Analysis of Gluten in a Wheat-Gluten-Incurred Sorghum Beer Brewed

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Analysis of gluten in a wheat gluten incurred sorghum beer brewed in the presence of proline endopeptidase by LC-MS/MS Katherine L. Fiedler, Rakhi Panda, and Timothy R Croley Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04371 • Publication Date (Web): 12 Jan 2018 Downloaded from http://pubs.acs.org on January 12, 2018

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Analytical Chemistry

Analysis of gluten in a wheat gluten incurred sorghum beer brewed in the presence of proline endopeptidase by LC-MS/MS

Katherine L. Fiedler*, Rakhi Panda, and Timothy R. Croley

Center for Food Safety and Applied Nutrition U.S. Food and Drug Administration 5001 Campus Drive College Park, Maryland 20740, United States

*Corresponding author – Phone: 1 (240) 402 -5055, Fax: 1 (301) 436-2624 Email: [email protected] 1 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract (80-250 words) Most gluten-reduced beers are produced using an enzyme called proline endopeptidase (PEP), which proteolyzes the gluten by cleaving at proline residues. However, the gluten content of beers brewed in the presence of PEP cannot be verified since current analytical methods are not able to accurately quantitate gluten in fermented foods. In this work, mass spectrometry was used to qualitatively characterize the gluten in a wheat gluten incurred sorghum model beer brewed with and without the addition of PEP. Hydrolyzed gluten peptides and chymotryptic gluten peptides produced from intact gluten proteins were detected in beer brewed in the presence of up to six times the manufacturer’s recommended dosage of PEP. The observation of chymotryptic gluten peptides indicates that some gluten proteins remained, at least partially, intact after fermentation and enzymatic treatment. Less intact gluten was observed in beer brewed in the presence of PEP, but more hydrolyzed gluten peptides were consequently observed in PEP containing beer. Gluten peptides that contained immunogenic sequences known to be associated with celiac disease were detected in PEP containing beer.

Keywords: celiac disease, mass spectrometry, proteomics, gluten-reduced, hydrolyzed gluten, crafted to remove gluten

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Celiac disease (CD) is an autoimmune disorder that is triggered by the ingestion of gluten proteins from wheat, barley, rye, and their crossbred varieties.1 For the millions of Americans afflicted with CD, a diet devoid of gluten is the only successful treatment option. Consumers with CD must rely on truthful and accurate food labels to avoid dietary exposure to gluten. A number of barley-based beers that are labeled “crafted to remove gluten” have recently become commercially available. These beers are usually produced using an enzyme called proline endopeptidase (PEP), which cleaves at the C-terminus of proline residues.2-5 Gluten proteins from barley, known as hordeins, are unusually high in proline content (14-23%).6 Thus, it is presumed that proteolysis of gluten with PEP during the brewing process leads to a reduction in the gluten concentration of beer. When analyzed by competitive enzyme-linked immunosorbent assay (ELISA), beer brewed in the presence of PEP generally test for gluten concentrations of less than 20 ppm,4,7,8 which is the internationally accepted threshold for gluten in foods that can be tolerated by most people with CD.9,10 However, there is uncertainty in interpreting competitive ELISA results for the quantitation of hydrolyzed gluten in terms of equivalent amounts of intact gluten in hydrolyzed or fermented foods, such as beer.3,11-14 A study performed by Allred et al. demonstrated that sera from a small subset of active-CD patients reacted to gluten that is present in beer brewed in the presence of PEP.15 This study suggests that some individuals in the celiac population could be sensitive beers brewed in the presence of PEP. The main challenge facing the accurate quantitation of hydrolyzed gluten is the lack of an appropriate reference material. Even though a pepsin/trypsin digest of wheat, barley, and rye prolamins is commonly used as a reference material for hydrolyzed gluten in beer, Panda et al. demonstrated that the peptide profile of a pepsin/trypsin digested calibrant is inconsistent with the peptide profile generated during fermentation and in the presence of PEP.7 Additionally, the



