applied sciences Article  
**Peptide Mapping with LC-HRMS and In Silico Tools for Evaluation of Potential Allergenicity of Commercially Available Whey Protein Hydrolysates**  
[PERSON],*, [PERSON], [PERSON], [PERSON], [PERSON], [PERSON] and [PERSON] Center of Food and Fermentation Technologies (TFTAK), Maealuse 2/4, 12618 Tallinn, Estonia; [EMAIL_ADDRESS] (T.L.)  
can explain the allergic reaction of children toward IF [11; 12; 13]. It has been shown that only extensively hydrolyzed IF has significantly reduced cow's milk protein-induced allerggenic symptoms [14], and only pure amino acid formulas are considered to be nonallergenic [15]. For infants at a high risk of allergy, the extensively hydrolyzed formula is recommended to prevent CMA, as the partially hydrolyzed formula can still induce allergic reactions [15; 16].  
The size of peptides (the length and the MW) obtained after the hydrolysis of proteins is crucial to the assessment of residual allergenicity, and the development of optimal IF [17; 18]. The IgE-binding allergenic epitope, which is able to bind with the corresponding Ig molecule, is usually 6-15 amino acids long [19]. Previous studies have shown that \(\beta\)-LG is widely covered by numerous allergenic epitopes, distributed regularly along the molecule, including those with the highest IgE-binding capacity, e.g., f25-40, f41-60, f75-86, f102-124, and f124-134, showing immunoreactivity toward the serum of patients with a milk allergy [20; 21; 22; 23; 24; 25; 26; 27]. In addition, several IgE-binding epitopes have been identified in \(\alpha\)-LA [20; 24]. During the hydrolysis of proteins, the potential allergenic epitopes are cleaved, preventing the binding of IgE [5]. Indeed, not only the extent of hydrolysis, but also the proteolysis of the specific epitopic areas, is an important factor in reducing the allergenicity of proteins.  
Numerous studies have investigated the reduction of the molecular weights of the peptides of enzymatically treated cow's milk proteins, using sodium dodecyl sulfate-polyacrylamideamide gel electrophoresis [4; 28; 29]. Several works have measured the peptide profiles of milk protein hydrolysates, using reversed-phase high performance liquid chromatography, evaluating the disappearance of native proteins, and the occurrence of their degradation products, by the action of various enzymes [4; 30]. Later research papers have also applied liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques to the identification of peptides in milk protein hydrolysates [31; 32], during gastrointestinal proteolysis [27] and bacterial degradation of \(\beta\)-LG [33; 34], on a lab scale, in order to locate certain IgE-binding epitopes related to these peptides. Moreover, in recent years, LC-MS/MS techniques have been applied to the analysis of allergens in milk and eggs [35; 36], peanuts [35], soybeans [35], sesame [37], and peaches [38], in order to overcome the limitations of antibody-based methods (false-positive or -negative results, due to the cross reactivity of proteins, or structural modifications of proteins). Nevertheless, reliable data on the characterization of the peptide composition of commercially available whey protein hydrolysates (WPHs) in relation to their potential allergenicity, are not available.  
The aim of this study was to provide an insight into the sequence-based characterization of the commercially available WPHs, applying liquid chromatography-high resolution mass spectrometry (LC-HRMS), and to investigate the potential IgE-binding epitopes related to identified peptides, using in silico tools.