Capillary electrophoresis of phosphoamino acids with indirect photometric detection Original Research Article

The reversible phosphorylation of proteins is important in intercellular communication. The most frequently phosphorylated amino acid in proteins is phosphoserine (P-Ser); however, phosphothreonine (P-Thr), phosphotyrosine (P-Tyr), and phosphoarginine (P-Arg) are also found in decreasing frequency. The detection of phosphorylated amino acids in the presence of amino acids is important to an understanding of the role of many proteins, some of which are involved in signal transduction pathways. We have shown that separation of these phosphoamino acids in the presence of similarly charged amino acids, such as aspartic acid (Asp) and glutamic acid (Glu) can be effectively done by capillary electrophoresis (CE) with indirect detection using adenosine monophosphate (AMP). Baseline resolution of P-Tyr, P-Thr, and P-Ser, without interference of Asp and Glu or residual phosphate, in about 18 min with detection limits of 1 mg/l (3–6 μM) for the phosphoamino acids is possible. This CE method has been used to follow the acid hydrolysis of phosvitin, a P-Ser containing protein, and the number of residues found correlates statistically to the molybdate colorimetric method.