Determination of phosphorylation levels of tyrosine hydroxylase by electrospray mass spectrometry

A novel approach has been developed to quantify the extent of phosphorylation of tyrosine hydroxylase (TH). The strategy consists of a chemical cleavage and characterization of the products using electrospray mass spectrometry (ESMS). The chemical cleavage involves selective hydrolysis of the aspartyl-peptide bond. Of the peptides formed, an 8-kDa NH2-terminus fragment is found to accurately duplicate the phosphorylation of TH using standard mixtures of TH-P/TH. The calibration yields a straight line with an R2 of 0.996, which is valid within the 10-90% range. The ESMS protocol has been used to determine the extent of phosphorylation of TH in the presence of CaM-PKII. The experimental conditions were designed to produce low levels of phosphorylation. Nevertheless, the ESMS analysis yielded single, double, and nonphosphorylation forms of TH. With respect to in vivo measurements, this ESMS protocol may be a generic procedure for determining the extent of phosphorylation of proteins.