorghum and tef and flour samples of rye, sorghum, buckwheat, soy, oats and millet were obtained from local health food stores. All grains were milled using a Metefem Hungarian Mill (model FQD2000, Hungary). Fine flour was obtained by sieving the wholemeal with a 300 μm sieve (Endecotts Pty Ltd. Sieves, London, England). Table 1. Protein and peptide identifications (at 1% global false discovery rate, FDR) in rye cultivars following protein extraction with 55% isopropyl alcohol/2% dithiothreitol (IPA/DTT) buffer and digestion with trypsin. #

Cultivar

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Saskatoon Everest Petka Petkuser Somerroggen King II Gazelle Carsten Roggen Castelo Branco Emory Ratborske M67-170 Pamirskaja Svalofs Ponsi Frontier Saratovskaja 5 Dacold Centeio BR1 Bevy Eho Westwood

AUS Identity 17681 17682 17709 17710 16010 18358 19064 19440 19504 20234 20434 20453 20912 24526 25913 26288 26882 36000 36395

Country Australia France South Africa South Africa Sweden Canada Germany Portugal United States Czechoslavakia* Soviet Union Sweden Canada Soviet Union United States Brazil Australia Austria Australia

Proteins @ 1% FDR 128 131 150 153 146 107 160 164 133 127 165 162 140 140 127 144 139 156 141

Peptides @ 1% FDR 833 863 917 1044 963 822 879 934 834 801 955 940 993 835 958 913 874 861 901 4

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20

Kenya 12 90481 Australia 113 Combined Results 204 *Name of the country at the time when accession was deposited into GenBank.

691 1457

Breakfast cereals and snacks A selection of breakfast cereals and snacks were obtained from a local supermarket based on the primary ingredients denoted by an asterisk in the ingredient list. The cereal grain ingredients of the breakfast cereals (BC) were: (BC1) wheat* and barley malt; (BC2) corn* and barley malt; (BC3) barley*, amaranth; (BC4) rice*, corn, sorghum, buckwheat and psyllium; (BC5) corn*, rice, millet and amaranth; (BC6) oats*; (BC7) oats*, barley, triticale, wheat, rye and rice; (BC8) wheat*, oats, triticale, barley, rye, rice and quinoa; (BC9) sorghum*; (BC10) oats*, wheat, barley, rye, quinoa, rice, corn and triticale. The cereal grain ingredients of the snack food (SF) were: (SF1) rice*; (SF2) corn* and wheat; (SF3) wheat*, rye and rice; (SF4) wheat*, rye and rice; (SF5) rye*; (SF6) wheat*; (SF7) wheat*, barley, rye and corn. All the BC and SF were milled as described for the cereal grains to produce a fine flour.

Gluten extraction The gluten-enriched fraction was prepared by dissolving grain or food-derived flour (20 mg) in 200 μL 55% (v/v) propan-2-ol (IPA), 2% (w/v) dithiothreitol (DTT) with incubation at 50°C for 30 min (33). The solutions were centrifuged for 10 min at 20,800 xg and the supernatant was used for subsequent analysis.

Protein digestion Gluten-enriched extracts (100 μL, n=4) were applied to a 10 kDa molecular weight cut-off filter (Millipore, Australia), washed with two 200 μL volumes of 8 M urea, 100 mM Tris-HCl (pH 8.5) with centrifugation (20,800 xg, 15 min). For cysteine alkylation, 100 mM iodoacetamide in 8 M urea, 100 mM Tris-HCl was added (100 μL) and incubated at RT in the dark for 20 min. The filters were centrifuged (20,800 xg, 10 min) to remove excess iodoacetamide and washed with 200 μL volumes of 5 ACS Paragon Plus Environment


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8 M urea, 100 mM Tris-HCl with centrifugation (20,800 xg, 15 min). The buffer was exchanged using 100 mM ammonium bicarbonate (pH 8.0) by two consecutive wash/centrifugation steps. Sequencing grade porcine trypsin (Sigma-Aldrich, Sydney Australia) at a concentration of 250 μg/mL in 100 mM ammonium bicarbonate (200 μL, ~20:1 protein to enzyme ratio) was added to the protein on the 10 kDa filters and incubated for 16 h at 37°C in a wet chamber. The filters were transferred to fresh centrifuge tubes and the filtrates (digested peptides) were collected following centrifugation (20,800 xg, 15 min). The filters were washed with 200 μL of 100 mM ammonium bicarbonate and the filtrates were combined and lyophilised. The tryptic peptides were resuspended in 100 μL of 1% formic acid and stored at 4°C until analysis.

