THE ANALYSIS OF MULTIPLE PHOSPHOSERYL-CONTAINING CASEIN PEPTIDES USING CAPILLARY ZONE ELECTROPHORESIS

Multiple phosphoseryl-containing sequences of peptides and proteins st abilize amorphous calcium phosphate at neutral and alkaline pH and hav e been implicated in the nucleation/regulation of biomineralization. I n an approach to analyze these peptides using capillary zone electroph oresis (CZE) we have attempted to relate the absolute electrophoretic mobility of various casein phosphopeptides to their physicochemical pr operties. Multiple phosphoseryl-containing peptides were selectively p recipitated from enzymic digests of sodium caseinate and further purif ied using RP-HPLC and anion-exchange fast protein liquid chromatograph y. Purified fractions were then analyzed by CZE. Absolute electrophore tic mobilities of 13 peptides were determined by measurement of migrat ion times relative to that of a neutral marker, mesityl oxide. A linea r relationship (r2 = 0.993) was obtained between absolute electrophore tic mobility and q/M(r)2/3 where q is the net negative charge of the p eptide calculated using relevant pK(a) values and M(r) is the molecula r mass. M(r)2/3 is a measure of the surface area of a sphere that has a volume proportional to the M(r) of the peptide and relates to the fr ictional drag exerted on the peptide during electrophoretic migration. As absolute electrophoretic mobility is influenced by charge and size CZE can be used to monitor peptide phosphorylation, dephosphorylation , deamidation and truncation. This technique therefore would be suitab le for quantitative analysis of peptide substrates in kinase and phosp hatase studies. In conclusion CZE is a rapid and efficient technique f or the resolution of multiple phosphoseryl-containing peptides from en zymic digests of casein.