Bovine milk has long been the primary milk source for human consumption. However, hypersensitivity to bovine milk proteins is a major food allergy, which affects primarily infants, but may also persist throughout adulthood and can be very severe (Lara-Villoslada et al. Reference Lara-Villoslada, Olivares and Xaus2005). Most children suffering from bovine milk protein allergy synthesise antibodies principally against the αs-caseins and β-lactoglobulin (Restani et al. Reference Restani, Gaiaschi, Plebani, Beretta, Cavagni, Fiocchi, Poisei, Velona, Ugazio and Galii1999; Bevilacqua et al. Reference Bevilacqua, Martin, Candalh, Fauquant, Piot, Roucayrol, Pilla and Heyman2001; Ametani et al. Reference Ametani, Sakurai, Katakura, Kuhara, Hirakawa, Hosoi, Dosako and Kaminogawa2003). Human milk has long been recognised as the optimal form of nutrition for the neonate (Work Group on Breastfeeding, 1997) and it is well known that the allergenicity of its primary alternative, i.e., infant formula is primarily based on its bovine milk-derived protein fraction. At present, the most common hypoallergenic alternative to conventional bovine milk-based formula is formula containing protein hydrolysates, containing peptides with a molecular mass around 3 kDa, as these do not normally act as allergens (El-Agamy, Reference El-Agamy2007). A drawback of this approach is that formulae containing milk protein hydrolysates often suffer from bitterness and require the inclusion of additional emulsifiers.
In recent years, there has been an increasing focus on the use of proteins from non-bovine milks as an alternative protein source for hypo-allergenic bovine milk alternatives. There are several reports that equine, camel and caprine milk might be preferable to bovine milk with respect to allergenicity, particularly for infants and elderly people. Sanz Caballos et al. (Reference Sanz Caballos, Morales, de la Torre, Diaz, Perez and Sanz2009) showed that caprine milk protein is more digestible and more tolerable than bovine milk protein, while Egito et al. (Reference Egito, Miclo, López, Adam, Girardet and Gaillard2002) suggested that equine milk may be a curative agent for digestive and cardiovascular diseases.
The gross composition of the protein fraction of milks of common dairying species have been characterised, but nutritional suitability is not only related to gross composition, but is also strongly affected by the microheterogeniety of the protein fraction; hence, further stages in the evaluation of the potential suitability of non-bovine milks in human/infant nutrition include a detailed characterisation of their proteinaceous constituents. Proteomic techniques are ideally suited to obtaining such information and have been applied to the study of bovine milk and milk products (Holt & Zeece, Reference Holt and Zeece1987; Chin & Rosenberg, Reference Chin and Rosenberg1998; Holland et al. Reference Holland, Deeth and Alewood2004; Lindmark-Månsson et al. Reference Lindmark-Månsson, Timgren, Aldén and Paulsson2004; Fong et al. Reference Fong, Norris and Palmano2008; Pappa et al. Reference Pappa, Robertsona, Rigbya, Mellona, Kandarakisb and Mills2008). However, only a few studies have examined non-bovine milks using the proteomic approach. The aim of the present study was to apply proteomic tools, i.e., two-dimensional electrophoresis (2-DE) followed by MALDI-ToF mass spectrometry, to characterise the protein system in buffalo, caprine, equine, camel and bovine milk. The outcomes of these investigations can be applied as an initial step to evaluate the suitability of non-bovine milk proteins as suitable source for hypoallergenic nutritional products.
Material and Methods
Milk supply
Raw fresh bovine milk was obtained from a local dairy company. Caprine milk was obtained from a commercial source. Raw fresh buffalo milk was obtained from North Wales Buffaloes (Midlist Farm, Halkyn, Holywell, Flintshire, UK). Raw fresh camel milk was obtained from Kamelenmelkerij Smits (Cromvoirt, The Netherlands). Raw fresh equine milk was obtained from Orchid's Paardemelkerij, (Zeeland, NBr., The Netherlands).
