Hordein and alcohol soluble protein levels in flour
The total protein content of flour from the four test lines varied from 12.9% (w/w; or 129 mg/g) for cv Sloop to a high of 15.4% for Risø 1508. Malt contained a slightly lower range of protein concentrations ranging from a low of 9.0% for cv Sloop to a high of 12.7% from ULG 2.0, presumably because a proportion of protein reserves were mobilised during germination. Wort contained approximately 3 mg/mL of protein and beer contained from 0.7 (ULG 2.0) to 2.8 (Sloop) mg/mL protein. The average protein content of the four test beers was 1.85±0.23 mg/mL (Table S1 in Information S1
The pattern of proteins in flour, malt, wort and beer
Staining of total protein extracts from flour samples following SDS-PAGE visualised a large number of proteins ranging from 10 kDa to 100 kDa (: flour). Similar analysis of malt samples identified fewer protein species with an increased intensity observed for smaller peptides (less than 10 kDa), indicating that significant proteolysis had occurred during germination and malting (: malt). In contrast SDS-PAGE of wort and beer, together with in-gel protein digestion and analysis, indicated that the protein composition had been dramatically enriched for two protein families, serpin Z4 at 43 kDa and LTP1 at approximately 9 kDa, accounting for the bulk of the protein in these fractions (; 1 & 2 respectively, Supplementary Results in Information S1, and Fig. S1 & S2 in Information S1
). There were differences in the serpin Z4 proportion of the beer produced by different lines, with the most Z4 produced by beer made from cv Sloop, intermediate levels produced by Risø 56, and relatively lower proportions of serpin Z4 produced by Risø 1508 and ULG 2.0.
Figure 1 Coomassie stained protein gels (16.7 µg total protein loaded per lane) of flour, malt, wort, and beer produced from: cv Sloop (A); Risø 56 (B); Risø 1508 (C); or ULG 2.0 (D); were calibrated against Benchmark 10 kDa protein standards (more ...)
Analysis of a replicate anti-gliadin western blot identified the main hordein families (: flour). These westerns were deliberately loaded with a high protein load to maximise the detection of hordeins in wort and beer. In all cases hordein bands in wort and beer were almost absent, indicating very low hordein levels in all wort and beer fractions (; wort and beer). The relative lack of hordeins in flour extracts from the hordein double-null line ULG 2.0 can be seen when anti-hordein western blots are compared with those from parental lines Risø 56, Risø 1508 and the wild type cv Sloop. Only two significant bands corresponding to known hordeins, D- and γ-3-hordein (, 7 & 8), were seen in blots of total protein from ULG 2.0. At this high protein loading other proteins, in addition to hordeins, were detected presumably due to a weak homology to the epitopes detected by the antibody. This is particularly clear in the extracts of ULG 2.0 flour, where faint western bands are seen at approximately 15, 20, 32, 48, 50, and 60 kDa. Previous protein sequencing of ULG 2.0 has shown that ULG 2.0 only accumulates γ-3 hordein and D-hordein 
. Additional bands due to serpin Z4 and LTP (; 1 and 2 respectively) were also seen in all wort and beer fractions, however, no hordein bands were seen in these fractions. The location of known proteins was confirmed by protein sequencing of replicate gels (: 1, serpin Z4; 2, LTP 1 and 2; 3, B-hordeins; 4, C-hordeins; 5, γ-1-hordein; 6, γ -2-hordein; 7, γ-3-hordein and 8, D-hordein 
Western blot of 8.3 µg of total protein per lane visualised with 1/2000 diluted anti-gliadin-HRP from flour, malt, wort, and beer produced from: cv Sloop (A); Risø 56 (B); Risø 1508 (C); and ULG 2.0 (D).
Response of sandwich ELISA and MS to beer
Standard curves of total hordein preparations from the test grains Sloop, Risø 56, Risø 1508 and ULG 2.0 were used to calibrate the hordein content of beers produced from these malted grains (). All other beers were diluted, so that the response was in the linear region of the H2
quenched Sloop total hordein standard curve, with A450 values between 0.5 and 1.5 (Fig. S3 in Information S1
). The Sloop standard curve was used to calculate the hordein concentration in all other beers ().
Comparison of hordein detection by MS vs ELISA.
Our ELISA results show that cv Sloop beer had a higher hordein level than expected at 130 ppm (), as the hordein concentration in barley beers has been reported to vary from 19–45 ppm 
. Beer from Risø 56 had an intermediate hordein level of 16.4 ppm (12.6% of Sloop), while ULG 2.0 and Risø 1508 beers were lower at 12.7 and 0.8 ppm respectively (9.7% and 0.3% respectively of Sloop) when measured by Elisa.
