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This study aimed to analyze metabolite abundances and proteome differences between buffalo milk from Binglangjiang (BBM) and Dehong (DBM). Untargeted UPLC-MS/MS, label-free proteomics approach, and bioinformatics analyses including GO, KEGG, and PPI were performed. The results revealed that catechol, L-proline, γ-aminobutyric acid, hippuric acid, creatinine, and betaine were detected in BBM and DBM as the major metabolites. One hundred thirteen proteins, mainly within the molecular weight range of 20–80 kDa, were identified, and the differential protein numbers of BBM were higher than those of DBM. These proteins were primarily involved in cholesterol metabolism, Staphylococcus aureus and Salmonella infections, the peroxisome proliferator-activated receptor signaling pathway, and coagulation cascades. Relative abundances of α-s1-casein, α-s2-casein, and β-casein were plentiful in BBM, while κ-casein, albumin, and α-lactalbumin were in DBM. PPI network analysis indicated that the identified proteins were highly interconnected. These findings promote a better understanding of quality biomarkers in dairy product improvements. [ABSTRACT FROM AUTHOR]

The recent findings on β-casein (β-CN) A2 cattle’s milk variant have triggered global research interest, as it is claimed to be a safer choice for human health. Only a few studies have been performed to investigate the presence of β-CN A1 and A2 alleles of the CSN2 gene in dual-purpose Simmental and Angus cattle in Yunnan province, China. This present study aimed to genotype cattle producing A2 milk, predict the physicochemical properties, and quantify the isoform concentration of β-CN A2 protein. Blood samples were collected from 286 cattle (201 Simmental and 85 Angus breeds). Genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) coupled with the sequencing method. Physicochemical properties were predicted using several web-based bioinformatics tools. A sandwich enzyme-linked immunosorbent assay (ELISA) was used to measure β-CN A2 protein isoform concentration in fresh A2 milk. Allele frequencies of β-CN A2 were more dominant in Simmental (0.642) and Angus (0.835) than A1. The β-CN A1A2 genotype was most frequent in Simmental (44.8%), while A2A2 genotype was primarily found in Angus (70.6%). The predicted monoisotopic mass, molecular weight, isoelectric point (pI), net charge at pH 7, total number of atoms, instability index, and grand average of hydropathicity (GRAVY) of β-CN A2 were 23,567.23 Da, 23,582.30 Da, 5.24, –6.5, 3,357, 96.53, and –0.355, respectively. The β-CN A2 protein isoform concentration in Angus (17.8 ng/ml) was higher than in Simmental (16.7 ng/ml). Identifying β-CN A2 milk provides new insights for screening favorable alleles and establishing breeding programs based on marker-assisted selection. [ABSTRACT FROM AUTHOR]