Global proteomic profiling

Gluten-enriched fractions (5 μL) of 20 rye cultivars were analysed with chromatographic separation using a nano HPLC system (Shimadzu Scientific, Rydalmere, Australia) directly coupled to a 6600 TripleTOF MS (SCIEX, Redwood City, USA). The peptides were desalted for 5 min on a ChromXP C18 (3 μm, 120 Å, 10 × 0.3 mm) trap column at a flow rate of 10 μL/min solvent A and separated on a ChromXP C18 (3 μm, 120 Å, 150 mm × 0.3 mm) column at a flow rate of 5 μL/min. The solvents used were (A) 5% DMSO, 0.1% formic acid, 94.9% water and (B) 5% DMSO, 0.1% formic acid, 90% acetonitrile, 4.9% water. A linear gradient from 5 to 45% solvent B over 40 min was employed followed by 45−90% B over 5 min, a 5 min hold at 90% B, return to 5% B over 1 min, and 14 min of re-equilibration. The eluent from the HPLC was directly coupled to the DuoSpray source of the TripleTOF 6600 MS. The ion spray voltage was set to 5500 V; the curtain gas was set to 138 kPa (20 psi), and the ion source gas 1 and 2 (GS1 and GS2) were set to 103 and 138 kPa (15 and 20 psi). The heated interface was set to 200°C. Data were acquired in information-dependent acquisition (IDA) mode. The IDA method consisted of a high-resolution time-of-flight (TOF)-MS survey scan followed by


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30 MS/MS scans, each with an accumulation time of 40 ms. First stage MS analysis was performed in positive ion mode over the mass range of m/z 350−1800 with a 0.25 s accumulation time. Tandem mass spectra were acquired on precursor ions that exceeded 200 counts/s with charge state 2−5. Spectra were acquired over the mass range of m/z 100−2000 using the manufacturer’s rolling collision energy (CE) based on the size and charge of the precursor ion and a collision energy spread (CES) of 5 V for optimum peptide fragmentation. Dynamic ion exclusion was set to exclude precursor ions after one occurrence with an 8 s interval and a mass tolerance of 50 ppm, and peaks within 6 Da of the precursor mass were excluded. Protein identification was undertaken using ProteinPilot™ 5.0 software (SCIEX) using the parameters: Sample type: Identification; Cys Alkylation: Iodoacetamide; Digestion: Trypsin; Instrument: TripleTOF 6600; ID Focus: Biological Modifications; and, Search Effort: Thorough ID. Tandem mass spectrometry data collected for the 20 rye cultivars was searched against in silico tryptic digests of Poaceae proteins of the Uniprot database (version 2019/01; 1,795,884 sequences) appended with the Common Repository of Adventitious Protein (cRAP). Protein identification reports, based on a combined database search of all samples, were exported to Excel: Supp. Table 1. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD013459 and are available in CSIRO’s Data Access Portal via https://doi.org/10.25919/5cafc86e5362f.

Identification of prototypic peptides for cereal contamination Peptide summaries generated by ProteinPilot were used to select rye peptides that yielded intense peaks and were fully tryptic, i.e. no unusual/variable or missed cleavages. The peptides were subjected to homology searching using the UniProt BLASTp server limited to the taxonomy Poaceae to identify peptide markers that are found in other grains. Three MRM transitions were designated for each peptide where the precursor ion (Q1) m/z and the fragment ion (Q3) m/z values were determined from the data 7 ACS Paragon Plus Environment


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collected in the discovery experiments. The initial 14 rye peptides (Supp. Table 2) were refined to nine peptides with three transitions each for use in the final method.

Targeted MS Reduced and alkylated tryptic peptides (5 μL or 20 μL for contamination confirmation) were chromatographically separated on a Shimadzu Nexera UHPLC and analysed on a 6500 QTRAP mass spectrometer (SCIEX) as described previously (25). Peptides were chromatographically separated on a on a Kinetex C18 column (2.1 mm x 100 mm, Phenomenex) using a linear gradient of 5–45% acetonitrile (in 0.1% formic acid) over 10 min at a flow rate of 400 μL/min. The eluent from the HPLC was directed to a QTRAP 6500 mass spectrometer (SCIEX) equipped with a TurboV ionisation source operated in positive ion mode for data acquisition and analysis. The MS parameters were as follows: ion spray voltage, 5500 V; curtain gas, 35; GS1, 40; GS2, 50; source temperature, 500°C; declustering potential, 70 V; and entrance potential, 10 V. Peptides were fragmented in the collision cell with nitrogen gas using rolling collision energy dependent on the size and charge on the size and charge of the precursor ion. Quantitation was achieved using scheduled MRM scanning experiments using a 40 s detection window for each MRM transition and a 0.3 s cycle time. Analyst 1.7™ software was used for data acquisition. Peaks were integrated using MultiQuant v3.0 (SCIEX) wherein all three transitions were required to co-elute at the same retention time (RT, min) with a signal-to-noise (S/N) > 3 for detection and a S/N > 5 for relative quantitation. Graphs were generated in Graphpad Prism v6 (San Diego, CA, USA).