Sandwich ELISA results for all beers varied from zero (for four known gluten-free beers) to concentrations of 40,800–46,500 ppm for three wheat beers. The mean ±SE hordein content of all beers was 5,400±1,760 ppm. The large range meant that the median value of all beers was only 198 ppm. The beers with the highest hordein level by ELISA were all wheat beers (: beers 6, 30, 33, 45, 55, 56) with a mean hordein ± S.E. content of 31,900±5,000 ppm with a median value of 40,800 ppm. The beers with a zero hordein level by ELISA included beers labelled as gluten-free (: beers 17, 47, 49, 50, 51, 52, 58, 60) as well as some that were brewed using barley (beers 11, 13, 22, 35, 53, 54). The mean hordein content of all gluten-free beers, excepting beers 47 and 49, was 0.08±0.005 ppm (median 0.15 ppm), identical to the (no addition) blank reading. Two designated gluten-free beers (47 and 49) had small, but significant ELISA readings (p<0.05) and MS analysis revealed the presence of avenin like-A protein, B3- and D-hordein (>1% of the average of all beers), most likely indicating contamination by barley. Two beers with a low gluten claim (beers 57 and 59) had a zero ELISA reading, but MS revealed they contained substantial levels of hordeins with at least one peptide with a value ~100% of the average hordein content for all non-zero beers ().
Relative hordein peptide composition of designated low gluten and gluten-free beers.
Commercial beers 11, 13, 22, 35, 53, and 54 did not react with the ELISA Systems kit (mean ELISA reading 0.1±0.01 ppm) suggesting that the hordein content of these beers was zero. Subsequent MS analysis showed these beers contained substantial hordein levels with values for at least one peptide greater than 50% of the average (). Thus we conclude that these beers generated false negatives by ELISA. The failure of ELISA to detect hordein in these beers may be explained by protein degradation as untargeted MS analysis revealed up to a 14-fold increase in hordein peptide fragments in the sub-10 kDa filtrate from undigested beers (data not shown). Beers 11, 13, 53 and 54 showed the highest levels of these relatively small (10–12 amino acid) hordein remnants.
Relative hordein peptide composition of apparently zero hordein beers (11, 13, 22, 35, 53 and 54).
In addition, there was another group of ten apparently low hordein beers (10, 19, 20, 23, 26, 28, 29, 39, 41 and 42) that had a very low ELISA reading of <1 ppm (mean 0.38±0.08, median 0.28 ppm), but a significant MS level with several hordein peptide values close to the average (). The mean hordein content by MS for avenin-like protein, B1-, B3-, D- and γ-hordein were 67, 178, 107, 120 and 145% respectively of the level relative to the average hordein content. These beers represent a large group where the ELISA reaction was suppressed.
Relative hordein peptide composition of apparently low (<1 ppm) hordein beers (10, 19, 20, 23, 26, 28, 29, 39, 41, and 42).
Stouts had similar hordein content by ELISA (mean 360±33 ppm, median 342 ppm, excluding stout 37), but highly variable content by MS (). Beers 1, 2, 3, and 4 of were good examples of the variation encountered. All had moderate hordein levels by ELISA (mean 307±26 ppm, median 328 ppm), however, for dark lager 1, the hordein peptides measured by MS varied from 140–270% of the average hordein content of all beers (). This contrasts markedly with stout 3, which had very low hordeins by MS, of approximately 10% of the average hordein content. Stouts 2 and 4 had relative hordeins by MS which varied from 40 to 152% of the average.
Relative hordein peptide composition of stouts (beers 1, 2, 3, 4, 34, 44).
Lagers were the largest group accounting for 39% of all beers assayed. In general they had lower but highly variable hordein levels with a mean ELISA content of 62.7±25 ppm (median 0.5 ppm, with a range of 0.067–341 ppm). The mean hordein content by MS for avenin like-A protein, B1-, B3-, D- and gamma-hordein were 90, 149, 118, 110 and 129% respectively of the level relative to the average hordein content ().
Relative hordein peptide composition of lagers (10, 11, 12, 13, 15, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 39, 40, 42, 46, 48, 53, 54).
Ales were highly variable compared to cv Sloop, when the hordein content was measured by ELISA (mean 3,100±1,700 ppm; median 289 ppm), however, the mean hordein content by MS was less variable at about 70% of the average hordein content, e.g. means for avenin like-A protein, B1-, B3-, D- and gamma-hordeins were 72, 30, 85, 62 and 60% respectively of the level relative to the average hordein content ().
Relative hordein peptide composition of ales (8, 9, 16, 31, 32, 35, 36, 38, 43).
