## 3 Results
### Determination of Peptides with LC-HRMS
Peptide coverage profiles allow the visualization of hydrolysis by mapping all detected peptides on the corresponding protein sequence. Figure 4 shows the peptide coverage profiles of \(\beta\)-LG in the hydrolysate samples, obtained by the processing of LC-HRMS data, with the script written in Python. The Immune Epitope Database [40] was used for the search of the IgE-binding allergenic epitopes related to the identified peptides. Epitopes found in the WPHs were also mapped on the peptide coverage profiles of \(\beta\)-LG and \(\alpha\)-LA proteins, using Python script (Python Software Foundation, version 3.5, available at [[http://www.python.org](http://www.python.org)]([http://www.python.org](http://www.python.org)) (accessed on 11 April 2023)) (Figures 4 and 5). Figure 4: \(\beta\)-LG derived peptides identified using LC-HRMS in the H1, H2, H3, and H4 hydrolyses. \(X\)-axis: the amino acid sequence of \(\beta\)-LG. \(Y\)-axis: the number of peptides. The intensity of peptides is shown in the scale on the right. Entire IgE-binding allergenic epitopes included in the sequences of identified peptides, and derived from the Immune Epitope Database, are marked with blue bars on the peptides of each hydrolysate, and mapped separately on the bottom pattern of \(\beta\)-LG sequence. Figure 3: Distribution of the length of peptides identified in the H1, H2, H3, and H4 hydrolyses using LC-HRMS. In the H3 and H4 hydrolysates, most of the peptides originated from the N-terminal part of \(\beta\)-LG f1-60, whereas peptides from H1 and H2 were more evenly distributed along the whole protein sequence. In the case of \(\alpha\)-LA, a few peptides identified in H4 and H3 originated from the central part of the \(\alpha\)-LA sequence, while the coverage profiles of the H1 and H2 hydrolysates were covered by several peptides uniformly (Figure 5). Overall, 78 epitopes from \(\alpha\)-LA, and 209 IgE-binding allergenic epitopes from \(\beta\)-LG (T- and/or B-cell responses), are listed in the Immune Epitope Database [40]. In total, 44 different epitopes from \(\beta\)-LG were detected in the WPHs: 17 in H1, 30 in H2, 31 in H3, and 23 in H4 (Figures 4 and 5, Table 3). Among the IgE-binding epitopes from \(\alpha\)-LA found in the Immune Epitope Database, only two were found in the H1 and H2 hydrolysates. Figure 5: \(\alpha\)-LA derived peptides identified using LC-HRMS in the H1, H2, H3, and H4 hydrolysates. \(X\)-axis: the amino acid sequence of \(\alpha\)-LA. \(Y\)-axis: the number of peptides. The intensity of peptides is shown in the scale on the right. Entire IgE-binding allergenic epitopes included in the sequences of identified peptides, and derived from the Immune Epitope Database, are marked with blue bars on the peptides of each hydrolysate, and mapped separately on the bottom pattern of \(\alpha\)-LA sequence. Altogether, 31 unique peptides from H1, 45 from H2, 27 from H3, and 13 from H4 were found to be related to these epitopes (Table 3). \begin{table}
\begin{tabular}{c c c c c c c} \hline
**Protein (UniProt Database ID)** & **Identified Peptides** & **WPH** & **Epitope Sequence** & **Epitope Location** & **Immun Epitope** & **References** \\  & **Deptades** & **WPH** & **Epitope Sequence** & **in Protein Chain** & **Database ID** & **References** \\ \hline \(\alpha\)-LA (P00711) & f79-90 & H1 & KFLDDDLTDD & f79-88 & 115308 & [24] \\ \hline  & f81-90 & H1, H2 & & & & \\  & f80-90 & H1 & LDDDLTDDIM & f81-90 & 115340 & [24] \\  & f79-90 & H1 & & & & \\ \hline \(\beta\)-LG (P02754) & f25-40 & H3, H4 & AASDISLLDAQSAPLR & f25-40 & 426 & [23] \\ \hline  & f9-14 & H2 & & GLDIQK & f9-14 & 20801 & [23] \\  & f84-91 & H3, H4 & & & & \\  & f84-93 & H3 & IDALNENK & f84-91 & 25535 & [23] \\  & f84-94 & H4 & & & & \\ \hline  & f46-56 & H1, H2 & & & & \\  & f46-57 & H1, H2, H3 & & & & \\  & f47-57 & H2 & & & & \\  & f45-57 & H2, H3 & & & &