Quantification of Casein in Baked Food Products by Selective Analysis of Phosphorylated Peptides Using Fluorous Derivatization with Liquid Chromatography-Tandem Mass Spectrometry Method

Casein is one of the allergen proteins present in milk. Therefore, a quantification method for the selective analysis of casein using fluorous derivatization with LC-tandem mass spectrometry (LC-MS/MS) was developed. After two allergen proteins (α_S1-casein and β-casein) extracted from baked sugar cookies were tryptic digested, the obtained phosphorylated peptides were selectively derivatized by β-elimination with Ba(NO₃)₂ under basic condition and Michael addition with perfluoroalkylthiol (1H,1H,2H,2H-perfluorooctanethiol, PFOT). In this study, YKVPQLEIVPN(pSer)AQQR (104-119 fragment from α_S1-casein) and FQ(pSer)EEQQQTEDELQDK (33-48 fragment from β-casein) obtained by tryptic digestion were selected as target peptides. The phosphorylated serine residue in each peptide was converted to a perfluoroalkyl group by derivatization. The obtained fluorous-derivatized peptides were analyzed by LC-MS/MS, to which a fluorous LC column was connected. Therefore, it was possible to analyze casein without being affected by the matrix components in the baked food sample. When the present method was applied to cookies with arbitrary amounts of α_S1-casein and β-casein, the obtained quantification values were in good agreement with the arbitrary amounts spiked. The quantification limits of α_S1- and β-casein in cookie analysis were 246 and 152 ng/g, respectively. Hence, this method can be used to analyze trace amounts of allergen proteins present in the baked food.