Protein fingerprinting and quantification of β-casein variants by ultra-performance liquid chromatography-high-resolution mass spectrometry

Infant formulations arc constantly evolving as novel protein ingredients are added to make them more closely mimic the protein profile of human milk; however, precise analytical methods for characterizing and quantifying the major milk proteins in such formulations are currently lacking. This article describes an ultra-performance liquid chromatography-high-resolution mass spectrometry method for intact proteins that can efficiently detect, identify, and characterize the major milk proteins and their proteoforms (phosphorylation status, degree of glycation, genetic variants among others) in ingredients and final products, with an emphasis on detecting and quantifying specific genetic variants of beta-casein in infant formulas. Method sensitivity allows detection of beta-casein A1 in A2-based infant formulas with a limit of detection of 2% (grams of beta-casein A1 per 100 g of total beta-casein). Protein glycation affects signal intensity in a linear fashion, which permits proteins to be quantified from their mass spectrometry signals after correction according to their measured glycation index. The method was validated for the quantification of beta-casein in infant formulas. Repeatability ranged from 2 to 3% and intermediate reproducibility from 5 to 9%. Calculated beta-casein amounts ranged between 77 and 110% of the values based on formulations and published protein profiles for milk. Altogether, this method can be used for general fingerprinting as well as specific characterization and quantification of individual major milk proteins in dairy-based ingredients and products.