Determination of phosphorylation levels of tyrosine hydroxylase by electrospray mass spectrometry

A novel approach has been developed to quantify the extent of phosphorylati on of tyrosine hydroxylase (TH). The strategy consists of a chemical cleava ge and characterization of the products using electrospray mass spectrometr y (ESMS). The chemical cleavage involves selective hydrolysis of the aspart yl-peptide bond. Of the peptides formed, an 8-kDa NH2-terminus fragment is found to accurately duplicate the phosphorylation of TH using standard mixt ures of TH-P/TH. The calibration yields a straight line With an R-2 Of 0.99 6, 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-PKI I. The experimental conditions were designed to produce low levels of phosp horylation. Nevertheless, the ESMS analysis yielded single, double, and non phosphorylation forms of TH. With respect to in vivo measurements, this ESM S protocol may be a generic procedure for determining the extent of phospho rylation of proteins. (C) 2000 Academic Press.