antibodies in commercially available competitive ELISAs target only the prolamin portion of gluten and are not able to accurately detect glutelins. Antibodies which target different gluten proteins/peptides have been recently incorporated into a multiplex competitive ELISA.13 The nine different gluten-specific antibodies used in the assay provided a range of quantitative results for gluten in fermented-hydrolyzed foods, including gluten-reduced barley beers, depending on their target. In previous work using the same wheat gluten incurred model sorghum beer that is used in the current study, Panda et al. showed that the detectable gluten concentration was reduced in PEP containing beer compared to beer brewed in the absence of PEP when analyzed by a commercial competitive ELISA.7 The competitive ELISA had a higher LOQ (10 mg/kg) compared to sandwich ELISAs and displayed a high coefficient of variation. Mass spectrometry (MS) has been used to characterize the beer proteome and identify hordein proteins and peptides present in beer.16-19 A multiple reaction monitoring (MRM) MS method has also been developed for the relative quantitation of gluten in beer.20,21 Colgrave et al. employed selected hordein peptide markers to show that the B- and D-hordeins are more susceptible to hydrolysis during the brewing process compared to C- and γ-hordeins. Unfortunately, the absolute quantitation of gluten in beer via LC-MS/MS analysis is not currently possible. Multiple research groups have successfully utilized mass spectrometry to observe gluten in PEP containing beers that have shown no ELISA response for gluten.4,7,14,22 Hordein fragments > 10 kDa in size have recently been detected in barley-based PEP containing beers by Colgrave et al., suggesting that PEP digestion was incomplete in a number of commercially available gluten-reduced beers.14 In their analysis by LC-MS/MS, Akeroyd et al. proposed that PEP treatment of a barleybased beer degraded all known immunogenic epitopes, which are the sequences within gluten


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proteins and peptides that are responsible for eliciting a celiac response.22 However, additional immunogenic sequence containing gluten peptides which could not be detected or identified with the employed method could have been present in the PEP treated beers. Not all of the immunogenic sequences associated with CD are known due to the complex nature of gluten proteins and incomplete understanding of the pathogenesis of CD.15,23 Of the immunogenic sequences that are known, only a few are derived from barley.24 Even though mass spectrometrybased proteomics is a powerful tool, the identification of every protein and peptide in a given sample is not generally possible.25 Consequently, it is an effective approach to confirm the presence of an analyte; however, rigorous controls are needed when attempting to prove the absence of an analyte. The detection of certain peptides can be hindered by matrix effects, dynamic range, and/or physiochemical properties that are not amicable to the technique used.26 For example, peptides may not be observed if they are too hydrophilic or hydrophobic, too big or small, or do not ionize well. In addition, mass spectrometry-based proteomics relies on comparing experimentally acquired data to a database of known protein sequences. Unfortunately, publicly available databases of gluten protein sequences are incomplete and poorly curated due to the complex nature of the polyploid wheat genome. For example, not all of the known immunogenic sequences in the AllergenOnline database are in the commonly used UniProt protein database; therefore, if present in the beer, these sequences would not be identified with this technique. The goal of this study was to determine the effects of PEP on gluten during the brewing process by using mass spectrometry to characterize the gluten present in beers brewed with and without PEP. Since the immunological properties of wheat in CD have been studied more extensively compared to barley, a model system of sorghum beer incurred with wheat gluten was



used to determine how PEP treatment impacts the profile of immunogenic CD-related epitopes in beer. Sorghum beer incurred with either 0 or 200 mg/L (ppm) wheat gluten was brewed with and without the addition of PEP at the start of fermentation. The amount of gluten was controlled by the use of a commercially available wheat gluten standard instead of malted grains. This model system also provided the opportunity to analyze the gluten before and after fermentation in beer brewed in the presence or absence of PEP. The extensive hydrolysis of gluten proteins by proteinases and carboxypeptidases during malting and mashing is well characterized,27 but little is known about the hydrolysis of gluten proteins during fermentation.3,7,28 The beer samples were filtered to isolate the peptides produced via hydrolysis during fermentation and enzymatic processing, if PEP was present. The hydrolytic products were then analyzed directly by LCMS/MS. Intact gluten proteins were also precipitated from the beer samples in a separate analysis and then digested with chymotrypsin in a traditional bottom-up proteomics approach. This two pronged method differs in the approach typically employed for the analysis of gluten in beer in which chymotrypsin is added directly to the beer sample.4,20,22 By isolating the intact proteins separately, it was possible to distinguish chymotryptic peptides originating from intact gluten proteins from hydrolyzed gluten peptides that were present in the sample as a result of hydrolysis during fermentation and PEP activity.

