Bovine rod-outer-segment (ROS) (0.5 mg/mL) containing 1 mM ATP was incubated in HEPES buffer (pH 7.5) with or without 2 pmol PKC and 100 nM phorbol myristate acetate. Following the separation of ROS proteins by SDS-PAGE, rhodopsin gel-bands were excised, reduced, alkylated and digested by trypsin. The tryptic digest was analyzed by 2-D LC-nanospray MS/MS using a high resolution Qq-TOF (Q-star, Sciex) or an ion trap mass spectrometer (LC-MSD, Agilent). The peptide mixture was injected onto a cation exchange column (SCX PolySulfoethyl A, 30x0.3mm, 5um, PolyLC) and was fractionated by successive injections of ammonium acetate solution of increasing concentration (0, 5, 50, 500, and 1000mM). Subsequently, each eluted fraction was carried directly into a reversed phase column (PicoTip silica C18, New Objective) via a C-18 trapping column. Peptides were eluted using a mobile phase consisting of water and acetonitrile each containing 1% formic acid with 300 nL/min flow rate.

We identified multiple PKC phosphorylation in rhodopsin. Phosphorylation was identified at the residues Ser-334, Thr-335, Thr-336, and Ser-338 in the C-terminal end of the rhodopsin. Mono-, di- and tri-phosphoryated forms were also identified. All of these phosphorylated products were eluted without salt injection, while majority of non-phosphorylated tryptic peptides eluted with the injection of various salt concentrations, facilitating the analysis of phosphorylated peptide ions with low intensity. In the absence of PKC, phosphorylation was observed only at Ser-338. In addition to the MS/MS analysis, the assignment of phosphorylation at this site was further confirmed by the fact that trypsin did not cleave at the adjacent Lys-339. The data concerning the phosphorylation of rhodopsin at these PKC sites in relation to light exposure will be presented.