Development of a trifluoroacetic acid-free liquid chromatography-mass spectrometry method for protein profiling of bovine milk and authentication of A2 milk

Milk proteins, particularly caseins and whey proteins, are essential to the nutritional and functional properties of milk. However, understanding their detailed composition and genetic variations requires precise and reliable analytical techniques. In this study, we present a novel method for protein profiling of bovine milk using liquid chromatography with ultraviolet absorption and mass spectrometry detection (LC-UV and LC-MS, respectively). Initially, a reversed-phase LC-UV method was developed, using a wide-pore C4 column and acetonitrile: water mobile phases with 0.1 % ( v /v) formic acid (HFor), replacing the commonly used ion-pairing agent trifluoroacetic acid (TFA). TFA is not recommended in best practices for LC-MS, as it interferes with on-line electrospray ionization (ESI) and contaminate the mass spectrometer due to its ion-suppression effects. By adopting HFor in LC-UV, the total analysis time was reduced by 10 min, and separation was slightly affected. Moreover, the TFA-free method was fully compatible with LC-MS, demonstrating consistent performance with low relative standard deviations (%RSDs) in retention time and peak area across extended sequences of analyses, with no mass spectrometer contamination. LC-MS analysis was performed using an accurate mass, high-resolution time-of-flight (TOF) mass spectrometer, enabling accurate identification and quantification of key bovine milk proteins from whey and casein fractions, including proteoforms with subtle structural differences, such as β-casein (β-CN) A1 and A2. This allowed differentiation between various β-casein genotypes (A1A1, A1A2, and A2A2) and the quantification of low levels of contamination of A2A2 milk with A1A1 milk. These findings highlight the method's potential for high-throughput, reliable, and efficient dairy research, quality control, and authentication, without compromising the mass spectrometer performance. • A TFA-free LC-UV method for bovine milk protein profiling is developed. • The formic acid-based method is fully compatible with best practices in LC-MS. • Accurate mass, high-resolution TOF-MS enables detailed profiling of intact proteins. • β-casein A1 and A2 proteoforms can be discriminated. • Low levels of A1A1 milk contamination in A2A2 milk can be quantified.