A Tyrosine Kinase Assay Using Reverse-Phase High-Performance Liquid Chromatography

Abstract

Reverse-phase HPLC can be used as a very precise and accurate routine assay for peptide phosphorylation by protein kinases that has advantages over other methods. In particular, peptides with native amino acid sequences can be used without the need for radioisotopes. However, reaction conditions that are employed can often present difficulties in recovery and quantitation of phospho- and apo-peptides. Two general problems were encountered: First, variation in the retention times of peptides and an increasing width of the injection front which can interfere with quantitation both resulted from repeated sample injections. These were caused mostly by the presence of carrier bovine serum albumin used to reduce loss of peptides during the reaction and by high concentrations of ATP used to study the kinetics of enzyme catalyzed reactions. These problems were solved by regular washing of the reverse-phase column, thus allowing a broad range of peptide and ATP concentrations to be used. Second, the stability of peptides used in the assay was affected by dithiothreitol in combination with manganese. The former is a common reagent of kinase purifications and the latter is often the metal cofactor used in kinase reactions. Minimizing the concentration of dithiothreitol or using magnesium resolved these difficulties. Consideration of these factors is therefore important when using reverse-phase HPLC to monitor peptide phosphorylation in protein tyrosine kinase assays.