Results and Discussion

Defining rye peptide markers using discovery proteomics Mass spectrometry (MS) has shown great potential as an alternative technology to antibody-based approaches for the detection and quantification of gluten (25, 33). The development of a MS-based 8 ACS Paragon Plus Environment

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approach relies upon the accurate characterisation of the proteins that exist in the gluten-containing cereals and the identification of peptide markers that are specific for the gluten proteins from each cereal and sensitively detected in complex matrices. In this study, 20 rye cultivars from 12 different countries (Table 1) have been comprehensively characterised using discovery proteomics to identify candidate rye-specific peptide markers that are useful across a wide variety of commonly used rye lines. The gluten-enriched fraction extracted from each rye cultivar was reduced with dithiothreitol, alkylated with iodoacetamide to prevent re-oxidation of cysteines and subjected to tryptic digestion to identify the proteins and the peptides showing the highest response in the LC-MS/MS system. The numbers of proteins and peptides identified (at a 1% false discovery rate, FDR) in the gluten-enriched fraction of each cultivar is summarised in Table 1 with the complete protein list presented at Supp. Table 1. Those proteins that were identified included a suite of secalins (γ, ω-) alongside protease inhibitors (α-amylase/trypsin inhibitors), lipid transfer proteins, defensins, peroxiredoxins, oleosins, serpins and various enzymes (e.g. dehydroascorbate reductase, protein disulfide isomerase). The tryptic peptide products identified in the global LC-MS/MS analyses were assessed to identify prototypic peptides that were unique to rye and that yielded high response in the MS as judged by the peak area/intensity.

Selection of rye peptide markers An initial set of 20 rye peptide markers (RPMs, Table 2) was selected based upon peptide intensity in the discovery experiments and these were subjected to preliminary MRM analysis. Peptide transitions were selected based on their intensity and m/z values wherein product ions with m/z values (Q3) > precursor ion m/z (Q1) were preferred as they tend to be free of interferences, i.e. isobaric compounds with identical precursor-product ion m/z values. Peptide MRM transitions that showed interference, broad peaks and/or low intensity peaks were excluded from further analysis leaving 14 RPMs.



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The peptide sequences were subjected to homology searching (using the UniProt BLASTp server) against all other cereals to assess their specificity to rye (Table 2). For example, the peptide EGVQILLPQSHK (RPM-B) from the 75k γ-secalin, (UniProt: E5KZQ5), was detected with high precursor intensity and did not yield 100% BLASTp matches against any proteins other than from the taxonomy Secale. In contrast, the peptide QCSTIQAPFASIVTGIVGH (RPM-I) from the same protein also mapped to the wheat protein E5KZQ7. Potential orthologous matches for eight of the 14 RPMs (Fig. 1G-N) were found. For example, the peptides IETPGPPYLAK and SRPDQSGLMELPGCPR deriving from the α-amylase/trypsin inhibitor (Q45FA6, Fig. 1M-N) were also noted to be present in barley (as ATI CMb, P32936) or wheat (as ATI CM16, P16159). These peptides (RPMs G-N) were retained for experimental validation of their non-specificity to rye.


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Table 2. Candidate peptide markers identified in a combined database search of all 20 rye cultivars.

Peptide sequence EHDVQEGQVGTTGAFPR EGVQILLPQSHK QHVGQGALAQVQGIIQPQQLSQLEVVR NVLLQQCSPVALVSSVR SVGGQCVPGLAMPHNPLGACR

Ranka 12 1 13 14 18

Uniprot accession C3VWV8 E5KZQ5 E5KZQ5 E5KZQ6 Q7M220

Unique by BLASTp Yes Yes Yes Yes Yes

Protein name Dimeric α-amylase inhibitor 75k γ-secalin 75k γ-secalin 75k γ-secalin Trypsin inhibitor Major Baker’s Asthma allergen SEC C 1 75k γ-secalin 75k γ-secalin 75k γ-secalin Omega-secalin Dimeric α-amylase inhibitor Monomeric α-amylase inhibitor Cereal-type amylase inhibitor Cereal-type amylase inhibitor Alpha-gliadin Alpha-gliadin 75k γ-secalin Avenin-like A protein Dimeric α-amylase inhibitor Trypsin inhibitor

