Purification and identification of β-casein phosphopeptide (1-25)

Abstract

The β-casein phosphopeptide 1–25 (βCPP) is involved in calcium binding, cellular transduction, and dental remineralization. The objective of this work was to improve upon the original protocol commonly used for isolation of this phosphopeptide from β-casein. This method exploits the isoelectric point of β-casein fragments to selectively precipitate βCPP. The highest βCPP extraction yield reported to date with this protocol is 14.4 ± 0.5% of theoretically available βCPP. The present work optimizes 2 steps in this procedure, namely the length of trypsin digestion and incorporation of cold acetone precipitation, to increase the yield to 32.3 ± 5.4%. Reverse-phase HPLC indicated high purity of the isolate, whereas mass spectrometry confirmed 2 forms of the phosphopeptide: fragments 1–25 (87%) and 2–25 (13%). The adaptation of the existing protocol represents a significant improvement in extraction yield and facilitates preparation of larger amounts of high-purity βCPP for subsequent analysis and use in functional foods and other applications.