title: 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.
authors: Department of Chemistry and Biology, Ryerson University, Toronto, Ontario, Canada, M5B 2K3.
authors: Muhammad Ali
authors: MA
authors: Naqvi
authors: Department of Chemistry and Biology, Ryerson University, Toronto, Ontario, Canada, M5B 2K3.
authors: Jasjit
authors: J
authors: Singh
authors: Department of Chemistry and Biology, Ryerson University, Toronto, Ontario, Canada, M5B 2K3.
authors: Eugene
authors: E
authors: Han
authors: Department of Chemistry and Biology, Ryerson University, Toronto, Ontario, Canada, M5B 2K3.
authors: Koushan
authors: K
authors: Farshad
authors: Department of Chemistry and Biology, Ryerson University, Toronto, Ontario, Canada, M5B 2K3. Electronic address: rousseau@ryerson.ca.
authors: Dérick
authors: D
authors: Rousseau
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D013058:Mass Spectrometry
chemical_list: D002364:Caseins
keywords: HPLC; casein; isolation; mass spectrometry; phosphopeptide