Experimental Section Reagents Wheat gluten was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Sorghum syrup (Cargill Malt Americas, 100% sorghum, lot #H022836) was provided by Cargill (Wayzata, MN). Brewers Clarex (PEP), activity ≥5.0 Proline Protease Units (PPU)/g, was


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obtained from DSM food Specialties USA, Inc. (Parsippany, NJ). Wyeast American Ale II # 1272 (Wyeast Laboratories, Inc. Odell, OR) yeast was used for the brewing process.

Sorghum Beer Production Wheat gluten incurred sorghum beer was produced as previously described by Panda et al.7 In summary, four independent brew replicates (1-4) of wheat gluten incurred sorghum beer were produced with and without the addition of PEP at the start of fermentation. Brew replicates 1 and 2 were 3.8 L batches of beer brewed in the absence or presence of 34 µL PEP/L of wort, which is the recommended dosage of PEP from the manufacturer. Brew replicates 3 and 4 were 400 mL batches of beer brewed in the absence or presence of PEP at two different levels, the standard dose of 34 µL PEP/L of wort and a higher dose of 212 µL PEP/L of wort. All four of the replicate brews contained either 0 or 200 mg/L wheat gluten added as a suspension in sorghum syrup and were brewed without hops. Samples were collected before and after fermentation. Fermentation was considered complete when the alcohol by volume (ABV) was >3.2% after either 2 or 3 weeks, depending on the brew replicate.

Sample Preparation Two aliquots of each beer sample were prepared using different protocols to separately analyze the intact proteins and the products of hydrolysis and enzymatic processing. The hydrolytic products were isolated as previously described by Panda et al.7 Briefly, 100 µL of each sample was filtered through a pre-rinsed 30 kDa molecular weight cut off (MWCO) filter and 50 µL of the flow-through was desalted using a Pierce C18 spin column according to the manufacturer’s instructions and dried. Samples from the MWCO preparation, which contained



the hydrolytic products, were reconstituted in 50 µL of sample buffer. The sample buffer consisted of 0.1% formic acid (FA), 5% acetonitrile (ACN), 500 fmol/µL angiotensin I, and 10 fmol/µL of the following isotopically labeled wheat gluten peptides: RPQQPYPQPQPQ[13C15

N-Y] and LQLQPFPQPQLP[13C-15N-Y]. The intact proteins were isolated using acetone precipitation. Cold acetone (600 µL, -

30°C) was added to 100 µL of each sample, vortexed for 20 sec, and then stored at -30°C overnight. Each sample was then centrifuged at 10,000 g for 10 min, the supernatant was decanted, and the precipitate was allowed to air dry for 10-15 mins. The protein pellets were washed according to the classic Osborne procedure for gluten extraction to remove any lingering hydrolyzed peptides and water soluble proteins, such as albumins and globulins.29 First, 100 µL of 0.5 M NaCl was added to each sample and then the samples were vortexed for 10 sec. Each sample was shaken at 1500 rpm for 1 min in an Eppendorf ThermoMixer and then centrifuged at 10 kg for 10 min. Each sample was then washed in the same manner with 100 µL water. The resulting protein pellet was reduced, alkylated, and digested with chymotrypsin as previously described by Fiedler et al.29 RapiGest SF surfactant (Waters) was used during digestion to improve the solubilization of the gluten proteins. The digested samples were desalted using Pierce C18 spin columns (Thermo) according to the manufacturer’s instructions and dried. Samples from the digested preparation, which contained chymotryptic peptides, were reconstituted in 100 µL of the sample buffer described above. Sample loss from the isolation procedure for intact proteins is assumed30 that could be avoided by the chymotryptic digestion of whole beer;4,20,22 however, the isolation procedure enables the distinction of chymotryptic peptides generated from intact, or partially intact, gluten proteins from hydrolytic products.


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The MWCO and digested preparations of each sample were performed and analyzed in duplicate. Only gluten peptides that were identified in both technical replicates were used.

