Fig. 1: Established signalling pathway of rho, experimental conditions for preparation of vesicles for GPCR signalling and representative mass spectrum.
From: Capturing a rhodopsin receptor signalling cascade across a native membrane
a, Following absorption of a photon of light hv (1), 11-cis-retinal of rho isomerizes to the all-trans isomer (2). The activated states of rho (rho*) engage with transducin (Gt), consisting of Gαt•GDPβγt, and exchange GDP for GTP. Gt dissociates to form Gαt•GTP and Gβγt; loss of retinal from rho* leads to the formation of opsin (3). α-Subunits of Gt interact with the γ-subunits in the PDE6 enzyme, with γ-subunits undergoing a conformational change, relieving inhibition and thereby activating PDE6 to cause hydrolysis of cGMP (4). Depletion of cGMP then closes the ion channel and the ‘dark current’ is terminated (5). The resulting change in the membrane potential produces the sensation of light. b, Disc membranes of rod cells are homogenized to form a heterogeneous distribution of vesicles that are introduced directly into the mass spectrometer (MS). The spectrum shown was recorded using the parameters stated above, which led to the dissociation of lipids from proteins. c, An LED light source is configured to apply timed light intervals before the electrospray ionization of the vesicles into an Orbitrap mass spectrometer under the conditions noted. HCD, higher-energy collisional dissociation. d, Following the addition of a soluble fraction containing PDE6 and Gt, all proteins along the signalling pathway were detected. The proteins were ejected intact as rho/opsin (red), trimeric Gt•GDP (cyan) and tetrameric PDE6•cGMP (pink). The mass spectrum shown from m/z 4,000 to m/z 8,000 represents the raw data. The experiment was repeated three times.
