Identification of Milk Protein Polymorphism in Indian Goats by 2D Gel Electrophoresis

Casein, the main protein fraction of ruminant milk, is one of the valuable components of milk due to its nutritional value and processing properties. Genetic variants of αs1, αs2, β, and κ-casein have been characterized both at protein and DNA level in different goat breeds [1,2]. The caprine casein analysis is quite complex due to large number of mutations in the coding region due to interallelic recombination [3]. The αs1 casein exhibited extensive polymorphism and is also associated with differential level of protein synthesis [4,5]. Similarly αs2-casein locus also showed seven alleles and they are associated with differential synthesis level [6]. κ-casein locus also showed polymorphism and associated with milk quality and cheese making properties [7,8]. At CSN3 locus, 15 different polymorphic alleles have been identified by analysing isoelectro focussing, PCR-SSCP and PCR-RFLP [9,10]. The whey protein, β-lactoglobulin and α-lactalbumin, also showed polymorphism and the distribution of different variants have been investigated in different breeds and population [9,10].


Introduction
Casein, the main protein fraction of ruminant milk, is one of the valuable components of milk due to its nutritional value and processing properties. Genetic variants of αs 1 , αs 2 , β, and κ-casein have been characterized both at protein and DNA level in different goat breeds [1,2]. The caprine casein analysis is quite complex due to large number of mutations in the coding region due to interallelic recombination [3]. The αs 1 casein exhibited extensive polymorphism and is also associated with differential level of protein synthesis [4,5]. Similarly αs2-casein locus also showed seven alleles and they are associated with differential synthesis level [6]. κ-casein locus also showed polymorphism and associated with milk quality and cheese making properties [7,8]. At CSN3 locus, 15 different polymorphic alleles have been identified by analysing isoelectro focussing, PCR-SSCP and PCR-RFLP [9,10]. The whey protein, β-lactoglobulin and α-lactalbumin, also showed polymorphism and the distribution of different variants have been investigated in different breeds and population [9,10].
It is also not possible to analyze the variation of casein cluster by any single approach, therefore various approaches have been applied to determine variability at casein loci. The protein variability has been characterized by SDS-PAGE analysis, however the effect of deletion/ substitution have no effect on net charge of proteins. Two-dimensional polyacrylamide gel electrophoresis (2-DE) is a powerful technique that separates the proteins on the basis of their isoelectric point (pI) in the first dimension and on the basis of their molecular weight in the second dimension [11]. It allows the separation of individual proteins that are defined by their size and chemical structure [12]. Milk casein presents some interesting findings with multiple forms present at both high and low amount. In the present study, we analyzed the major variants of goat milk casein complex by SDS-PAGE and the resolution of different goat milk casein forms using 2-DE.

Genetic stock and experimental design
The six goat breeds namely Jamunapari, Barbari, Jakhrana, Ganjam, Beetal and Sirohi have been included in this study. The total number of samples 160, 337, 72, 42, 30 and 16 were collected from Jamunapari, Barbari, Marwari, Jakhrana, Sirohi, Beetal and Ganjam goats, respectively. The details of the goat breeds and their utility have described in table 1.

Sample collection and processing of samples
Milk samples were collected from six Indian goat breeds from their natural habitat and transported to the laboratory and stored at -20°C for further analysis.

Milk samples
The milk samples stored at -20°C were allowed to thaw and then centrifuged at 12000 rpm for 10 min at 4°C. The milk serum was

Abstract
Analysis of casein and whey protein was carried out in six Indian goats by both SDS-PAGE and 2-DE analysis. The variation was observed mainly in αs 1 , αs 2 , β, κ-casein and β-lacto globulin locus in these breeds. Proteome analysis showed the presence of high number of spots in αs 1 -casein, and κ-casein showed highest number of spots in all the goat breeds. The gels were showing remarkably both variability and similarity suggesting that the heterogeneity in protein forms in individual milk samples exist, and milk protein represents a common pattern of post-translational modification. Large size known for meat and milk aspirated out from below the surface creamy layer and was used for all further analysis

Protein determination
Protein concentration of the samples was determined by Folinphenol method [13], using BSA as the standard.

