The concern for the genetic variants of the different milk proteins has been intriguing in the dairy sector. The phenotypes of these proteins have been intensively studied because of their known relation to milk processing characteristics (such as cheese-making ability) and other compositional aspects. The Gangatiri is a dual-purpose zebu breed of Bos indicus origin and is one of a number of indigenous cattle breeds in India. It is a relatively obscure indigenous dairy animal with its primary use in milk and draught. It has always been an elite bovine breed for small and marginal farmers because of its low input management, high disease resistance and better survival in harsh climatic conditions (Singh et al., Reference Singh, Kumar, Nimmanapalli and Hooda2023).
Beta-lactoglobulin (β-Lg) is a typical globular protein consisting of 162 amino acids that typically comprises more than half of the total whey protein in milk (Gharedaghi et al., Reference Gharedaghi, Shahrbabak and Sadeghi2016). The most common polymorphic products of β-Lg are A and B, with eleven different less frequent genotypes. In bovine, exon IV is the point of mutation for variant B that differs from the A variant in that it substitutes a glycine and an alanine for an aspartic acid and a valine (Keppler et al., Reference Keppler, Heyse, Scheidler, Uttinger, Fitzner, Jandt, Heyn, Lautenbach, Loch, Lohr, Kieserling, Günther, Kempf, Grosch, Lewiński, Jahn, Lübbert, Peukert, Kulozik, Drusch, Krull, Schwarz and Biedendieck2021). The aim of this study is the phenotyping of β-Lg protein in Gangatiri cow milk by applying polyacrylamide gel electrophoresis (PAGE) and then proteome sequence analysis with the sophisticated technology of a high-resolution accurate mass spectrometry system (HRAMS).
Materials and methods
All the consumables and markers were procured from SRL chemicals.
Milk samples collection
A total of 24 raw milk samples (100 ml) were drawn individually from native Gangatiri cows, mostly kept in the animal shed of the Faculty of Veterinary and Animal Sciences, Banaras Hindu University, Mirzapur and some from the State Animal Farm, Shahanshahpur, Varanasi, Uttar Pradesh. All the selected animals were healthy and had no clinical signs of mastitis. Raw milk samples were stored at −20°C until analysis (Nguyen et al., Reference Nguyen, Solah, Busetti, Smolenski and Cooney2020). Samples were pooled into eight milk samples because of the high fat content, making them easier to handle and process.
Whey protein extraction and electrophoresis
As Vincent et al. (Reference Vincent, Ezernieks, Elkins, Nguyen, Moate, Cocks and Rochfort2016) described, a modified whey precipitation method was used to fractionate the whole whey portion. Briefly, the raw milk samples were heated to 60°C before centrifugation at 2600 × g for 20 min at 30°C to separate the fat. Then, TCA precipitation was carried out with 10% TCA (1.5 ml volume) for the whey sample preparation followed by centrifugation at 13 000 rpm for 10 min at 5°C. Sample pellets were resuspended in 2% DEA (diethylamine) and 1 × PBS and the pH was maintained at 7.5. The protein quantification was done using the Lowry assay.
The method of Andrews (Reference Andrews1983) was applied to perform the gel electrophoresis of Gangatiri whey protein. Samples were added 1 : 1 (v/v) with a 125 mM tris HCl, 4% SDS, 0.4% bromophenol blue, 10% 2-mercaptoethanol and 10% glycerol at pH 6.8. At 95°C, the working solutions of whey were heat-denatured for 10 min. In the run of the experiment, 10 μg of isolated whey protein from different milk samples was run into the PAGE wells. The pre-stained protein marker (6 μl) was loaded in the first well between all the runs. The stacking gel solution was made up of 5% acrylamide and 10% SDS at pH 6.8, whereas the resolving gel contained 15% acrylamide and 10% SDS at pH 8.8. A Mini PROTEIN cell (Bio-Rad) was filled with 5 × running buffer with 0.025 M Tris, 0.192 M glycine, and 10% concentration of SDS, pH 8.3. Both the electrophoretic runs were completed at ambient temperature using the voltage stepped method: 30 V potential difference was maintained until the samples were entirely removed from the stacking gel. Then the voltage was set to 100 V by increasing 15 V per minute four times until the tracking dye had reached the bottom. The gel staining was performed with Coomassie R-250 and documentation on the Gel Doc Go image system.
