Simultaneous quantification of α-lactalbumin and β-casein in human milk using ultra-performance liquid chromatography with tandem mass spectrometry based on their signature peptides and winged isotope internal standards

• An new UPLC–MS/MS method for quantification of human milk α-Lac & β-CN was developed.
• The winged IS could release isotope-labeled signature peptide after tryptic digestion.
• The winged IS can sufficiently track the enzymatic digestion.
• The method provided a new reference strategy for analysis of other human milk proteins.

In recent years, there is an increasing need to measure the concentration of individual proteins in human milk, instead of total human milk proteins. Due to lack of human milk protein standards, there are only few quantification methods established. The objective of the present work was to develop a simple and rapid quantification method for simultaneous determination of α-lactalbumin and β-casein in human milk using signature peptides according to a modified quantitative proteomics strategy. The internal standards containing the signature peptide sequences were synthesized with isotope-labeled amino acids. The purity of synthesized peptides as standards was determined by amino acid analysis method and area normalization method. The contents of α-lactalbumin and β-casein in human milk were measured according to the equimolar relationship between the two proteins and their corresponding signature peptides. The method validation results showed a satisfied linearity (R2 > 0.99) and recoveries (97.2–102.5% for α-lactalbumin and 99.5–100.3% for β-casein). The limit of quantification for α-lactalbumin and β-casein was 8.0 mg/100 g and 1.2 mg/100 g, respectively. CVs for α-lactalbumin and β-casein in human milk were 5.2% and 3.0%. The contents of α-lactalbumin and β-casein in 147 human milk samples were successfully determined by the established method and their contents were 205.5–578.2 mg/100 g and 116.4–467.4 mg/100 g at different lactation stages. The developed method allows simultaneously determination of α-lactalbumin and β-casein in human milk. The quantitative strategy based on signature peptide should be applicable to other endogenous proteins in breast milk and other body fluids.