2.4.1. Analysis of A1 β-Casein/A2 β-Casein Using Liquid Chromatograph Triple Quadrupole Mass Spectrometer (LC-MS/MS)
SEPARATION, AIDS
One hundred microliters (100 µL) of each milk sample (conventional milk and A2 milk) were denatured using 400 µL of 8 M urea and 10 mM DTT (dithiotheitol), and vortexed for 15 min at room temperature. Denaturation with urea aids in the separation of fat from protein. Fat removal was followed by the process of centrifugation. The proteins extracted from the solution were alkylated with 20 mM IAA (iodoacetamide) and digested with pepsin for one hour at room temperature using a 1:20 enzyme-to-substrate ratio. After digestion, peptides were desalted using C