Cloud point extraction applied to casein proteins of cow milk and their identification by mass spectrometry

This work describes the optimization of a cloud point extraction (CPE) method for casein proteins from cow milk samples. To promote phase separation, polyoxyethylene(8) isooctylphenyl ether (Triton® X-114) and sodium chloride (NaCl) were used as nonionic surfactant and electrolyte, respectively. Using multivariate studies, four major CPE variables were evaluated: Triton® X-114 concentration, sample volume, NaCl concentration, and pH. The results show that surfactant concentration and sample volume were the main variable affecting the CPE process, with the following optimized parameters: 1% (w/v) Triton® X-114 concentration, 50 μL of sample volume, 6% (w/v) NaCl concentration and extractions carried out at pH 7.0. At these conditions, 923 ± 66 and 67 ± 2 μg mL−1 of total protein were found in the surfactant-rich and surfactant-poor phases, respectively. Finally, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was then used to evaluate those target proteins (αs1-casein, αs2-casein and β-casein) separation as well as to check the efficiency of the extraction procedure, making a fingerprint of those target proteins possible.