% variation across cultivars 25.5 37.4 47.6 48.0 41.3

RPMb (A) (B) (C) (D) (E)

SISNNPVPACR 5 Q9S8H2 Yes 30.1 (F) NVLLQQCSPVALVSSLR 7 E5KZQ2 No 42.6 (G) SLVLQNLPTMCNVYVPR 6 E5KZQ2 No 33.4 (H) QCSTIQAPFASIVTGIVGH 15 E5KZQ2 No 89.9 (I) QLNPSEQELQSPQQPVPK 9 Q43639 No 36.8 (J) LTAASITAVCR 2 C3VWV8 No 29.1 (K) LTAASVPEVCK 3 C4P627 No 53.6 (L) IETPGPPYLAK 11 Q45FA6 No 34.0 (M) SRPDQSGLMELPGCPR 10 Q45FA6 No 28.0 (N) DVMLLQCDIITPSFK 20 F4ZL26 No 48.1 (O) QYSWDVGTFK 17 F4ZL26 No 46.9 (P) SLVLQNLPTMCNIYVPR 19 H6ULI8 No 87.7 (Q) QQCCQPLAQISEQAR 8 Q2A781 No 23.7 (R) EGTEVFPGCR 4 A0A3B6PE52 No 53.2 (S) ILMDGVVTQQGVFEGGYLK 16 Q7M220 No 51.1 (T) a. Rank is according to peptide peak area in LC-MRM-MS experiments. b. Rye peptide markers were selected based on theoretical specificity to rye after homology searching: (A-F) are theoretically unique to Secale species; (G-T) are common to orthologous proteins in the Poaceae subset of the UniProt database.



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Experimental assessment of the rye peptide markers These 14 potential peptide markers (RPMS A-N) were reassessed across the 20 commercial rye varieties (Table 1), to determine their potential suitability for more general use in food applications where different rye varieties are used. To compare the peptide expression pattern, the response (peak area) of each peptide was converted to a percentage relative to the average peak area of that peptide across all tested rye cultivars (Supp. Table 3). In this way two peptides that had peak areas that differed by orders of magnitude could be graphically compared. The technical variation (i.e. peak area variation between identical replicates) for this experiment was on average below 13%. The variation (biological) of the peptide peak area across the rye cultivars ranged from 26% up to 90% (Table 2). In addition to the peak area itself, the MRM peak area ratios (comparing each of the three transitions) were used as an additional quality control measure to ensure correct peak annotation. Based on these data, the Cterminal peptide of E5KZQ2 (Fig. 1I) was excluded owing to high technical variation (~31%) and high biological variation observed between cultivars (90%). This variation is likely due to exoprotease trimming of the C-terminus of the protein. In a few instances, two peptides deriving from a single protein (i.e. E5KZQ5, Fig. 1B-C; Q45FA6, Fig. 1M-N) were monitored and it was noted that the peptide levels followed the same pattern, allowing the deduction that the peptides were representative of their parent protein expression levels.


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Figure 1. Assessment of peptide markers across diverse rye varieties. The 14 candidate rye peptide markers were assessed across 20 commercial rye cultivars lines. The protein accession, peptide sequence, ranking by the average MRM peak area of all 20 cultivars (indicated in parentheses), and variation across 20 cultivars (expressed as a percentage) and are annotated in the header. Columns are coloured according to theoretical/experimental specificity to rye: black, theoretically and experimentally specific; grey, experimentally specific; white, theoretically and experimentally nonspecific. The panels are annotated (A) to (N) according to the RPM label.



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To experimentally verify the specificity of the RPMs, a collection of 16 cereal and pseudocereal grains (Fig. S1, Table 3) were analysed. Three of the RPMs (labelled K, L and N) showed very high levels in spelt and wheat. Two RPMs (labelled M and N) also showed high responses in barley (Q45FA6) confirming the previous BLASTp result. The six RPMs that were rye-specific according to the homology searches were notably absent in all grains other than spelt flour wherein the level detected was ~2% (Table 3). This suggested that the spelt flour contained a low level of rye and warranted further investigation. This batch of spelt flour was re-tested alongside a newly sourced spelt flour, together with rye (Fig. S2). The six specific RPMs were not detected in the second batch of spelt flour, but were detected in the original batch, confirming the suspicion that the first spelt flour under exam was contaminated with ~2% rye flour, but also demonstrating that the six RPMs were rye specific. From a consumer health perspective, the presence of rye in spelt is of no concern as both commodities contain gluten and are not suitable within the diet of people with CD, but this detection highlights the commonality of agricultural co-mingling. A single peptide (RPM-B) was detected in wheat (~0.3%, Figure S1) but this was notably the most sensitive of the RPMs capable of detecting trace levels of rye.


