The analysis of multiple phosphoseryl-containing casein peptides using capillary zone electrophoresis.
Multiple phosphoseryl-containing sequences of peptides and proteins stabilize amorphous calcium phosphate at neutral and alkaline pH and have been implicated in the nucleation/regulation of biomineralization. In an approach to analyze these peptides using capillary zone electrophoresis (CZE) we have attempted to relate the absolute electrophoretic mobility of various casein phosphopeptides to their physicochemical properties. Multiple phosphoseryl-containing peptides were selectively precipitated from enzymic digests of sodium caseinate and further purified using RP-HPLC and anion-exchange fast protein liquid chromatography. Purified fractions were then analyzed by CZE. Absolute electrophoretic mobilities of 13 peptides were determined by measurement of migration times relative to that of a neutral marker, mesityl oxide. A linear relationship (r2 = 0.993) was obtained between absolute electrophoretic mobility and q/M(r)2/3 where q is the net negative charge of the peptide calculated using relevant pKa values and M(r) is the molecular 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 frictional 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 suitable for quantitative analysis of peptide substrates in kinase and phosphatase studies. In conclusion CZE is a rapid and efficient technique for the resolution of multiple phosphoseryl-containing peptides from enzymic digests of casein.
Adamson
N
N
Biochemistry and Molecular Biology Unit, School of Dental Science, University of Melbourne, Australia.
Riley
P F
PF
Reynolds
E C
EC
D000595:Amino Acid Sequence; D002364:Caseins / Q000032:analysis*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004586:Electrophoresis; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology; D010748:Phosphopeptides / Q000032:analysis*; D010768:Phosphoserine / Q000032:analysis*; D014357:Trypsin
D002364:Caseins; D010748:Phosphopeptides; D010768:Phosphoserine; D014357:Trypsin