Information S1 - Measuring Hordein (Gluten) in Beer – A Comparison of ELISA and Mass Spectrometry

Supplementary Results and Methods, Figures and Tables. Table S1: Total protein content of flour, malt wort and beer from test grains. Table S2: Peptides used for MRM Quantification. Table S3: ANOVA analysis of (log10 ELISA) transformed content of gluten free and low gluten beers. Table S4: ANOVA analysis of (log10 hordein +1) transformed peptide content of beers by MS. Figure S1: Western blot of 8.3 µg of total protein per lane visualised with 1 in 2000 diluted rabbit polyclonal anti-serpin Z4 antibody, followed by 1/2000 diluted donkey-anti-rabbit from flour, malt, wort, and beer produced from: cv Sloop (A); Risø 56 (B); Risø 1508 (C); and ULG 2.0 (D). Serpin Z4 is the dominant band seen at approximately 43 kDa in each blot (*1), the triple bands seen in flour samples are presumably native serpin Z4 isoforms (*1a, *1b, *1c). Pre-stained molecular weight markers (M, Invitrogen), were calibrated against a 10 kDa unstained protein ladder (Invitrogen) and used to determine the migration of protein bands on the western blot. Figure S2. Western blot of 8.3 µg of total protein pre lane visualised with 1/2000 diluted rabbit polyclonal anti-LTP1 antibody, followed by 1/2000 diluted donkey-anti-rabbit from flour, malt, wort, and beer produced from: cv Sloop (A); Risø 56 (B); Risø 1508 (C); and ULG 2.0 (D). The faint band at 43 kDa (*1) was due to pre-existing serpin Z4 conjugate that was not completely denatured during the membrane stripping. The dominant band seen at 9 kDa was due to LTP (2*). The faint band seen in the flour samples at 9 kDa was due to native LTP. Pre-stained molecular weight markers (M, Invitrogen), were calibrated against a 10 kDa unstained protein ladder (Invitrogen) and used to determine the migration of protein bands on the western blot. Figure S3. A typical standard curve of total Sloop hordeins generated using the ELISA systems kit showing the A450 vs hordein content in µg/kg (ppb). The A450 is shown for triplicate hordein concentrations. A sigmoidal curve of best fit was calculated using GraphPAD Prism 5.04 with an r2 of 0.9685. Figure S4. The lack of correlation between ELISA values for 60 beers and the relative level determined by MS for avenin (A), B1-hordein (B), B3-hordein (C), D-hordein (D), γ3-hordein (E), or the sum of all relative levels of peptides determined by MS (F), using GraphPAD Prism 5.04. No figures had an R2 value greater than 0.06 confirming a lack of any meaningful correlation. Figure S5. Comparison of the response of the two D-hordein derived tryptic peptides across the range of beers tested. The values represent the average determined for two individual beer bottles; for clarity the error bars have been omitted however the variation between the bottles was <12% for all gluten-containing beers. The peptide common to wheat showed an increase in the percentage peak area (relative to average) when compared to the peptide unique to barley in beers 5, 6, 9, 16, 30, 45, 55 and 56 (all wheat beers). Figure S6. The effect of lyophilisation and dissolution of wort and beer in Urea/DTT, compared with direct sampling on SDS-PAGE. Solutions from cv Sloop (Sloop) or Risø 56 (Risø 56), wort (W) or beer (B) were either sampled directly, the protein measured and 20 µg added directly to Urea/SDS (D), or lyophilised, redissolved in Urea/DTT, the protein measured and 20 µg added to Urea/SDS (Ly). In each case the intensity of protein bands after SDS-PAGE was more intense in the solutions which had been lyophilised, indicating that this treatment was more successful in denaturing proteins, and produced a higher concentration of homogenous protein bands which ran as a coherent band during SDS-PAGE.
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