A rapid microdetermination of phosphoserine, phosphothreonine, and phosphotyrosine in proteins by automatic cation exchange on a conventional amino acid analyzer.

A cation-exchange chromatographic method for the separation and determination of phosphoserine, phosphothreonine, and phosphotyrosine in proteins after partial acid hydrolysis is described. The short column (0.6 X 8 cm) of an automatic amino acid analyzer was used and elution was carried out isocratically with 10 mM trifluoroacetic acid. The method is highly sensitive and each of the three O-phosphoamino acids can be accurately determined down to the 50-pmol level. Higher sensitivity may be obtained by the use of [32P]phosphate-labeled proteins. A correction factor for the decomposition of phosphoserine or phosphothreonine during acid hydrolysis can be deduced from the amount of inorganic phosphate recovered at the column void volume. The method is sensitive enough to be used for 32P-labeled proteins isolated by two-dimensional gel electrophoresis.