One- and two-dimensional gel electrophoresis
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Urea-PAGE, were performed as described by Hinz et al. (Reference Hinz, Huppertz, Kulozik and Kelly2007) and Larsen et al. (Reference Larsen, Hinz, Jørgensen, Møller, Wellnitz, Bruckmaier and Kelly2010), respectively. 2-DE was performed as described by Larsen et al. (Reference Larsen, Hinz, Jørgensen, Møller, Wellnitz, Bruckmaier and Kelly2010). Stained gels were then scanned using a calibrated GS-800 densitometer (Bio-Rad, Hercules, CA, USA)
In-gel digestion and matrix-assisted-laser-desorption/ionization time-of-flight (MALDI-ToF) analysis
Relevant protein spots were excised from the 2D-gels and 50 μl acetonitrile were added to each gel spot and left at room temperature for 15 min. Acetonitrile was then removed and 25 μl of 25 mm ammonium bicarbonate was added for 10 min to rehydrate the spots. This procedure was repeated twice more, after which the spots were dehydrated with 50 μl acetonitrile. The acetonitrile was subsequently removed and the spots were dried at 37°C for 20 min in a heating block.
Sequencing-grade modified trypsin (Promega Corporation, Madison, WI, USA) was dissolved in 25 mm ammonium bicarbonate at a final concentration of 10 ng per μl. A 3 μl aliquot of trypsin solution was added to each spot and this was allowed to absorb fully into the spot, before further 3 μl aliquots of trypsin were added until the spot was fully rehydrated. Subsequently, 10 μl of 0·5 mm ammonium bicarbonate was added to cover the spots. Each gel spot was kept on ice during rehydration with trypsin to prevent trypsin autodigestion peaks. Gel spots were digested in a Discover Microwave (CEM Corporation, Matthews, NC, USA) at 50 W at 55°C for 15 min. Digests were allowed to cool and the supernatant was transferred to a fresh vial. To each gel spot, 10 μl of 25 mm ammonium bicarbonate in 50% acetonitrile was added, after 15 min this was added to the original supernatant.
Mass spectrometry was performed on the gel spot extracts with an Axima ToF2 MALDI-ToF mass spectrometer (Shimadzu Biotech, Manchester, UK). A 0·5 μl aliquot of matrix solution (10 mg/ml, α-cyano 4-hydroxy cinnamic acid, in 50% (v/v) acetonitrile-0·1% (v/v) trifluoroacetic acid) was deposited onto the target and left for 5 s before being removed. The residual solution was allowed to air-dry and 0·5 μl of the sample solution was deposited onto the pre-coated sample spot. A 0·5 μl aliquot of matrix solution was added to the deposited sample and allowed to air-dry. The sample was subsequently analysed in positive-ion reflectron mode.
Protein identification was carried out via peptide mass fingerprinting (PMF) using the Mascot search engine (http://www.matrix-science.com). The monoisotopic, positive ion data ±0·25 Da, was searched using the following parameters: NCBInr database or Swiss Prot, taxonomy mammalian, trypsin digest with one missed cleavage. Variable modifications including cysteine modified by carbamidomethylation and methionine modified by oxygen were also checked.
Tandem mass spectrometry (MS/MS) was carried out on peaks from spots that did not score well using PMF. The MS/MS positive ion averaged data,±0·8 Da, was again searched using the Mascot engine and the following parameters: NCBInr database or Swiss Prot, taxonomy mammalian trypsin digest with one missed cleavage or semi trypsin with one missed cleavage; variable modifications including cysteine modified by carbamidomethylation, methionine modified by oxygen and acetyl N-terminal were also checked.
Results and Discussion
SDS-PAGE and Urea-PAGE analysis of milk samples
Urea-PAGE patterns of proteins from the five types of milk are shown in Fig. 1. Bovine β- and αs1-casein had about the same migration position as their counterparts in caprine or buffalo milk, whereas the migration of equine β-and αs1-casein was slightly slower. The slowest migration for β- and αs1-casein was found in camel milk. Since separation of proteins by Urea-PAGE is determined primarily by the net-negative charge of the proteins, the results presented in Fig. 1 suggest that equine and camel caseins may have a lower net-negative charge than their bovine, buffalo and caprine counterparts.
Urea-PAGE electrophoretogram of (1) bovine milk; (2) camel milk; (3) equine milk; (4) buffalo milk or (5) caprine milk. Protein identification is based on the bovine sample lane.
SDS-PAGE gels of bovine, buffalo, caprine, camel and equine milk casein (Fig. 2) highlighted a unique electrophoretic pattern for the milk of each species. The principal bands in all samples corresponded to αs1-casein, β-casein and, with the exception of equine milk, κ-casein (Merin et al. Reference Merin, Bernstein, Bloch-Damti, Yagil, van Creveld, Lindner and Gollop2001; Park, Reference Park2001; Haenlein, Reference Haenlein2004; El-Agamy et al. Reference El-Agamy, Nawar, Shamsia, Awad and Haenlein2009). The migration positions of αs1-, β- and κ-casein in bovine, buffalo, caprine and equine milk were comparable, but a slower migration rate was observed for the camel milk caseins. Bovine, buffalo, caprine and equine milk showed a band for β-lg, while camel milk did not contain a band at the expected position of β-lg. α-Lactalbumin was observed in all samples.