β-Lg variant identification by HRAMS sequencing
After the tricine SDS-PAGE in-solution digestion process, gel fractions were excised and cut into small pieces before destaining. The reduction treatment was carried out using 0.005 M TCEP, followed by additional alkylation using 0.05 M IAA and then trypsin degradation for 16 h at 37°C (1 : 50, trypsin/lysate ratio). Peptides were then vacuum dried when the reaction was ended with 10% trifluoroacetic acid. Buffer A contained 2% acetonitrile by volume and 0.1% formic acid by volume, and B (80% acetonitrile, 0.1% formic acid). The Thermo-scientific Ultimate-TM 3000 RSLC-nano system coupled with QE Plus was used for the mass spectra analysis of the peptide mixture. In the C-18 column, gradients of chromatography were performed for 100 min. The orbitrap was used to acquire the mass spectra at 70 K resolution. All charged states of the precursor had been eliminated after a 10 s dynamic exclusion. All samples were evaluated, a file was produced and a proteome discoverer was utilized to compare it to the UniProt custom proteome reference database. The protein false discovery rate (FDR) was adjusted to 0.02 after matching the peptide spectra (Singh et al., Reference Singh, Kumar, Nimmanapalli and Hooda2023).
Results and discussion
In the present study of the variant detection of β-Lg of the Indian zebu breed Gangatiri, the optimized TCA/acetone precipitation procedure was used to obtain the aqueous form of whey protein. This isolation method is best for identifying a high number of proteins in the mass spectroscopy and better electrophoresis profile (Chopra et al., Reference Chopra, Ali, Bathla, Rawat, Vohra, Kumar and Mohanty2020). In the run of SDS-PAGE of Gangatiri whey protein with increasing loading concentration in the six consecutive wells of the gel after the protein marker, all presented a similar type of separation of different whey fractions in all the wells (Fig. 1). The separation of whole whey protein into different fractions showed uniformity in each lane with an average retention factor (Rf) value of 0.44 with a standard deviation of 0.5%. The bands of varied whey protein fractions were reliably found in all the triplicate experiments.
Figure 1. Illustration of the representative mass spectrum obtained after the accurate mass spectroscopy using Amanda 2.0 and Sequest HT search engines for the identification of the Gangatiri β-Lg amino acids chain.
The isolated protein fractions of the Gangatiri zebu milk whey were digested with in-gel tryptic digestion and applied into nano-LC/MS run (Fig. 1). Using the data processing modules of MS Amanda 2.0 and Sequest HT the proteomic analysis was carried out and showed the sequence of β-Lg on the high FDR confidence with 23% coverage. The detailed sequencing result of β-Lg is highlighted in the boxes with serial numbers, showing their coverage percentage, total unique peptides, the number of amino acids, identified molecular weight and gene IDs (Table 1 and in online Supplementary Table S1). Some other minor whey proteins were seen including ubiquitin, histones, heat shock proteins and others. The scatter density plots were plotted for the quality pattern analysis of the obtained whey proteins with highlighted β-Lg, showing the range of unique peptides (0–22), Sum posterior error probability (PEP) score (0.842–277.44), peptide spectrum matches (1–966) and peptides score (1–23) (Fig. 2).
Table 1. Sequencing result of beta-lactoglobulin obtained from accurate mass spectroscopy highlighting glycine amino acid at 64th and alanine at 118th among the 162 residues
Sum PEP Score, sum posterior error probability scores; PSM, peptide spectrum matches.
Figure 2. Score plot highlighting the Gangatiri milk beta-lactoglobulin among the other whey protein from the HRAMS proteome data. Sum PEP Score, sum posterior error probability scores; PSM, peptide spectrum matches.
