Dark adaptation requires timely deactivation of phototransduction and efficient regeneration of visual pigment. No previous study has directly compared the kinetics of dark adaptation with rates of the various chemical reactions that influence it. To accomplish this, we developed a novel rapid-quench/mass spectrometry-based method to establish the initial kinetics and site specificity of light-stimulated rhodopsin phosphorylation in mouse retinas. We also measured phosphorylation and dephosphorylation, regeneration of rhodopsin, and reduction of all-trans retinal all under identical in vivo conditions. Dark adaptation was monitored by electroretinography. We found that rhodopsin is multiply phosphorylated and then dephosphorylated in an ordered fashion following exposure to light. Initially during dark adaptation, transduction activity wanes as multiple phosphates accumulate. Thereafter, full recovery of photosensitivity coincides with regeneration and dephosphorylation of rhodopsin.

On stimulation, rhodopsin, the light-sensing protein in the rod cells of the retina, is phosphorylated at several sites on its C terminus as the first step in deactivation. We have developed a mass spectrometry-based method to quantify the kinetics of phosphorylation at each site in vivo. After exposing either a freshly dissected mouse retina or the eye of an anesthetized mouse to a flash of light, phosphorylation and dephosphorylation reactions are terminated by rapidly homogenizing the retina or enucleated eye in 8 M urea. The C-terminal peptide containing all known phosphorylation sites is cleaved from rhodopsin, partially purified by ultracentrifugation, and analyzed by liquid chromatography coupled with mass spectrometry (LCMS). The mass spectrometer responds linearly to the peptide from 10 fmole to 100 pmole. The relative sensitivity to peptides with zero to five phosphates was determined using purified phosphopeptide standards. High pressure liquid chromatography (HPLC) coupled with tandem mass spectrometry (LCMS/MS) was used to distinguish the three primary sites of phosphorylation, Ser 334, Ser 338, and Ser 343. Peptides monophosphorylated on Ser 334 were separable from those monophosphorylated on Ser 338 and Ser 343 by reversed-phase HPLC. Although peptides monophosphorylated at Ser 338 and Ser 343 normally coelute, the relative amounts of each species in the single peak could be determined by monitoring the ratio of specific daughter ions characteristic of each peptide. Doubly phosphorylated rhodopsin peptides with different sites of phosphorylation also were distinguished by LCMS/MS. The sensitivity of these methods was evaluated by using them to measure rhodopsin phosphorylation stimulated either by light flashes or by continuous illumination over a range of intensities.

In this study, large-scale qualitative and quantitative proteomic technology was applied to the analysis of the opportunistic bacterial pathogen Pseudomonas aeruginosa grown under magnesium limitation, an environmental condition previously shown to induce expression of various virulence factors. For quantitative analysis, whole cell and membrane proteins were differentially labeled with isotope-coded affinity tag (ICAT) reagents and ICAT reagent-labeled peptides were separated by two-dimensional chromatography prior to analysis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) in an ion trap mass spectrometer (ITMS). To increase the number of protein identifications, gas-phase fractionation (GPF) in the m/z dimension was employed for analysis of ICAT peptides derived from whole cell extracts. The experiments confirmed expression of 1331 P. aeruginosa proteins of which 145 were differentially expressed upon limitation of magnesium. A number of conserved Gram-negative magnesium stress-response proteins involved in bacterial virulence were among the most abundant proteins induced in low magnesium. Comparative ICAT analysis of membrane versus whole cell protein indicated that growth of P. aeruginosa in low magnesium resulted in altered subcellular compartmentalization of large enzyme complexes such as ribosomes. This result was confirmed by 2-D PAGE analysis of P. aeruginosa outer membrane proteins. This study shows that large-scale quantitative proteomic technology can be successfully applied to the analysis of whole bacteria and to the discovery of functionally relevant biologic phenotypes.

Phosphorylation and regeneration of rhodopsin, the prototypical G-Protein Coupled Receptor, each can influence light-and dark-adaptation. To evaluate their relative contributions we quantified rhodopsin, retinoids, phosphorylation and photosensitivity in mice during 90 minutes of illumination followed by dark-adaptation. During illumination, all-trans retinyl esters and, to a lesser extent, alltrans retinal, accumulate and reach steady state within an hour. Each major phosphorylation site on rhodopsin reaches a steady state level of phosphorylation at a different time during illumination. The dominant factor that limits dark adaptation is isomerisation of retinal. During dark adaptation dephosphorylation of rhodopsin occurs in two phases. The faster phase corresponds to rapid dephosphorylation of regenerated rhodopsin present at the end of the illumination period. The slower phase corresponds to dephosphorylation of rhodopsin as it forms by regeneration. We conclude that rhodopsin phosphorylation has three physiological functions, it quenches phototransduction, it reduces sensitivity during light-adaptation and it suppresses bleached rhodopsin activity during dark adaptation.The temporal demands of vision require that photo-activated rhodopsin be inactivated quickly. Phosphorylation of the C-terminus of photo-activated rhodopsin by rhodopsin kinase (GRK1) serves this purpose. Together with arrestin binding, phosphorylation of rhodopsin provides a fast and effective mechanism to quench phototransduction (1,2).Vision also requires a mechanism to restore photosensitivity to rhodopsin. On a time scale slower than phosphorylation photoactivated rhodopsin decays spontaneously to form opsin and all-trans retinal. Opsin with all-trans retinal and even opsin alone weakly stimulate phototransduction (3), so it is essential to inactivate the opsin, eliminate the all-trans retinal and ultimately regenerate rhodopsin with 11-cis retinal. The all-trans retinal generated in response to light is reduced in the photoreceptor to retinol then transferred to the retinal pigment epithelium (RPE) where it is esterified to a fatty acid. The retinyl ester is isomerized and deesterified by a single protein in the RPE, an isomerohydrolase known as RPE65. This reaction generates 11-cis retinol, which is then oxidized to the aldehyde. The 11-cis retinal then returns to the photoreceptor outer segment to regenerate rhodopsin (4-6).Following an intense but brief flash of light several important biochemical changes occur in photorecepors of living rodents. The most distal serines on the C-terminal tail of rhodopsin are 3Corresponding author, James B. Hurley, 357350,