SDS-PAGE electrophoretogram of (1) bovine milk; (2) camel milk; (3) equine milk; (4) buffalo milk or (5) caprine milk. Protein identification is based on the bovine sample lane.
Comparison of the 2-DE patterns and identification of protein spots from different milks
The protein patterns of each milk sample were also analysed by 2-DE. A typical 2-DE electrophoretogram of bovine milk is shown in Fig. 3; as expected the most abundant proteins of bovine milk were the caseins αs1-casein, αs2-casein, β-casein and κ-casein and the whey proteins β-lg, α-la and bovine serum albumin. There were clear differences between the 2D electrophoretograms of buffalo, caprine, camel and equine milk (Fig. 4), compared with that of bovine milk. Mass spectrometry was performed on peptic digests of spots excised from these gels to identify the principal proteins, as well as proteolysis products in these milks.
Two-dimensional gel electrophoretogram of bovine milk. The gels show molecular mass (M r) and IEF at pH 4 to 7.
Two-dimensional gel electrophoretograms of (a) camel milk; (b) equine milk; (c) buffalo milk or (d) caprine milk. The gels show molecular mass (M r) and IEF at pH 4 to 7.
Mass spectrometry identifications of the relevant spots highlighted in Figs 4a–d are shown in Table 1. For camel milk, 13 spots were identified, arising from β-casein (spots 4A, 4A, 5A and 7A), αs1-casein (spots 2A, 8A and 11A) κ-casein (spots 9A, 10A, 11A, 12A and 13A), α-la (spots 6A) and serum albumin (spot 1A). The camel κ-caseins were identified in the pI region 4·1–4·6, which has not been reported previously. Indeed, some studies have not detected bands for κ-casein at all in SDS-PAGE electrophoretograms (Farah & Farah-Riesen, Reference Farah and Farah-Riesen1985) but, in this study five isoforms of κ-casein were identified using 2-DE. Several isoforms of κ-casein are known to be present in 2-DE electrophoretograms of bovine milk (Holland et al. Reference Holland, Deeth and Alewood2004). The low pI region where the camel κ-caseins were observed is not in agreement with their theoretical pI of 8·03 (Table 1). However, it should be taken into account that the theoretical pI values in Table 1 are based on the primary structure of the protein only and do not take into account post-translational modification. κ-Casein, however, is a phosphoglycoprotein, and the carbohydrate groups are attached to κ-casein via O-glycosidic linkages to serine and threonine residues within the C-terminal portion of the molecule (West Reference West1986; Rasmussen et al. Reference Rasmussen, Johnsen, Tsiora, Sørensen, Thomsen and Nielsen1999). Glycosylation occurs post-translationally, and a possible explanation for the lower-than-predicted pI of camel κ-casein could be a strong negative charge introduced through glycosylation and phosphorylation; this has not been studied to date, and requires experimental conformation. The observation that a decrease in pI for the 5 κ-casein spots in Fig. 4a is accompanied by increases in molecular mass, and that the molecular mass is considerably higher than that predicted based on its amino acid sequence (Table 1) support the possibility of strong glycosylation of camel κ-casein. Studies have also shown that human κ-casein is more highly glycosylated than the bovine protein and that carbohydrate residues may account for up to 55% of the weight of the molecule (Ginger & Grigor, Reference Ginger and Grigor1999).
