Identification of phosphorylated amino acids in cardiac PR65A.

MALDI/TOF spectra for PR65A extracted from Dahl R rat hearts show that S303 (Panel A), T268 (Panel B), and S314 (Panel C) are phosphorylated. Methods: Phosphoproteins from control Dahl R rats were separated in 2D-DIGE. The protein spot corresponding to PR65A was excised from the gel, subjected to tryptic digestion and phosphopeptide enrichment using Supel-Tips (Sigma-Aldrich, Inc). The phosphopeptides were spotted on an eMALDI plate followed by mass spectrometry and database search. The spectra of all peptides were manually evaluated for the loss of phosphate which is shown in red circles and the mass and sequence of the peptides are shown. X-axis represents mass and Y axis represents intensity (see General Experimental Methods for details).