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Table 3. Rye detection in flour derived from cereal grains. A selection of 16 cereals and pseudocereals were assessed revealing contamination of rye in spelt and wheat. No rye was detected in quinoa, chia, sorghum, buckwheat, teff, millet, soy, rice, amaranth, corn, oats, or green wheat Freekeh and as such these are not listed herein. The detection is expressed as a percentage relative to rye (100%). The table is segregated by peptides markers that are theoretically (BLASTp analysis) specific and experimentally (LC-MRM-MS) unique, and non-specific RPM

Rye Mean ± SD

CV (%)

A B C D E F Mean

100 ± 9.2 100 ± 5.1 100 ± 15.5 100 ± 14.1 100 ± 4.6 100 ± 2.8

9.2 5.1 15.5 14.1 4.6 2.8

G H I J Mean

100 ± 16.5 100 ± 7.9 100 ± 35.5 100 ± 8.2

16.5 7.9 35.5 8.2

K L M N

100 ± 4.2 100 ± 6.5 100 ± 6.1 100 ± 5.9

4.2 6.5 6.1 5.9

Barley Spelt Mean ± SD Mean ± SD Specific peptide markers 0.00 ± 0.00 N/A 1.55 ± 0.17 0.00 ± 0.00 N/A 1.97 ± 0.13 0.00 ± 0.00 N/A 1.78 ± 0.25 0.00 ± 0.00 N/A 2.27 ± 0.09 0.00 ± 0.00 N/A 2.13 ± 0.21 0.00 ± 0.00 N/A 2.53 ± 0.15 0.00 ± 0.00 2.04 ± 0.17 Experimentally-unique peptide markers 0.00 ± 0.00 N/A 3.42 ± 0.33 0.00 ± 0.00 N/A 3.39 ± 0.21 0.00 ± 0.00 N/A 0.00 ± 0.00 0.00 ± 0.00 N/A 0.00 ± 0.00 0.00 ± 0.00 1.70 ± 0.14 Non-specific peptide markers 0.11 ± 0.02 16.1 96.4 ± 8.9 0.24 ± 0.04 16.5 547.8 ± 3.5 50.5 ± 8.40 16.6 1.8 ± 0.17 51.1 ± 10.7 20.9 193.2 ± 12.3

CV (%)

Wheat Mean ± SD

CV (%)

10.8 6.8 14.1 4.1 9.7 5.9

0.00 ± 0.00 0.34 ± 0.04 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 < 0.34 ± 0.04

N/A 12.4 N/A N/A N/A N/A

9.7 6.1 N/A N/A

0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

N/A N/A N/A N/A

9.2 3.5 9.4 6.4

99.9 ± 2.1 56.5 ± 6.8 0.00 ± 0.00 211.7 ± 23.7

2.1 12.0 N/A 11.2



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A further four RPMs (labelled G-J) that database analysis suggested were common to wheat were not detected in the wheat sample, and thus were retained as candidate markers, with the exception of RPM-I which had been earlier deemed unsuitable due to high technical and biological variation. A limitation of this experiment was in the fact that only a single cultivar of each cereal or pseudocereal was examined, and thus it is possible that these theoretically non-specific peptide markers, while absent in the studied varieties may be present in other cultivars used globally. One of these peptide markers (RPM-G, NVLLQQCSPVALVSSLR) was also identified as a rye-specific marker in a recent study wherein the authors aimed to develop a multi-allergen LC-MS/MS method (34). To further examine the utility of RPMs G-J, the complete suite of 14 RPMs were analysed across 14 wheat cultivars (26) and 12 barley cultivars (24) (Figure S3A). This analysis affirmed the absence of these peptides (RPM A-J) across all cultivars, and confirmed that RPM-K, -L and -N were also found in wheat (14/14 cultivars) and RPM-M (10/12 barley cultivars) and RPM-N (12/12 barley cultivars) were also found in barley (Figure S3B).