SDS-PAGE and image analysis
SDS-PAGE was carried out in a Vertical Gel Electrophoresis apparatus (plate size: 160 mm×160 mm) (ATTO Corp., Japan) using 5% (w/v) stacking gel and 12% (w/v) separating gel [14]. After electrophoresis, the gels were stained with Coomassie blue R-250 for visualization of the proteins. Molecular weight of the protein bands was determined with reference to standards (SIGMAMARKER, M-4038). Densitometric scanning of the gels was carried out using a Gel Analysis Software, Gene Tool (Mascon Global Ltd.)

2-DE
The samples which showed more variability in casein loci in SDS-PAGE were examined by 2-DE. For 2-DE [15], isoelectric focusing (first dimension) was performed in a Mini-Protean II electrophoresis cell (Bio-Rad Laboratories, Richmond, CA) according to the manufacturer's instructions. The gel solution was prepared as follows: 9.2 M urea, 4% acrylamide, 2.0% (w/v) CHAPS, 1.6% Bio-Lyte 5/7 ampholyte, 0.4% Bio-Lyte 3/10 ampholyte. Polymerization was initiated by 0.01% ammonium persulfate and 0.1% (v/v) TEMED. The upper chamber was filled with cathode electrode buffer (100 mM NaOH). The anode electrode buffer was 10 mM ortho-phosphoric acid. First-dimension gels were prefocussed at 200 V for 10 min, 300 V for 15 min, and 400 V for 15 min. The electrode buffers were exchanged against fresh and degassed buffers. Samples were prepared by adding an equal volume of sample buffer (final conc.: 9.5 M urea, 2.0% CHAPS, 5% β-mercaptoethanol, 1.6% Bio-Lyte 5/7 ampholyte, 0.4% Bio-Lyte 3/10 ampholyte) and incubated at room temperature for 10-15 min after which sample proteins (80-100 µg) were loaded into the sample reservoirs. The samples were overlaid with 20-40 µl of sample overlay buffer (final conc.: 9M urea, 0.8% Bio-Lyte 5/7 ampholyte, 0.2% Bio-Lyte 3/10 ampholyte, and 0.05% bromophenol blue). 2D SDS-PAGE Standards (Bio-Rad) were co-run parallel to samples. The proteins were electrophoresed at 500 V for 10 min followed by 750 V for 3.5 hr. After the first dimension run, the gels were pressed out of the tubes with the help of a tube gel ejector filled with second dimension sample buffer and were equilibrated for 10-15 min at room temperature. They were loaded on to the slab gel for second-dimension run. The IEF gels not used immediately were stored at -70°C in sample equilibration buffer. The second dimension run (SDS-PAGE) was carried out in a Mini-Protean 3 electrophoresis cell (Bio-Rad Laboratories) using 5% (w/v) stacking gel and 12% (w/v) separating gel, as described above. The gels were silver stained.

2-D Gel image analysis
Analysis of gel samples and matching between gels were carried out by Gel Fox 2D V.3.01 (Alpha Innotech, USA). The 2-DE of bovine milk proteins was used as reference gel [16].

Analysis of milk protein variants by SDS-PAGE
SDS-PAGE was carried out to analyze the milk sample for genetic polymorphism of six Indian goat breeds. The SDS-PAGE profile of milk protein variants of αs 1, αs 2, β-Cn, β-LG and α-LA are presented in figure 1. In αs 1 -casein, the A variant was most frequent in all the breeds and majority of goat were found as homozygous AA. The B variant was observed in heterozygous from (AB). The F variant was found as heterozygous AF in some of the samples. In αs 2 _ casein, the most prevalent variant was A in all the samples and mostly observed as homozygous AA. The B variant was found as homozygous (BB). The monomorphic pattern was observed in the κ-casein locus and found as homozygous AA in all the samples. The SDS-PAGE pattern of β-LG was almost similar in all the samples analyzed in this study. It exhibited homozygous AA, however, B variant was also observed in individuals in all the breeds as heterozygous AB. The electrophoretic pattern revealed that the variant 'A' was most frequent than variant 'B' and was observed as homozygous AA in all the samples at α-LA locus. The milk protein polymorphism indicates that each milk protein has two or more forms, which is genetically determined by autosomal gene and co-dominant alleles. The samples which showed more variability in SDS-PAGE were examined by 2-DE.

