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

摘要

Infant formulations are constantly evolving as novelprotein ingredients are added to make them more closelymimic the protein profile of human milk; however,precise analytical methods for characterizing and quantifyingthe major milk proteins in such formulationsare currently lacking. This article describes an ultraperformanceliquid chromatography–high-resolutionmass spectrometry method for intact proteins that canefficiently detect, identify, and characterize the majormilk proteins and their proteoforms (phosphorylationstatus, degree of glycation, genetic variants among others)in ingredients and final products, with an emphasison detecting and quantifying specific genetic variants ofβ-casein in infant formulas. Method sensitivity allowsdetection of β-casein A1 in A2-based infant formulaswith a limit of detection of 2% (grams of β-casein A1 per100 g of total β-casein). Protein glycation affects signalintensity in a linear fashion, which permits proteinsto be quantified from their mass spectrometry signalsafter correction according to their measured glycationindex. The method was validated for the quantificationof β-casein in infant formulas. Repeatability rangedfrom 2 to 3% and intermediate reproducibility from 5to 9%. Calculated β-casein amounts ranged between 77and 110% of the values based on formulations and publishedprotein profiles for milk. Altogether, this methodcan be used for general fingerprinting as well as specificcharacterization and quantification of individual majormilk proteins in dairy-based ingredients and products.