Assessment of the rye peptide markers in commercial foods In order to demonstrate the utility of the LC-MRM-MS approach for the detection of rye in real world food products, a selection of 10 breakfast cereals and 7 snack foods were obtained from a local supermarket. Breakfast cereals are typically produced using thermal and/or pressure treatments commonly in the presence of sugars. These processes can lead to modification of the proteins, potentially altering both the m/z and chemical properties of the peptides that are used for LC-MS/MS analysis (or as epitopes for antibody techniques). Figure 2 show the detection of the nine RPMs across the range of breakfast cereals (BCs) and snack foods (SFs). Two of the products that contained rye as the primary ingredient (BC7 and SF5) were used as references for each food type. The results of the previous grain analysis confirmed that the RPMs were useful for detecting contamination of minimally processed grain flours (Figure S1). The MRM peak area was expressed as a percentage relative to the


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food product containing the highest level of rye (BC7 or SF5) (Supp. Table 4). At least five of the nine RPMs were detected in the products wherein rye was a labelled ingredient (BC7-8, BC10; and SF3-5, SF7). In BC5, a cereal not labelled as rye-containing, a single peptide (RPM-D) was detected at a level of 0.2% relative the rye-based cereal BC7. Four of the nine rye peptide markers (B, D, G and H) were also detected in SF2 where rye was not specifically stated as an ingredient with signals from ~0.1% to ~0.3% compared to SF5. The four detected peptide markers were among the five highest responding peptides, both in raw commodities but also in processed foods, adding weight to their detection despite the absence (below limit of detection, LOD) of the remaining four RPMs that yielded lower LC-MS responses.

Figure 2. Analysis of breakfast cereals (BC) and snack foods (SF). The 9 peptide markers were tested for their utility in processed food. All 9 peptide markers were detected in rye-containing breakfast cereals (BC7, BC8) and snack foods (SF3-5, SF7). The panels are annotated (A) to (J) according to the RPM label, noting RPM-I was excluded based on higher variation.

As the database analysis (BLASTp searches) indicated that RPMs G-J were common to wheat and the experimental data revealed the presence of RPMs K, L and N in wheat and of RPMs M and N 17 ACS Paragon Plus Environment


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in barley, these RPMs were explored in the processed foods (Figure 3). Two of the theoretically and experimentally-verified rye-specific peptide markers are presented in Fig. 3A-B and show a similar pattern of abundance across the food products. The two RPMs that appeared to be experimentally ryespecific but had sequences listed within annotated wheat proteins (Fig. 3C-D) also showed the same pattern of abundance. The peptide markers that were detected in wheat (Table 3) were detected not only in the products containing rye, but additionally in BC1, BC10, SF6 and at higher levels in SF3, SF4 and SF7, with all listed products labelled as wheat containing. Notably, that despite RPM-K, RPM-L and RPM-N revealing high levels of wheat in BC1 and BC10, there was no signal detected in these cereals using RPM-G and RPM-H, giving further evidence that annotation in a wheat protein in the public database should not be an absolute means of excluding peptides in studies such as this. The two peptide markers showing commonality to barley protein isoforms (Fig. 3G-H, Table 3) were detected uniquely in BC3, a cereal wherein barley was the primary ingredient. The results generated herein thus agreed with the ingredients as listed and the RPMs show promise as a means of rye detection.


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Figure 3. Analysis of breakfast cereals (BC) and snack foods (SF). Two peptide markers theoretically specific to rye that were experimentally-verified (A, B) and two experimentally-verified peptide markers that mapped to wheat protein isoforms (C, D) are shown. Three peptide markers that also detect wheat are shown (E, F, H) and two peptide markers that also detect barley are shown (G, H).



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Four of the breakfast cereals and a single snack food did not contain wheat, barley or rye according to the listed ingredients. BC4 listed rice, corn, sorghum, buckwheat and psyllium as ingredients and did not contain rye, nor evidence of wheat or barley using the common peptide markers. Likewise, BC6 listed as containing only oats; BC9 containing only sorghum, and SF1 made from rice, were all absent of rye, wheat and barley according to the LC-MRM-MS method applied. BC5, however, listed only corn, rice, millet and amaranth as ingredients, yet a single RPM (RPM-D from 75K γ-secalin, Supp. Table 4) was detected at a trace level of 0.22%. Additionally, the peptides that were common to wheat/barley (RPM K-N) were detected at levels ranging from 0.35-0.83%, indicating the presence of gluten-containing grains in this product, albeit at low levels. A conservative estimation of the gluten content was performed making the following assumptions: (1) 50% of the food product is grain (w/w, based on the ingredient listing); (2) 12% of rye grain is protein; (3) 50% of rye protein is gluten; and using 0.22% as the detected level, then the gluten content would be ~66 mg/kg (based only on rye). In a similar manner, if the value of 0.35-0.83% (for wheat) is used, for which there is a higher proportion of gluten in total protein (wheat is estimated to be ~75%), then such an estimate would also place this product above the 20 mg/kg threshold. The method applied in this study was based on relative quantitation and verification of the gluten-free status (or not) of this product would rely on the use of isotopically-labelled internal standards to determine the absolute level of rye/gluten in the finished product. Moreover, validation experiments wherein rye is spiked into complex matrices (with/without common processing techniques) to enable validation of the methods described herein are underway and will provide further support of the utility of LC-MS/MS for gluten detection in foods. The lack of globally available reference materials for rye (and other gluten-containing grains) remains a challenge, but progress towards the generation of gluten protein fractions has been made (35). The method developed in this study should be useful in validating such reference materials. This study differs from recent applications in the use of LC-MS/MS for gluten quantitation (31) wherein chymotrypsin was used as the digestion enzyme and seven rye peptide markers were selected and absolute quantitation was performed using a single heavy labelled peptide as the internal standard. In the previous study, a combination of four rye cultivars was used for peptide selection, whereas in


