## 3 Results
### Determination of Peptides with LC-HRMS  
In addition to the robust analysis of the MW distribution of protein fractions, the analysis of peptides was carried out using LC-HRMS, to define the peptide composition more \begin{table}
\begin{tabular}{c c c c c c} \hline \hline  & & \multicolumn{4}{c}{**\% of Total Area (Mean \(\pm\) SD)**} \\ \hline
**MW, Da** & **RT, Min \({}^{\star}\)** & **H1** & **H2** & **H3** & **H4** \\ \hline \(>\)13,500 & 0–4.4 & 0.0 \(\pm\) 0.0 & 6.0 \(\pm\) 0.4 & 35.4 \(\pm\) 0.7 & 34.0 \(\pm\) 1.0 \\
3500–13,500 & 4.4–5.0 & 17.8 \(\pm\) 0.1 & 19.5 \(\pm\) 0.2 & 24.4 \(\pm\) 0.8 & 20.3 \(\pm\) 0.0 \\
500–3500 & 5.0–5.9 & 54.2 \(\pm\) 0.2 & 42.8 \(\pm\) 1.0 & 21.5 \(\pm\) 0.2 & 21.1 \(\pm\) 0.1 \\
150–500 & 5.9–6.5 & 16.9 \(\pm\) 0.1 & 15.7 \(\pm\) 1.1 & 7.5 \(\pm\) 0.3 & 8.1 \(\pm\) 0.1 \\ \(<\)150 & 6.5–15.0 & 11.1 \(\pm\) 0.0 & 16.1 \(\pm\) 0.3 & 11.2 \(\pm\) 1.0 & 16.5 \(\pm\) 0.8 \\ \hline \hline \end{tabular} \({}^{\star}\) retention time.  
\end{table} 
Table 1: Molecular weight (MW) distribution of protein fractions in WPHs, determined using SE-UPLC. Figure 1: The SE-UPLC elution profiles of the WPHs. UV detection was at 220 nm. The blue line is H1, the orange line is H2, the gray line is H3, and the yellow line is H4. Using the standards, the calibration curve was constructed for setting the tentative MW boundaries for different protein fractions. precisely, and to identify potential IgE-binding allerggenic epitopes in the WPHs. Overall, 318, 233, 111, and 58 peptides were identified in the H1, H2, H3, and H4 hydrolyses, respectively (Figure 2). The summed intensity of all identified peptides was the highest in the sample with the highest degree of hydrolysis, H1, and it decreased gradually in the H2, H3, and H4 samples, accordingly (Figure 2). The majority of the identified peptides were derived from the predominant milk whey proteins \(\alpha\)-LA and \(\beta\)-LG (74.1-93.0% of the total intensity of peptides, depending on the sample). The exception to this observation was the hydrolysate H3, in which the peptides originating from \(\alpha\)-LA accounted only for 1% of the total intensity of peptides (Table 2). Peptides originating from other whey proteins comprised up to 25.9% of the total intensity of peptides. These proteins included BSA and lactotransferrin, which can also induce milk allergy [2]. However, minor relative amounts of peptides originating from BSA and lactotransferrin were obtained, due to the low quantities of these proteins in milk. Therefore, the peptides from minor proteins will not be considered in further detail in this study. The peptide length distribution showed that compared to the other hydrolyses, the H1 sample contained a higher amount of short (up to 4 amino acids long, approximately 450 Da) and average-length peptides (7-10 amino acids long, approximately 800-1100 Da) (Figure 3). In the H2 hydrolysate, the amount of identified peptides of 5-6 and 12-15 amino acids in length (approximately 1350-1650 Da) was higher than in the other hydrolyses. \begin{table}
\begin{tabular}{c c c c c} \hline \hline  & \multicolumn{4}{c}{**\% of the Total Intensity**} \\ \hline
**Protein** & **H1** & **H2** & **H3** & **H4** \\ \hline \(\alpha\)-LA & 12.8 & 6.7 & 1.0 & 13.0 \\ \(\beta\)-LG & 61.3 & 85.1 & 73.3 & 80.0 \\ BSA & 4.9 & 1.7 & 1.8 & 0.9 \\ Apolipoprotein & 1.5 & 0.8 & 3.9 & 1.3 \\ Complement C3 & 5.3 & 2.1 & 6.3 & 4.4 \\ Fibrinogen alpha & 1.5 & 0.5 & 2.0 & 0.0 \\ Lactoperoxidase & 2.5 & 1.0 & 8.3 & 0.0 \\ Lactotransferrin & 8.0 & 1.1 & 2.1 & 0.5 \\ Osteopontin & 2.4 & 0.9 & 1.4 & 0.0 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Distribution of peptides in WPHs (% of the total intensity of identified peptides). Figure 2: Total number and summed intensity of all identified peptides in the H1, H2, H3, and H4 WPHs, determined using LC-HRMS analysis. The summed intensity of peptides was normalized against the average protein content of each sample.