The sequence of whey proteins obtained from the mass spectroscopy shows the Gangatiri β-Lg sequence having homology with the variant B chain given in the UniProt KB proteome database (Table 1). According to the NCBI database, variant B has UniProt KB ID number P02754. This has confirmed the B variant of β-Lg in the Gangatiri milk. The protein is coded as the PAEP gene on chromosome number 11 (Gene ID B-Lg: 280838). The genetic polymorphisms of the PAEP gene in cattle are responsible for the different genotypes. A, B, C, and D are common with variants B and A being the most prevalent (Farrell et al., Reference Farrell, Jimenez-Flores, Bleck, Brown, Butler, Creamer, Hicks, Hollar, Ng-Kwai-Hang and Swaisgood2004). The difference among these variants is the change in the positioning of amino acids on the 64th and 118th numbers, with the specific difference between A and B being, for B, glycine in the place of aspartate and alanine in the place of valine, respectively. The presence of the A variant has been linked with a high amount of β-Lg protein concentration in the milk and also higher milk yield, protein content and solids-not-fat content, in contrast, variant B is associated with a high percentage of fat (Zaglool et al., Reference Zaglool, Awad, El, Araby and El-Bayomi2016).
In other cattle breeds, such as Girolando cows (Botaro et al., Reference Botaro, Lima, Aquino, Fernandes, Garcia and Santos2008), Turkish cattle breeds (Dinc et al., Reference Dinc, Ozkan, Koban and Togan2013), Sahiwal cattle (Mir et al., Reference Mir, Ullah and Sheikh2014) and Mexican Jersey cattle (Zepeda-Batista et al., Reference Zepeda-Batista, Alarcón-Zúñiga, Ruíz-Flores, Núñez-Domínguez and Ramírez-Valverde2015), the polymorphic products of the β-Lg gene were investigated. They discovered variant B in all of the studies. The present data show that the Gangatiri breed has a B variant by showing similarities with previous reports on the genotyping of indigenous cattle. Lukac et al. (Reference Lukač, Vidović, Nemeš, Stupar and Popović-Vranješ2013) reported the predominance of the A variant in Estonian dairy cattle and Holstein cattle. Most of the previous studies on the genotyping of the β-Lg for different dairy animals have been done with the use of PCR-RFLP analysis of blood samples (Maletic et al., Reference Maletic, Aleksić, Vejnović, Nikšić, Kulić, Đukić and Ćirković2016), and this is the first attempt at the detection of the variant of this whey protein with the application of gel electrophoresis and mass-spectrometry in milk. The biological function of this whey protein is in the transport of various metabolites and in the mammary gland it has a role in metabolism also (Zaglool et al., Reference Zaglool, Awad, El, Araby and El-Bayomi2016). Some studies have also investigated the correlation of this protein variant with cheese-forming (Meza-Nieto et al., Reference Meza-Nieto, González-Córdova, Piloni-Martini and Vallejo-Cordoba2013) and compositional attributes (Khaldi et al., Reference Khaldi, Nafti, Jilani and Souid2023). Genotypes of β-Lg have highly significant effects on casein number (B > A) and protein recovery (B > A) and also determine the yield of cheese dry matter (B > A) (Di Gregorio et al., Reference Di Gregorio, Di Grigoli, Di Trana, Alabiso, Maniaci, Rando, Valluzzi, Finizio and Bonanno2017).
In conclusion, this study has shown the Gangatiri breed of cow produces milk that contains the B variant of β-Lg rather than the A variant, which potentially has a number of advantages for milk processing and quality. As far as we are aware, this is the first report of genotype identification of β-Lg in the Gangatiri breed with the applied techniques of electrophoresis separation and sequencing of amino acids using accurate mass spectroscopy.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0022029923000481.
Acknowledgements
The authors acknowledge the help of the Principal Investigator, RKVY Project No. P-26/0124, Faculty of Veterinary and Animal Sciences, Rajeev Gandhi South Campus, Banaras Hindu University, for providing Gangatiri cows. We also acknowledge the funding support of the Institute of Eminence (IoE) Research Incentive Grant, Banaras Hindu University No. R/Dev/D/IoE/Seed and Incentive Grant-III/2022-2023/49243, and technical support of the Sophisticated Analytical and Technical Help Institute (SATHI), Central Discovery Center, Banaras Hindu University.
