Milk protein genetic variants and isoforms identified in bovine milk representing extremes in coagulation properties

A gel-based proteomic approach consisting of 2-dimensional gel electrophoresis coupled with mass spectrometry was applied for detailed protein characterization of a subset of individual milk samples with extreme rennet coagulation properties. A milk subset with either good or poor coagulation abilities was selected from 892 Danish Holstein-Friesian and Jersey cows. Screening of genetic variants of the major milk proteins resulted in the identification of common genetic variants of β-casein (CN; A(1), A(2), B), κ-CN (A, B), and β-lactoglobulin (LG; A, B), as well as a low frequency variant, κ-CN variant E, and variants not previously reported in Danish breeds (i.e., β-CN variant I and β-LG variant C). Clear differences in the frequencies of the identified genetic variants were evident between breeds and, to some extent, between coagulation groups within breeds, indicating that an underlying genetic variation of the major milk proteins affects the overall milk coagulation ability. In milk with good coagulation ability, a high prevalence of the B variants of all 3 analyzed proteins were identified, whereas poorly coagulating milk was associated with the β-CN variant A(2), κ-CN variant A or E, and β-LG variant A or C. The β-CN variant I was identified in milk with both good and poor coagulation ability, a variant that has not usually been discriminated from β-CN variant A(2) in other studied cow populations. Additionally, a detailed characterization of κ-CN isoforms was conducted. Six κ-CN isoforms varying in phosphorylation and glycosylation levels from each of the genetic variants of κ-CN were separated and identified, along with an unmodified κ-CN form at low abundance. Relative quantification showed that around 95% of total κ-CN was phosphorylated with 1 or 2 phosphates attached, whereas approximately 35% of the identified κ-CN was glycosylated with 1 to 3 tetrasaccharides. Comparing isoforms from individual samples, we found a very consistent κ-CN isoform pattern, with only minor differences in relation to breed, κ-CN genetic variant, and milk coagulation ability.