Separation of goat milk proteins by two dimensional gel electrophoresis (2-DE)
The There are number of protein spots resolved in milk protein area and large spots identified as α S1 -casein and β-casein dominated the gel. The two-dimensional gel electrophoresis profile of milk protein variants showed sharp, distinct bands for whey protein and their more diffuse, wavy bands could recognize the casein. The 2-DE patterns of individual milk sample are presented in figure 3 in different Indian breeds. The high degree of heterogeneity with goat milk protein explains the different band intensities. The α S1 -casein focused anodic end of the gel. Each variant consists of two fractions i.e. the more concentrated α S1casein and diffuse fraction, which was not identified. The variant A and B could be clearly distinguished. α S2 -casein was found in two variants α-S2 csein α-S1 csein β -Casein κcasein A, B and each variant revealed one major band situated cathodically, and two minor fractions, which focused more anodally. Two large spots identified as β-casein, dominated the gel. κ-casein presents an even more complex picture with at least 6 forms present with pI values ranging from 5.81 to 4.47 (Figure 3). A similar gel obtained with milk from a single goat electrophoretically typed as having both the A and B variants of κ-casein. The major whey proteins, β-lactoglobulin and α-lactalbumin, were also observed. Ganjam goat showed the absence or very low intensity spot of αs 1 -casein locus, where as β-casein and κ-casein is showing higher number of spots in the sample. κ-casein locus showed less number of spots in Jamunapari goats. It is clear that the vast majority of protein spots on the 2-D gels of milk samples occurred due to proteolysis, phosphorylation or glycosylation. The gels showed some similarity suggesting that milk protein represents a common pattern of post-translational modification among individuals ( Figure  3). With its high resolution, 2-DE allows separation of protein isoforms containing the slightest differences, a key in the comprehensive analysis of individual milk samples.

Discussion
The important contribution of milk to human nutrition and its availability in large amounts have made it the subject of biochemical studies for many decades. The protein in milk is just the product of six genes, which constitutes about 95% of milk protein. The proteome of milk is showing complicated spots with the presence of numerous low-abundance gene products and a high level of post-translational modifications including phosphorylation and glycosylation. There has been considerable interest in goat milk protein due to availability of different bioactive peptide derived from milk proteins, which have pharmaceutical and nutraceutical applications and have future commercial importance [17,18]. Secondly, goat milk is used for treatment of different disease, allergy and heat stress and can have future health application. Therefore, the milk proteome analysis appears to be timely and required for future industrial application with respect to human health and nutrition [19].
Milk proteome is complex and this complexity is the consequence of post-translational modifications and due to the presence of numerous genetic variants of protein and bioactive peptides in milk. The 2-DE gel analysis presented the complex heterogeneity in goat milk  proteins in different breeds. The variation was observed between SDS-PAGE and 2-DE analysis mainly at κ-casein, α S1 -casein and β-LG and α-LA loci. Despite the presence of hundreds of proteins, no study was able to identify any proteins other than caseins, β-LG, α-LA. It is also clear that numerous protein spots in 2-DE gels of milk samples are due to proteolysis, phosphorylation and glycosylation. Attempts have been made to analyze different low abundance protein through different approaches [20][21][22].

Conclusion
2-DE is one of the powerful techniques used for characterizing milk proteome in goats. A large variation in protein spots was observed in gel analysis indicating differential expression due to the presence of hundreds of protein and bioactive peptide in milk.