Comparison of ELISA and MS determinations
The ELISA results did not correlate with the relative content of individual hordein peptides as determined by MS (Fig. S4 in Information S1
). The relative concentrations of the individual proteins (avenin, B1-hordein, B3-hordein, D-hordein or γ-3-hordein) determined by MS did not correlate with the total hordein content determined by ELISA with r2
values for the lines of best fit less than 0.06. Nor was there correlation between the ELISA reading and the summed relative hordein concentrations for each beer. This was due, in part, to the fact that the MS analysis only considered barley proteins. Several of the beers examined in this study contained substantial amounts of glutenin and gliadin (wheat gluten).
Given the lack of correlation, we sought to find an explanation and to this end, we looked for possible post-translational modifications that would alter the ability of MS to accurately detect and quantify the hordein proteins. Tryptic digestion of wild-type (Sloop), the three deletion mutants and 60 commercial beers was undertaken and analysed by LC-MS/MS in a non-targeted manner. Typical peptide modifications, such as oxidation of methionine, were detected, however, this type of modification did not affect the outcome of the MS assay as the MRM-MS method used in the targeted approach incorporated two versions (non-oxidised and oxidised) for all Met-containing peptides. As glycosylation and glycation were expected, we also looked for the presence of these modifications. We found limited peptide evidence for glycation of lysine residues. Evidence of glycation was restricted to peptides from abundant proteins including LTP1 (Uniprot accession: P07597), LTP2 (P20145), α-amylase trypsin inhibitor CMb (P32936) and in a single peptide from D-hordein (Q84LE9). Where glycated lysine residues were present, the peptide was not cleaved by trypsin. The single instance of a glycated peptide (VAK*AQQLAAQLPAMCR) in D-hordein did not affect the MS results as an alternate tryptic peptide was selected for quantification.
Detection of wheat peptides in beer by MS
Examination of the complete lists of proteins identified, revealed that a number of the commercial beers not labelled as containing wheat did indeed contain traces of wheat gluten proteins (beers 9, 16, 31, 32 (all ales) and 37 (stout)). For example, beer 9 (an Australian ale) yielded α-gliadin (Q9M4L6) as the primary protein identification along with γ-gliadins (Q9M4L5, Q94G94) and a low molecular weight glutenin (Q75ZV8) as lesser abundant protein identifications. Further, a number of defensins, α-amylase trypsin inhibitors and non-specific lipid transfer proteins from wheat were also identified. A similar suite of proteins were identified in beers 5, 6, 30, 33, 45, 55 and 56, all declared wheat beers. The average ELISA measurement of non-wheat containing beers was 103.3±24.2 ppm. All of the beers found to contain wheat by MS gave ELISA values well in excess of 500 ppm with nine of the eleven samples yielding ELISA values >4,000 ppm.
Subsequently, we performed MRM analysis of the beers to look for the presence of peptides that mapped to known wheat gluten proteins (α-gliadin, Q9M4L6; low molecular weight glutenin, Q75ZV8; and high molecular weight glutenin, P10388). Using this more sensitive MS assay, wheat peptides were detected in beers 5, 6, 9, 16, 30, 31, 32, 33, 37, 45, 55 and 56 with at least two wheat peptides detected in each beer ().
Scheduled multiple reaction monitoring (MRM) mass spectrometry revealed the presence of wheat gluten proteins in beers 5, 6, 9, 16, 30–33, 45, 55 and 56.
The identification of wheat proteins in a number of the beers necessitated further MS analysis to differentiate between wheat and barley gluten. Each peptide candidate was interrogated by BLAST searching on the Uniprot and NCBI servers to check for species specificity. In the case of the avenin-like A protein (Uniprot accession F2EGD5), only a single peptide was detectable in beer under the experimental conditions and this peptide was found to be present in proteins from wheat (Triticum
), goatgrass (Aegilops
) and barley (Hordeum
). In the case of D-hordein (Uniprot accession Q84LE9), a suite of tryptic peptides were detected. MRM analysis was performed on the test and commercial beers using peptides derived from a tryptic digestion of D-hordein that were either: (a) unique to barley gluten (D-hordein, Uniprot accession Q84LE9); or (b) common to barley and wheat gluten (HMW-glutenin, Uniprot accession P10387). Table S2 in Information S1
lists the peptides that were investigated. The peak areas (relative to average) for 3 MRM transitions per peptide for the common (ELQESSLEACR) and unique (QYEQQTEVPSK) D-hordein derived peptides are shown in Fig. S5 in Information S1
. In contrast to the non-wheat beers, beers containing wheat (5, 6, 9, 16, 30, 31, 33, 45, 55 and 56) showed an increased proportion of the common peptide.