LC-MS/MS Analysis A Q Exactive mass spectrometer (Thermo, Waltham, MA) equipped with a nanoAcquity HPLC (Waters, Milford, MA) and Proxeon Nanospray Flex source was used for mass spectrometric analyses. Equal amounts of each sample from the two different preparations were injected onto a Symmetry C18 M class trap column (Waters, Milford, MA, 5 µm particle size, 180 µm i.d. x 20 mm length at 25°C) using an isocratic gradient at 0.5% B for 3 min at 5 µL/min (solvent A, Optima LC/MS 0.1% formic acid in water; solvent B, Optima LC/MS 0.1% formic acid in ACN). Peptides were then separated on a BEH130 C18 PicoFRIT column (New Objective, Woburn, MA, 1.7 µm particle size, 100 µm i.d. × 100 mm length at 25°C) using a gradient of 3−40% B over 60 min at 300 nL/min. The Q Exactive (QE) was operated in a Top10 data dependent acquisition mode in which a full MS scan was acquired first and then the ten most abundant peaks were selected for MS/MS analysis. QE operation parameters include resolution, automatic gain control (AGC) target, and maximum injection times of 70,000, 1e6, and 50 ms, respectively, for the full MS scans and 17,500, 1e5, and 120 ms, respectively, for the MS/MS scans. A normalized collision energy of 20% was used as well as a 2 Da precursor ion isolation width. The minimum AGC target was set at 5e3 for an intensity threshold of 4.2e4.

Data Processing Proteome Discoverer (PD) 2.1 (Thermo, Waltham, MA) was used to perform a noenzyme SEQUEST HT search of the data against a protein sequence database containing all



Swiss-Prot and TrEMBL entries from Poaceae, Saccharomyces cerevisiae, and Cannabaceae (1,323,350 total entries downloaded Sep/Oct 2016 from UniProt). Mass tolerances of 10 ppm and 0.03 Da were used for precursor and fragment ions, respectively. The no-enzyme parameter was used for the samples digested with chymotrypsin to account for the possibility of partially intact proteins created from hydrolysis during fermentation and cleavage by PEP at prolines in the PEP containing beer. Additionally, chymotrypsin is a low specificity enzyme which preferentially cleaves C-terminally to Phe, Trp, or Tyr (except when followed by proline), but it also has low affinity to Leu, Met, and His.29 Only data from the digested samples were searched with a static modification of carbamidomethyl on cysteine residues. The .MSF files from the MWCO and digested preparations were further evaluated in PD 2.1 using a max delta Cn of 0.05 and a 1% false discovery rate (FDR) for peptide spectral matches (PSMs). The identified peptides were filtered to only include high confidence peptides (XCorr ≥ 1.9 for z=2, XCorr ≥ 2.3 for z=3, and XCorr ≥ 2.6 for z≥4) and then grouped into proteins using only peptides ranked first by the search engine (to prevent double counting) with strict parsimony principle applied and a 1% protein FDR. The parameters used for the processing and consensus workflows in PD 2.1 are shown in Supplementary Figure 1. Numerous peptides associated with yeast and sorghum proteins were identified; however, the results were filtered for only gluten peptides associated with proteins containing the following keywords: gliadin, glutenin, hordein, avenin, secalin, prolamin, LMW and HMW. Gluten proteins from the other gluten-containing grains were included because of the homology between gluten proteins. Also, the publicly available databases are poorly curated31 and some of the entries are misannotated. For example, some wheat entries are annotated as avenins (oat gluten proteins) or secalins (rye gluten proteins) when they should be annotated as gliadins.


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Additionally, to perform a comprehensive search for CD-related epitopes, gluten annotations from oats were included since avenins can elicit a celiac immune response in some individuals with CD32 and CD relevant T-cell epitopes from avenins have been identified.24,33 The database of native immunogenic sequences was downloaded from AllergenOnline (July 2017).33

Results and Discussion Mass spectrometry was used to characterize the effects of PEP on gluten during the brewing process. Wheat gluten incurred sorghum beer was brewed with and without the addition of PEP at either the standard dose recommended by the manufacturer or a higher dose of approximately six times the standard dose. A parallel sample preparation approach was employed to separately analyze the hydrolyzed gluten and intact, or partially intact, gluten proteins present in the beer samples. Gluten peptides identified in the non-PEP containing beer and beer brewed in the presence of the manufacturer’s recommended dosage of 34 µL PEP/L wort are shown in Figure 1. For each beer sample, chymotryptic gluten peptides generated from intact, or partially intact, gluten proteins during the digestion preparation are shown in blue. Hydrolyzed gluten peptides (

Analysis of Gluten in a Wheat-Gluten-Incurred Sorghum Beer Brewed

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