Proteomic analysis and cross species comparison of casein fractions from the milk of dairy animals

Abstract

Casein micelles contribute to the physicochemical properties of milk and may also influence its functionality. At present, however, there is an incomplete understanding of the casein micelle associated proteins and its diversity among the milk obtained from different species. Therefore, milk samples were collected from seven dairy animals groups, casein fractions were prepared by ultracentrifugation and their constituent proteins were identified by liquid chromatography tandem mass spectrometry. A total of 193 distinct proteins were identified among all the casein micelle preparations. Protein interaction analysis indicated that caseins could interact with major whey proteins, including β-lactoglobulin, α-lactalbumin, lactoferrin, and serum albumin, and then whey proteins interacted with other proteins. Pathway analysis found that the peroxisome proliferator-activated receptor signaling pathway is shared among the studied animals. Additionally, galactose metabolism pathway is also found to be commonly involved for proteins derived from camel and horse milk. According to the similarity of casein micelle proteomes, two major sample clusters were classified into ruminant animals (Holstein and Jersey cows, buffaloes, yaks, and goats) and non-ruminants (camels and horses). Our results provide new insights into the protein profile associated with casein micelles and the functionality of the casein micelle from the studied animals.

Figure 1

Protein-protein interaction network of the identified proteins from co-immunoprecipitation with antibody β-casein (a), casein micelle in Holstein (b) generated with STRING software. Each node represents a protein; different line colors represent the types of evidence for the association: pink lines from experimental study, the blue lines from databases, and the yellow lines from abstracts of articles published in PubMed.

Figure 2

Western blot analysis of β-casein (a) and ‘far-western blot’ analysis of β-casein interacted with major whey proteins (b). M shows protein marker; Lanes 1, 2, and 3 represent samples with 9, 18 and 36 μg milk proteins; Lanes 4 shows β-casein standard.

Figure 3

Identified proteins of casein fractions grouped into biological process (a), cellular component (b) and molecular function (c) according to the annotated functions.