Identification of spots from the 2D gels of milk from different animal species, by peptide mass fingerprinting (PMF) using MALDI-ToF†
† Protein reference (Ref.) correspond to the Swiss-Prot/NCBI accession number; % cov. refers to sequence coverage. Theoretical molecular mass and isoelectric point (pI) of proteins are as according to the amino acid primary sequence and without consideration and/or degradation modifications. Observed molecular mass and isoelectric point (pI) are as observed with the position of the corresponding spots on the 2-DE gels
‡ Tandem mass spectrometry (MS/MS) was carried out on peaks from spots that did not score well using PMF
β-Lg was not found in camel milk (Fig. 4a), which is in agreement with the SDS-PAGE electrophoretogram (Fig. 2a) and previous reports (Kappeler et al. Reference Kappeler, Farah and Puhan2003, El-Agamy et al. Reference El-Agamy, Nawar, Shamsia, Awad and Haenlein2009). It should be noted that, unlike the other common milk proteins, camel β-lg is not included in the Swiss Prot and UniProt database, as it has not been possible to confirm the presence of this protein in milk and thus allow its isolation and primary sequence determination if present. However, unlike serum albumin (see below), for which the sequence of the camel variant has thus far not been established either, no spot showing a high degree of sequence homology to β-lg from other species was found in camel milk. As β-lg is a common allergen in bovine milk and is absent from human milk, camel milk has been suggested to be a good substitute for people who suffer from bovine milk allergy, as this is free of β-lg; in addition, the ratio of casein to whey protein is low, which results in soft curds and therefore easier digestibility (El-Agamy, Reference El-Agamy and Park2006). The molecular weight of camel serum albumin estimated in this study was similar to data reported by Farah (Reference Farah1986) and Ereifej et al. (Reference Ereifej, Alu'datt, AlKhalidy, Alli and Rababah2011), who found that its molecular weight was 66 kDa. However, there seems to be a close taxonomic proximity between camel serum albumin and bovine serum albumin.
When comparing the masses identified by electrophoresis, to those of the parent proteins, it is apparent that for some spots, the observed mass was considerably lower than that of the full protein. This indicates that some of the spots, i.e., 3A, 4A, 5A, 7A and 8A in the case of camel milk (Fig. 4a), represent proteolysis products rather than intact proteins. Identification of the peptides covered in the MALDI-ToF and MS/MS spectra of the tryptic digests of the spots is shown in Table 2. Using these peptides, the minimum part of the parent protein covered in the peptide found in each spot can be reconstructed. As outlined in Table 2, for the peptide spots found in the 2D-gel of camel milk, the 4 peptides derived from β-casein all included its C-terminus. Plasmin is known to be present in camel milk, and hydrolysis of camel casein by plasmin results in the formation of hydrolysis products which are probably homologous to the γ-caseins in bovine milk (Baer et al. Reference Baer, Ryba and Farah1994). Hence, it is possible that the β-casein peptides identified in the 2-DE electrophoretogram of camel milk (Fig. 4a, Tables 1 & 2) represent peptides analogous to the γ-caseins produced in bovine milk as a result of plasmin-induced hydrolysis of β-casein. The αs1-casein peptide identified in camel milk (spot 8A) covered part of the middle part in the amino acid sequence (Table 2).
Peptide identification for selected spots from 2-DE gels by peptide mass fingerprinting (PMF) by using MALDI-ToF
In equine milk, 6 spots were identified, as β-casein (spots 1B and 6B), αs1-casein (spot 5B), β-lg (spots 2B and 3B) and α-la (spot 4B). In contrast to the other milks investigated, κ-casein was not identified in equine milk. Spots 5B and 6B were identified as peptides originating from the C-terminus of equine αs1-casein and β-casein, respectively. The two polypeptide fragments derived from αs1- and β-casein may be proteolysis products arising from the action of plasmin, as reported by Egito et al. (Reference Egito, Miclo, López, Adam, Girardet and Gaillard2002). Matéos et al. (Reference Matéos, Girardet, Molle, Dary, Miclo and Gaillard2009) reported that equine β-casein presents a complex 2-DE pattern, arising from variable degrees of phosphorylation, alternative splicing, and probable instability resulting from non-enzymatic deamidation, but this microheterogeniety was not observed in this study (Fig. 4b).
The 2-DE pattern of buffalo milk (Fig. 4c) is similar to that of bovine milk. The nine spots identified in buffalo milk consisted of β-casein (spots 4C and 9C), αs1-casein (spots 2C and 3C), κ-casein (spot 8C), β-lg (spot 5C), α-la (spots 2C and 3C) and serum albumin (spot 1C). Peptide spot 9C in buffalo milk covered the C-terminus of β-casein; Madkor & Fox (Reference Madkor and Fox1991) reported that the breakdown of buffalo casein by plasmin was generally similar to that in bovine milk, so it is possible that spot 9C represents a buffalo γ-casein. Spot 3C covered a large part of the middle of the amino acid sequence of buffalo αs1-casein. Serum albumin was identified by the database search as bovine serum albumin which suggests that buffalo serum albumin has a close taxonomic analogy to bovine serum albumin (Satija et al. Reference Satija, Rajpal, Pandey and Sharma1979; Buffoni et al. Reference Buffoni, Bonizzi, Paciullo, Ramunno and Feligini2011).