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this study the peptides were confirmed across 20 rye cultivars. The previous study has the benefit of being able to derive an absolute gluten level, but questions remain as to whether the correction factors employed will be useful across the genetic diversity of rye that exists globally. Additionally, the rye chymotryptic peptide used as the internal standard (ASIETGIVGH) is the C-terminal peptide of the 75K γ-secalin. In the present study, the C-terminal peptide of a 75K γ-secalin (E5KZQ2, Fig 1I) was notably the most variable, presumably due to the action of exoproteases, implying that terminal peptides may not be optimal choices as peptide markers. Of the seven rye peptide markers utilised in the previous study, five peptides were found in wheat orthologues within the UniProt database and four of seven contained (missed) chymotryptic cleavage sites, raising question about their suitability to uniquely and accurately quantify rye. Three of the RPMs used in this study also mapped to wheat orthologues, however, experimental validation indicated their suitability for rye detection. In fact, only one peptide from the previous study was theoretically unique to rye, was fully cleaved (by chymotrypsin) and the peptide showed low recovery (6.7%) (31).

Conclusions Using high resolution LC-MS/MS the protein profiles of 20 rye cultivars from 12 countries were generated providing a foundation for the identification and evaluation of rye-specific peptide markers. A number of potential rye peptide markers were assessed based on a range of criteria: uniqueness to rye; commonality to a wide range of rye varieties; sensitivity (high response by MS); and reproducibility (in digestion and analysis). The final peptide marker panel was demonstrated to be both highly specific and sensitive. The devised method revealed a 2% rye contamination in the original spelt flour under examination, yet a second spelt flour commercially purchased was free from contamination. The rye peptide marker panel (9/9 RPMs) showed utility in detecting rye in processed food products (breakfast cereals and snacks) where rye was a labelled ingredient and in detecting trace levels in food products not naming rye on the packaging. The panel of rye-specific peptide markers developed in this study will complement those previously developed for wheat and barley providing the ability to detect all glutencontaining grains in a sensitive and specific manner. LC-MS/MS represents a suitable technology for 21 ACS Paragon Plus Environment


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detecting gluten contamination which is critical for transparency in food labelling and ensuring the health and wellbeing of a subset of the population, including those affected by coeliac disease and noncoeliac gluten sensitivity.

Acknowledgements The authors thank the Molecular and Cellular Proteomics Facility at the University of Queensland for access to some of the mass spectrometers used in this study.

Supporting Information: The following supporting information is available free of charge at ACS website http://pubs.acs.org

Figure S1. Experimental validation of six theoretically specific and three theoretically non-specific rye peptide markers. Figure S2. Confirmation of rye peptide marker utility based on specificity. Figure S3. Assessment of rye peptide marker specificity across commercial wheat and barley cultivars. Table S1. Proteins identified in combined database search of spectra from 20 rye cultivars (at 1% global false discovery rate). Table S2. MRM transitions used for candidate peptide markers. Table S3. Variation of rye peptide markers amongst 20 commercial rye cultivars.. Table S4. Detection of nine rye peptide markers amongst 10 commercial breakfast cereals (BC) and 7 snack foods (SF).


