Capillary electrophoresis of phosphoamino acids with indirect photometric detection

The reversible phosphorylation of proteins is important in intercellular co mmunication. The most frequently phosphorylated amino acid in proteins is p hosphoserine (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 whic h are involved in signal transduction pathways. We have shown that separation of these phosphoamino acids in the presence o f similarly charged amino acids, such as aspartic acid (Asp) and glutamic a cid (Glu) can be effectively done by capillary electrophoresis (CE) with in direct detection using adenosine monophosphate (AMP). Baseline resolution o f 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 mu M) for t he phosphoamino acids is possible. This CE method has been used to follow t he acid hydrolysis of phosvitin, a P-Ser containing protein, and the number of residues found correlates statistically to the molybdate colorimetric m ethod. (C) 1999 Elsevier Science B.V. All rights reserved.