Separation and characterization of bovine milk proteins by capillary electrophoresis-mass spectrometry

Abstract

Protein profiling of major bovine milk proteins (i.e., whey and casein proteins) is of great interest in food science and technology. This complex set of protein proteoforms may vary with breed, genetics, lactation stage, health, and nutritional status of the animal. Current routine methods for bovine milk protein profiling at the intact level are typically based on capillary electrophoresis-ultraviolet, which does not allow confirming unequivocally the identity of the separated proteins. As an alternative, in this study, we describe for the first time a novel and simple capillary electrophoresis-mass spectrometry method in positive electrospray ionization mode. Under the optimized conditions, capillary electrophoresis-mass spectrometry allowed the separation and identification at the intact level of major bovine milk whey and casein proteins in less than 15 min. Furthermore, high-resolution mass spectrometry confirmed its importance in the reliable characterization of bovine milk protein proteoforms, especially those with slight molecular mass differences, such as β-casein A1 and A2, which are relevant to unequivocally identify milk with specific β-casein compositions (e.g., A2A2 milk, which is widely known as A2 milk). This differentiation was not possible by matrix-assisted laser desorption/ionization mass spectrometry, which provided rapidly and easily a rich but less accurate fingerprint of bovine milk proteins due to the lower mass resolution.

Keywords: capillary electrophoresis; food analysis; mass spectrometry; milk proteins; proteoforms.

FIGURE 1

CE‐UV electropherogram at 214 nm for an ultra‐high temperature (UHT) skim milk sample. Hydroxypropyl cellulose (HPC) coated capillary (58 cm L_T × 50 μm id × 365 μm od, 49.5 cm effective length). BGE: sodium citrate 10 mM, 0.05% (m/v) hydroxyethyl cellulose (HEC), 6 M urea, adjusted to pH 2.5 with citric acid. Sample injection: 10 s at 50 mbar. Temperature 45°C and voltage 30 kV

FIGURE 2

Extracted ion electropherograms (EIEs) of the bovine milk proteins detected by CE‐MS in an ultra‐high temperature (UHT) skim milk sample. Detection of the different proteins in the peaks labeled with an asterisk was confirmed by obtaining the experimental relative molecular mass (M_r) after deconvolution of the mass spectra (experimental M_r values are shown in Table 1). The summed intensity of the m/z values of the 5 most intense molecular ions in the mass spectra was considered for each EIE. Hydroxypropyl cellulose (HPC) coated capillary (58 cm × 75 μm id × 365 μm od). BGE: HAc 2 M (pH 2.2). Sample injection: 10 s at 50 mbar (sandwiched between two plugs of water of 20 s at 50 mbar). Temperature 25°C and voltage 15 kV

FIGURE 3

a) Experimental and b) deconvoluted mass spectra obtained by CE‐MS for the time window of the β‐casein A1 (βCN‐A1) and β‐casein A2 (βCN‐A2) peaks in the extracted ion electropherograms (EIEs) of Figure 2

FIGURE 4

MALDI‐MS mass spectrum for an ultra‐high temperature (UHT) skim milk sample (acquired over a range of 5000–30 000 m/z using the mid‐mass positive mode. The mass spectrum is shown in the m/z range where peaks were detected). Protein m/z values labeled with an asterisk correspond to lactose adducts from the Maillard reaction