Peptides generated from the 26-mer and 33-mer.

<p>Numbers and letters refer to peaks in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128065#pone.0128065.g002" target="_blank">Fig 2</a></p><p>Sequences that contain epitopes are underlined</p><p>*peptides shorter than eight amino acids are considered too short to contain an epitope</p><p>Peptides generated from the 26-mer and 33-mer.</p

Mass spectrometric analysis of the degradation products of 26-mer and 33-mer peptide by digestive enzyme supplements.

<p>Peptide was incubated at 37°C for 30 minutes at pH 6.0 in the presence of 1 capsule equivalent digestive enzyme supplements. As a control, 1/100 capsule equivalent of AN-PEP at pH 5.0 was also incubated. Peptide reaction products were analyzed by Q-TOF LC-MS and their MS spectra shown in this Figure. The identity of the prominent peptides 1–6 and a-g are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128065#pone.0128065.t004" target="_blank">Table 4</a>. In the case of AN-PEP, small amounts of epitope-containing peptides were observed under these conditions, but they disappear at higher enzyme concentration or prolonged incubation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128065#pone.0128065.g003" target="_blank">Fig 3</a>). The digestive enzyme supplements display comparable activities and remove at most two amino acids from the N-terminus of the 26-mer (peptide 1 (26-mer) is degraded to peptide 2, and then to peptide 3), and three amino acids at most from the 33-mer peptide (peptide a (33-mer) is degraded to b, to c, and then to peptide d).</p

Epitopes contained in the gastrointestinal-enzyme resistant 26-mer and 33-mer gluten peptides.

<p>The sequences of the 26-mer and 33-mer peptide are shown. Underneath, the position of the immunogenic epitopes is denoted</p><p>^*the epitopes DQ8-glia-γ1a and DQ8-glia-γ1b are identical to epitopes DQ2.5-glia-γ4c respectively DQ2.5-glia-γ3</p><p>Epitopes contained in the gastrointestinal-enzyme resistant 26-mer and 33-mer gluten peptides.</p

Protein compositon of digestive enzyme supplements.

<p>20 μg of digestive enzyme supplement was acid-inactivated, reduced, alkylated and digested with 2 μg of trypsin as specified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128065#pone.0128065.s008" target="_blank">S2 Text</a> Methods.Tryptic peptides were analyzed by LC-MS and the proteins identified using Mascot search engine. The numbers represent the number of unique peptides matching the protein.</p><p>¹Uniprot database</p><p>²alternative name, Lactase-N</p><p>Protein compositon of digestive enzyme supplements.</p

Ineffective detoxification by digestive enzyme supplements.

<p>33-mer was incubated for 60 minutes with 1 capsule equivalent of digestive enzyme supplements, or 1/10 capsule equivalent of AN-PEP as a control. The peptide reaction products were deamidated using tissue transglutaminase, purified by C18 solid phase extraction and tested for T-cell activation. As a negative control, T-cells were incubated without the 33-mer peptide or digestive enzyme supplements (blanc). As a positive control, T-cells were incubated with 33-mer peptide. The enzyme preparations, when tested separately, were not toxic for the T-cells (not shown). T-cells stimulated with control peptide (LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF) incorporated 40,000 cpm. T-cell proliferation ([³H]thymidine incorporation in cpm) is shown as a function of the input of 33-mer and its degradation products. Error bars represent standard deviation of triplicate experiments. The result shown is representative for two independent experiments.</p