As the primary source of nutrients for infants, children and also adults, milk has been largely consumed across the world. Milk is an essential food source for humans, and it has been proven to have been a part of the human diet since nearly 6000 years ago (Bleasdale et al., 2021). Milk is regarded as a complete food because of its versatility; it contains both macronutrients and micronutrients (Park and Haenlein, 2010). Cow's milk is commonly used to feed infants who do not have access to breast milk due to its long history of consumption. But in certain cases, cow's milk may be perceived as foreign in the gastrointestinal system, causing an allergic reaction by the immune system. Milk allergy is more prevalent among infants; as they have an underdeveloped gastrointestinal system, more often in infants as their main source of nutrients comes from milk (Yadav and Naidu, 2015). Although sensitization toward milk allergens among atopic children will subside or go as they age; as their gastrointestinal system developed, the persistence of milk allergy past adolescence has been reported (Skripak et al., 2007) posing a risk to adult consumers on the consumption of cow’s milk. An allergic reaction is usually categorised as IgE-mediated or non-IgE-mediated, its lethality varies depending on the IgE-reactivity of the patient’s serum IgE. It can be as simple as rashes or sneezing to anaphylaxis which can lead to casualty. IgE produced by the plasma cell upon foreign protein antigen entry played an important role in triggering the allergic reaction; IgE will attempt to bind with the foreign antigen if succeeded will trigger the mast cells to release histamine to initiate an allergic reaction (Sampson et al., 2018, Yu et al., 2016).

This circumstance has compelled people with an allergic reaction toward cow’s milk protein to opt for different dairy milk alternatives. Goat, sheep, horse, camel, and donkey milk are among the ruminant milk that has been studied for their hypoallergenic potential (Bernard et al., 1999; Hinz et al., 2012). As an alternative, plant-based milk such as soy milk and nut milk has also been considered. However, in terms of nutrients for infants' postnatal development, mammalians milk is a better choice because it naturally contains the essential nutrients for infant development (Vojdani et al., 2018). According to dairy milk product output statistics in Malaysia, dairy milk output seemed to increase steadily from 2013 to 2019, with cows having the highest livestock population, followed by goats. There are only a few large-scale dairy goat farms in Malaysia, as the majority of dairy goat farms are owned by small-scale farmers(Shahudin et al., 2018). Saanen, Jamnapari, Toggenburg, Alpine, and Shami are among the dairy goat breeds raised in Malaysia (Liang and Paengkoum, 2019). These breeds are known to be excellent at producing milk, and farmers practise cross-breeding among these breeds to increase milk yield and quality.

However, due to the general homology between the two species' milk proteins, goat's milk may pose some issues as an alternative to cow's milk for CMPA patients (Bernard et al., 2012). Milk protein from different phylogenetic species usually has the same set of proteins; casein and whey. The cross-reactivity of milk proteins from different species of ruminants has been documented (Bellioni-businco et al., 1999, El-Agamy et al., 2009, Goodman et al., 2007). However, there have been cases of goat's milk allergy in patients who have never had an allergic reaction to cow's milk, and vice versa (Ah-Leung et al., 2006, Bellioni-businco et al., 1999, Goh et al., 2019). According to a study on the cross-reactivity of casein fraction among different ruminant species, IgE responses to ewe’s and goat’s milk were weaker than those to cow’s and buffalo’s milk, implying that some CMPA patients may have the tolerance to ewe’s and goat’s milk (Clark and Mora García, 2017). Furthermore, previous research on the genetic differences between goat breeds has been shown to affect the yield and chemical composition of the goat's milk (Selvaggi et al., 2014, Taiwo Idowu and Olufunke Adewumi, 2017). Rather than dismissing goat's milk entirely, its hypoallergenic potential could be further evaluated using proteomic methods.

Allergenomic analysis remains a potent tool for the rapid and comprehensive identification and characterisation of allergens in specific sources, as well as for further direct comparison with other methods. Protein analytical techniques such as sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoassay techniques such as enzyme-linked immunosorbent assay (ELISA) and Western blotting have been used for allergen detection (Carrera et al., 2020, Sharma et al., 2017). Bottom-up proteomics studies subject a complex protein mixture to several processes such as SDS-PAGE separation and proteolytic cleavage before peptide products are analysed using mass spectrometry (MS). For allergen detection, 2DE separates proteins based on their molecular weight and isoelectric point, resulting in a pattern of protein dispersion. Western blotting can be used to further analyse the specific IgE reactive protein spots produced by SDS-PAGE. MS analysis, such as matrix-assisted laser desorption ionisation time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), can be used to identify the target protein. The integration of 2DE, 2D-immunoblotting, and MS analysis has been widely used in allergen detection due to its reliability when dealing with food proteins, which can have a wide range of isoforms and are susceptible to post-translational modification and structural changes during storage or processing (Carrera et al., 2018; Villa et al., 2018).

In this study, we aimed to perform a comparative mapping and identification of allergenic proteins in milk from different dairy goat breeds that cross-reacted with cow’s milk allergens utilising 2DE paired with IgE-immunoblotting, followed by protein identification via MALDI TOF/TOF MS/MS. Differences in the 2DE protein separation profiles of goat’s milk from different breeds; Saanen, Jamnapari and Toggenburg, were analysed on 2DE before proceeding with the IgE-immunoblotting; to determine the goat’s milk protein that cross-reacted with cow’s milk allergen. Although phylogenetically related ruminant milk proteins are known to be homologous, the genetic variance between the species or breed has shown to be affecting their milk composition and IgE-binding epitope, which should be further investigated.