Quantitative LC-MS/MS analysis of high-value milk proteins in Danish Holstein cows

Abstract

High-value milk proteins, which can be obtained by optimized fractionation procedures, are ideal ingredients in many food applications. Thus, a simple and robust analytical method is required for the identification and quantification of these individual milk proteins. Here, we present a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method using multiple reaction monitoring (MRM) to simultaneously detect and measure target peptides of two major milk proteins, α-lactalbumin (α-LA) and β-casein (β-CN), in raw milk samples from 662 Danish Holstein cows. The MRM quantification of α-LA and β-CN was achieved with limit of detection (LOD) of 0.14 and 0.16 g/L, respectively and reproducibility of the assay <15%. By this newly established MRM-based method, the concentration of α-LA and β-CN in an individual cow's milk ranged from 0.5 to 1.9 (average 1.1) g/L, and from 7.5 to 23.4 (average 15) g/L, respectively. There was no significant effect of parity, whereas significantly increasing concentrations of α-LA and β-CN were observed through lactation (P < 0.001). This shows a considerable biological variation of these two ingredient milk proteins, providing potential varying outputs of fractionation in the dairy streams.

Investigation of the Presence of Fibrillin in Sea Cucumber (Apostichopus japonicus) Body Wall by Utilizing Targeted Proteomics and Visualization Strategies

“…The calibration curves were fabricated by plotting the peak area ( y ) and mass concentration ( x , μg/mL) of each signature peptide. The established MRM-based method for quantifying fibrillin was subjected to validation (parameters as follows) according to a previous study with slight modifications . The limit of detection (LOD) and limit of quantification (LOQ) were regarded as 3.3 and 10 times the ratio of standard deviation and standard curve slope, respectively.…”

“…These results indicated that the established method has good sensitivity and high repeatability, which was similar to the previous investigations on other protein quantifications. 14,19 Hence, a feasible MRM-based method for the quantification of sea cucumber fibrillin has been established. The method has a variety of potential applications, such as the calculation of fibrillin content in A. japonicus and monitoring of fibrillin degradation during autolysis.…”

“…The established MRM-based method for quantifying fibrillin was subjected to validation (parameters as follows) according to a previous study with slight modifications. 14 The limit of detection (LOD) and limit of quantification (LOQ) were regarded as 3.3 and 10 times the ratio of standard deviation and standard curve slope, respectively. The relative standard deviation (RSD) was calculated by injecting the same sample three times.…”

Dairy Milk Protein–Derived Bioactive Peptides: Avengers Against Metabolic Syndrome

“…Likewise, when treated with aged rat skin fibroblasts, the bovine milk β-casein–derived peptide VLPVPQK increased cell migration by decreasing IL-6 and TNF-α, nuclear transmigration of the dormant Nrf2, and cell proliferation. The peptide reverses the growth arrest in aged fibroblast cells by lessening the activities of caspase-9 and -3, retaining nuclear integrity, and downregulating NF-κB/p38MAP kinase signaling by means of decreasing phosphorylated p38MAP kinases in the cytoplasm and by Nrf2 activation [ 63 , 64 ].…”

Genetic factors affecting the composition and quality of cow’s milk

“…Other functional important proteins include one of the major milk proteins, β-CN, as well as the less abundant but bioactive proteins, like osteopontin and lactoferrin. For β-CN and α-LA, large biological variations in their contents in individual cow's milk were revealed, showing potentials for exploiting this to elevate the levels of these valuable components through natural means (Le et al, 2020). Production of β-CN A 2 infant formulae has also gained interest, which may lead to an increase of β-CN A 2 cows/farms.…”