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21. Parker, C. E.; Borchers, C. H., Mass spectrometry based biomarker discovery, verification, and validation – Quality assurance and control of protein biomarker assays. Molecular Oncology 2014, 8, (4), 840-858. 22. Gillette, M. A.; Carr, S. A., Quantitative analysis of peptides and proteins in biomedicine by targeted mass spectrometry. Nature methods 2013, 10, (1), 28-34. 23. Croote, D.; Quake, S. R., Food allergen detection by mass spectrometry: the role of systems biology. NPJ Systems Biology and Applications 2016, 2, 16022. 24. Colgrave, M. L.; Byrne, K.; Blundell, M.; Howitt, C. A., Identification of barley-specific peptide markers that persist in processed foods and are capable of detecting barley contamination by LC-MS/MS. J Proteomics 2016, 147, 169-76. 25. Colgrave, M. L.; Goswami, H.; Blundell, M.; Howitt, C. A.; Tanner, G. J., Using mass spectrometry to detect hydrolysed gluten in beer that is responsible for false negatives by ELISA. J Chromatogr A 2014, 1370, 105-14. 26. Colgrave, M. L.; Goswami, H.; Byrne, K.; Blundell, M.; Howitt, C. A.; Tanner, G. J., Proteomic profiling of 16 cereal grains and the application of targeted proteomics to detect wheat contamination. J Proteome Res 2015, 14, (6), 2659-68. 27. Fiedler, K. L.; McGrath, S. C.; Callahan, J. H.; Ross, M. M., Characterization of grainspecific peptide markers for the detection of gluten by mass spectrometry. Journal of Agricultural and Food Chemistry 2014, 62, (25), 5835-5844. 28. Gomaa, A.; Boye, J., Simultaneous detection of multi-allergens in an incurred food matrix using ELISA, multiplex flow cytometry and liquid chromatography mass spectrometry (LC-MS). Food Chemistry 2015, 175, 585-592. 29. Lock, S., Gluten Detection and Speciation by Liquid Chromatography Mass Spectrometry (LC-MS/MS). Foods 2014, 3, (1), 13-29. 30. Schalk, K.; Koehler, P.; Scherf, K. A., Targeted liquid chromatography tandem mass spectrometry to quantitate wheat gluten using well-defined reference proteins. PLoS One 2018, 13, (2), e0192804. 31. Schalk, K.; Koehler, P.; Scherf, K. A., Quantitation of Specific Barley, Rye, and Oat Marker Peptides by Targeted Liquid Chromatography-Mass Spectrometry To Determine Gluten Concentrations. J Agric Food Chem 2018, 66, (13), 3581-3592. 32. Dawson, C.; Mendoza-Porras, O.; Byrne, K.; Hooper, T.; Howitt, C.; Colgrave, M., Oat of this world: Defining peptide markers for detection of oats in processed food. Peptide Science 2018, 110, (3), e24045. 33. Tanner, G. J.; Colgrave, M. L.; Blundell, M. J.; Goswami, H. P.; Howitt, C. A., Measuring hordein (gluten) in beer--a comparison of ELISA and mass spectrometry. PLoS One 2013, 8, (2), e56452. 34. Henrottin, J.; Planque, M.; Huet, A. C.; Marega, R.; Lamote, A.; Gillard, N., Gluten Analysis in Processed Foodstuffs by a Multi-Allergens and Grain-Specific UHPLC-MS/MS Method: One Method to Detect Them All. J AOAC Int 2019. doi: 10.5740/jaoacint.19-0057 35. Schalk, K.; Lexhaller, B.; Koehler, P.; Scherf, K. A., Isolation and characterization of gluten protein types from wheat, rye, barley and oats for use as reference materials. Plos One 2017, 12, (2), e0172819.


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TOC Graphic



Figure 1. Assessment of peptide markers across diverse rye varieties. The 14 candidate rye peptide markers were assessed across 20 commercial rye cultivars lines. The protein accession, peptide sequence, ranking by the average MRM peak area of all 20 cultivars (indicated in parentheses), and variation across 20 cultivars (expressed as a percentage) and are annotated in the header. Columns are coloured according to theoretical/experimental specificity to rye: black, theoretically and experimentally specific; grey, experimentally specific; white, theoretically and experimentally non-specific. The panels are annotated (A) to (N) according to the RPM label. 195x259mm (300 x 300 DPI)


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Figure 2. Analysis of breakfast cereals (BC) and snack foods (SF). The 9 peptide markers were tested for their utility in processed food. All 9 peptide markers were detected in rye-containing breakfast cereals (BC7, BC8) and snack foods (SF3-5, SF7). The panels are annotated (A) to (J) according to the RPM label, noting RPM-I was excluded based on higher variation. 274x192mm (300 x 300 DPI)



Figure 3. Analysis of breakfast cereals (BC) and snack foods (SF). Two peptide markers theoretically specific to rye that were experimentally-verified (A, B) and two experimentally-verified peptide markers that mapped to wheat protein isoforms (C, D) are shown. Three peptide markers that also detect wheat are shown (E, F, H) and two peptide markers that also detect barley are shown (G, H). 275x131mm (300 x 300 DPI)


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Catcher of the Rye: Detection of Rye, a Gluten-Containing Grain, by

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