Six spots were identified in caprine milk, i.e., β-casein (spots 3D and 6D), αs2-casein (spot 2D), κ-casein (spot 4D) and serum albumin (spot 1D). β-Casein is the major fraction in caprine casein, which is similar to human casein and different from bovine casein (El-Agamy, Reference El-Agamy and Park2006). Caprine milk lacks αs1-casein and this may explain why caprine milk is less allergenic than bovine milk (Lara-Villoslada et al. Reference Lara-Villoslada, Olivares and Xaus2005). The appearance of low molecular weight products in Fig. 4d are polypeptide fragments derived from β-casein (spots 6D). Caprine milk contains the entire plasmin system: plasmin, plasminogen, plasmin inhibitors, plasminogen activators, and inhibitors of plasminogen activators (Trujillo et al. Reference Trujillo, Guamis and Carretero1997). Santillo et al. (Reference Santillo, Kelly, Palermo, Sevi and Albenzio2009) reported that plasmin mainly contributed to hydrolysis of β-casein in caprine milk, analogously for bovine milk.
Two-DE studies have also been undertaken on the protein system in human milk (Armaforte et al. Reference Armaforte, Curran, Huppertz, Ryan, Caboni, O'Connor, Ross, Hirtz, Sommerer, Chevalier and Kelly2010). While caseins are the principal class of protein in milk from most species, human milk has a higher proportion of whey proteins, and β-lg is absent. The comparison of 2-DE gels of human milk with those of bovine, buffalo, camel, goat and equine milk showed that the protein expression of human milk and bovine/buffalo differs remarkably, whereas human milk is more similar to that of goat, camel and equine milk (D'Auria et al., Reference D'Auria, Agostoni, Giovannini, Riva, Zetterstroem, Fortin, Greppi, Bonizzi and Roncada2005; Armaforte et al. Reference Armaforte, Curran, Huppertz, Ryan, Caboni, O'Connor, Ross, Hirtz, Sommerer, Chevalier and Kelly2010). While the most abundant proteins in the bovine milk proteome are αs1-casein, αs2-casein, β-casein and the two genetic variants of β-lg, with α-la and high molecular mass proteins present in low concentrations, the most abundant protein in human milk was α-la, followed by groups of isoforms of β-caseins and α-caseins; moreover, high molecular mass proteins represented an important fraction of the total protein content. Several isoforms of κ-casein are known to be present in bovine milk, and several isoforms where found in camel milk, while only one spot was identified as κ-casein in human milk (Armaforte et al. Reference Armaforte, Curran, Huppertz, Ryan, Caboni, O'Connor, Ross, Hirtz, Sommerer, Chevalier and Kelly2010). Armaforte et al. (Reference Armaforte, Curran, Huppertz, Ryan, Caboni, O'Connor, Ross, Hirtz, Sommerer, Chevalier and Kelly2010) also identified a number of breakdown products in pre-term human milk, which derived mainly from αs1-and β-casein. The study also reported a proteomic comparison between bovine milk and infant formulae and, as expected, the qualitative protein profile of powdered infant formula was very similar to that of bovine milk.
Conclusion
A proteomic approach showed the differences in the protein composition of the milk of 5 different milk species. Several studies have evaluated the use of milk from different animals like camel, buffalo, goat and horse. The available data in the literature may suggest the use of animal milk as alternatives to human milk. However, proteomic tools highlighted significant differences in the protein profile between the animal species and can help further to find alternatives to human milk due to hypoallergenic properties of their proteins. The findings of the present study provide further evidence that the protein profile of bovine milk appears to be quite different, i.e., from camel, caprine, equine or buffalo milk. Camel milk might be a promising new protein source for children allergic to bovine milk protein, as β-lg the most dominant bovine milk allergen, could not be found in camel milk. These results could thus be useful in the further improvement of infant formulae. Another potential application of these results relates to adulteration of expensive milks (horse, camel, buffalo, goat) with cheaper milk types, which is not uncommon in various parts of the world and remains an issue of concern. In this respect, 2D electrophoresis in tandem with MS could prove a rapid way to identify the origin of individual proteins.
Funding for this research was provided under the National Development Plan 2007–2013, through the Food Institutional Research Measure, administered by the Department of Agriculture, Fisheries & Food, Ireland.
