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 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. (DOC)补充结果与方法、图表及表格。表S1：供试谷物制成的面粉、麦芽汁与啤酒的总蛋白质含量。表S2：用于多反应监测（MRM）定量的肽段。表S3：无麸质与低麸质啤酒经log₁₀转换的酶联免疫吸附测定（ELISA）检测含量的方差分析（ANOVA）。表S4：通过质谱（MS）检测的啤酒经log₁₀(醇溶蛋白+1)转换的肽段含量的方差分析（ANOVA）。图S1：以1:2000比例稀释的兔源多克隆抗丝氨酸蛋白酶抑制剂Z4（anti-serpin Z4）抗体孵育，再以1:2000比例稀释的驴抗兔二抗孵育，对每个泳道上样8.3 μg总蛋白质进行的蛋白质免疫印迹（Western blot），样品来自以下4个品种谷物制成的面粉、麦芽、麦芽汁与啤酒：Sloop品种（A）、Risø 56品种（B）、Risø 1508品种（C）以及ULG 2.0品种（D）。丝氨酸蛋白酶抑制剂Z4在每个免疫印迹中均为约43 kDa处的主条带（*1），面粉样品中出现的三条条带推测为天然丝氨酸蛋白酶抑制剂Z4同工型（*1a、*1b、*1c）。预染分子量标志物（M, Invitrogen）以10 kDa未预染蛋白分子量标准品（Invitrogen）校准，用于确定免疫印迹上蛋白条带的迁移位置。图S2：以1:2000比例稀释的兔源多克隆抗脂质转运蛋白1（anti-LTP1）抗体孵育，再以1:2000比例稀释的驴抗兔二抗孵育，对每个泳道上样8.3 μg总蛋白质进行的蛋白质免疫印迹（Western blot），样品来自以下4个品种谷物制成的面粉、麦芽、麦芽汁与啤酒：Sloop品种（A）、Risø 56品种（B）、Risø 1508品种（C）以及ULG 2.0品种（D）。43 kDa处的弱条带（*1）源于膜洗脱过程中未完全变性的预存丝氨酸蛋白酶抑制剂Z4结合物。9 kDa处的主条带源于脂质转运蛋白（2*）。面粉样品中9 kDa处的弱条带源于天然脂质转运蛋白。预染分子量标志物（M, Invitrogen）以10 kDa未预染蛋白分子量标准品（Invitrogen）校准，用于确定免疫印迹上蛋白条带的迁移位置。图S3：采用酶联免疫吸附测定（ELISA）试剂盒生成的典型Sloop醇溶蛋白标准曲线，以A450吸光度值对应以μg/kg（ppb）为单位的醇溶蛋白含量。展示了三个重复的醇溶蛋白浓度对应的A450吸光度值。采用GraphPAD Prism 5.04软件计算得到拟合的S型曲线，决定系数r²为0.9685。图S4：针对60份啤酒的酶联免疫吸附测定（ELISA）值，与通过质谱（MS）测定的燕麦球蛋白（A）、B1-醇溶蛋白（B）、B3-醇溶蛋白（C）、D-醇溶蛋白（D）、γ3-醇溶蛋白（E）的相对水平，或通过质谱（MS）测定的所有肽段相对水平总和（F）之间无相关性。采用GraphPAD Prism 5.04进行分析，所有图表的决定系数R²均未超过0.06，证实二者无显著相关性。图S5：两种D-醇溶蛋白来源的胰蛋白酶肽段在全部受试啤酒中的响应对比。数值为2个独立啤酒瓶样品的测定平均值；为便于展示已省略误差棒，但两瓶样品间的变异情况详见图S6。图S6：对比麦芽汁与啤酒经冻干后溶于尿素/二硫苏糖醇（Urea/DTT），与直接取样在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）上的结果。样品取自Sloop品种或Risø 56品种的麦芽汁或啤酒，处理方式分为两种：直接取样、测定蛋白浓度后取20 μg蛋白直接加入尿素/SDS缓冲液（D组）；或经冻干后溶于尿素/二硫苏糖醇、测定蛋白浓度后取20 μg蛋白加入尿素/SDS缓冲液（Ly组）。各组经SDS-PAGE电泳后的蛋白条带强度在冻干处理的样品中更高，表明该处理可更高效地使蛋白变性，且能获得浓度更高的均一蛋白条带，在SDS-PAGE电泳中呈现为整齐的单一条带。(DOC)