Post translational modifications of milk proteins in geographically diverse goat breeds

www.nature.com/scientificreports OPEN Post translational modifications of milk proteins in geographically diverse goat breeds P. K. Rout* & M. Verma Goat milk is a source of nutrition in difficult areas and has lesser allerginicity than cow milk. It is leading in the area for nutraceutical formulation and drug development using goat mammary gland as a bioreactor. Post translational modifications of a protein regulate protein function, biological activity, stabilization and interactions. The protein variants of goat milk from 10 breeds were studied for the post translational modifications by combining highly sensitive 2DE and Q-Exactive LC-MS/ MS. Here we observed high levels of post translational modifications in 201 peptides of 120 goat milk proteins. The phosphosites observed for CSN2, CSN1S1, CSN1S2, CSN3 were 11P, 13P, 17P and 6P, respectively in 105 casein phosphopeptides. Whey proteins BLG and LALBA showed 19 and 4 phosphosites respectively. Post translational modification was observed in 45 low abundant noncasein milk proteins mainly associated with signal transduction, immune system, developmental biology and metabolism pathways. Pasp is reported for the first time in 47 sites. The rare conserved peptide sequence of (SSSEE) was observed in αS1 and αS2 casein. The functional roles of identified phosphopeptides included anti-microbial, DPP-IV inhibitory, anti-inflammatory and ACE inhibitory. This is first report from tropics, investigating post translational modifications in casein and non-casein goat milk proteins and studies their interactions. Milk is the primary source of nutrition for mammals and serves as a major vehicle of maternal immunity transfer, thus, plays a vital role in inclusive development of the neonates. The milk proteome is extremely complex due to abundant post-translational modifications and various proteolytic processes1. Milk protein composition exhibited high heterogeneity due to numerous genetic variants, and isoforms with different degrees of posttranslational modifications such as phosphorylation and glycosylation in c aseins2,3. Milk proteins exhibit conformational structure due to post translational modifications and constitutive levels of proteolytic activity produce a range of significant peptides. The posttranslational modifications of the polypeptide chain occur in the Golgi apparatus of the mammary epithelial c ells4. Casein phosphorylation at amino acid serine or threonine is catalyzed by kinase enzymes5. Phosphorylation is affected by different factors such as protein sequence, efficacy of kinase enzymes, gene expression, substrate availability and access to phosphorylation site which is responsible for the specific protein conformation2,6. Protein functions such as binding, stabilization, biological activity, interactions with proteins and other biomolecules are regulated by phosphorylation-dephosphorylation of p rotein7. Phosphorylation stabilizes calcium phosphate nano clusters in casein m icelles8. The micellar structure of casein enables milk to carry calcium and phosphate to the neonate by channelizing the risk of mammary gland bio-calcification9,10. Phosphorylation state of caseins varies widely from 1P to 3P on CSN3, 4P to 5P on CSN2, 8P to 9P on CSN1S1, 10P to 13P on CSN1S26,11. In bovine milk, CSN1S1 accounts for about 35% of the total casein and has 2 common phosphorylation isoforms: CSN1S1-8P and CSN1S1-9P. Similarly, CSN1S2 accounts for about 10% of the total casein and is present with isoforms from 10 to 14P and occasionally with 9P or 1 5P2,12. The identification and analysis of phosphopeptides has been challenging because of the relatively low stoichiometry, inherent lower ionization efficiency and variation of phosphorylation sites13,14. However, advances in proteomics have largely enhanced the quotient of protein identification. Phosphorylation of milk proteins has been studied in llama15, camel16 and goat milk fat globule membrane proteins17 using MS/MS proteomic approach. A total of 8 phosphopeptides corresponding to 18 phosphorylation sites were identified in CSN1S1, CSN1S2 and CSN2 of goat milk using nLC-MS/MS18. Casein phosphopeptides in goat milk have been studied by Olumee-shabon and Boehemer18 and phosphoproteome of goat milk fat globule membrane have been reported by Henry and others17. Phosphorylation have been reported for bovine caseins using inductively coupled plasma Genetics and Breeding Division, ICAR-Central Institute for Research on Goats, Makhdoom, Farah, Mathura, Uttar Pradesh 281122, India. *email: Scientific Reports | (2021) 11:5619 | https://doi.org/10.1038/s41598-021-85094-9 1 Vol.:(0123456789) www.nature.com/scientificreports/ Protein name Gene ID Number of phosphosites identified Identified phosphorylation sites α-S1 casein CSN1S1 11 S27, D58, S61, S63, T64, D66, Y95, D100, Y106, S130, S138 α-S2-casein CSN1S2 13 S23, S24, S25, S145, T146, S147, S151, T154, D156, S159, T160, T161, T164 β-casein CSN2 17 S50, T56, D58, D62, Y129, T135, S137, S139, T141, T143, D144, S157, S167, T169, S181, D197, Y206 κ-casein (Fragment) CSN3 6 S24, Y16, S60, T64, T73, S95 α-lactalbumin LALBA 4 D82, D83, D116, Y122 β-lactoglobulin BLG 19 D29, T36, Y38, S39, S45, D46, S48, D51, S54, Y60, T67, T94, D114, T115, D116, Y117, T143, D147, D155 Table 1.  Identified phosphosites for the major milk proteins in Capra hircus reference database. mass spectrometry (ICP-MS)19, equine CSN1S1 and and CSN2 by nESI-MS/MS20,21, donkey CSN2 by MALDITOF and nESI-MS/MS22 and for CSN1S1 and CSN2 of water buffalo by M S23. Bovine milk is extensively used, due to its high biological value and plasticity as it can be transformed to cheese and several other dairy products. The functional knowledge of casein and whey proteins has been identified for the presence of bioactive p eptides24. These peptides show various biological activities and are released by proteolytic digestion of caseins and milk proteins in gut or during fermentation. This study was designed to gain an insight into molecular diversity of goat milk proteins and to identify various degree of PTM in casein and noncasein proteins. The characterization of high and low abundant milk proteins by gel based proteomic approach i.e. 2DE and nLC-MS/MS. The present study analysed post-translational modification such as phosphorylation, oxidation, and carbamidomethylation on casein and non-casein proteins in goat milk. As post-translational modifications (PTMs) are acting as the major means of intracellular communication, therefore, the interactions of various proteins involved in post translational modifications were analysed. Results The present study has analyzed the post translational modifications in low and high abundant proteins in goat milk of genetically diverse goat breeds/genotypes reared in varied ecological and grazing condition in India. The present study generated a comprehensive profile of PTM in goat milk and identif (...truncated)