Reverse phase high performance liquid chromatograph for analysis of casein phosphopeptides

Abstract

Milk intrinsically provides the essential calcium requirements in the diet. Upon examining the components of milk that make it a natural source of calcium, it was discovered that casein phosphopeptides are partially responsible for the bioavailability of calcium in milk. This study examined high performance liquid chromatography as a viable method for purifying casein phosphopeptides. Specifically, reverse phase HPLC (high performance liquid chromatography) was used to purify casein phosphopeptides. Initially, sodium casemate was enzymatically digested with trypsin at pH 8.0 for 24 hours. Following a calcium precipitation, preliminary purification was achieved using a reverse phase C-3 (containing propyl hydrocarbons attached to the silica) analytical column with a gradient of 0-70% acetonitrile over 49 minutes. Selected fragments of alpha and beta casein were collected and further resolved using a reverse phase C-1 8 (containing octadecyl hydrocarbons attached to the silica) analytical column. By extending the gradient to 1 5-85% over 70 minutes, the C1 8 column provided acute resolution of casein peaks. Peaks were individually collected and submitted to amino acid analysis for composition determination. Analysis containing multiple phosphorylated serine residues were compared to computer simulated casein phosphopeptides generated from the University of Wisconsin Genetics User Group (UWGCG). One of the peaks of the digested beta casein was found to have an amino acid composition matching that of the phosphopeptide fragment found in beta casein. Peaks from the digested alpha casein could not be conclusively matched to any of the phosphopeptide fragments expected. However, several of the peaks were found to have a significant number of serine residues. Protein sequence analysis via Edman degradation confirmed that the peak from the digested beta casein was in fact one of the phosphopeptides responsible for binding of calcium in caseins.