Non-radioactive Determination of Phosphorylation in Proteins or Peptides by Capillary Electrophoresis

A new method of capillary electrophoresis was described which allowed the non-radioactive determination of all O-phosphoamino acids in peptides or proteins. It involved a partial hydrolysis of the peptide bonds, the derivatization of an amino acid mixture with phenylisothiocyanate and the separation of all the PTH-phosphoamino acids from other PTH-amino acids by capillary electrophoresis. The correlation coefficients of the linear least-squares regression curves of all the phophoamino acids in concentration ranging from 25 to 250 pmol/&mgr;l, were greater than 0.992 (n=6). Detection limits were in the fmol range. The method had been applied to the analysis of the phosphorylation sites in several model polypeptides and two nature phosphorylated proteins beta-casein and phosvitin.